U.S. patent application number 09/860618 was filed with the patent office on 2002-03-14 for methods for diagnosing and treating hemostatic disorders by modulating p-selectin activity.
Invention is credited to Andre, Patrick, Hartwell, Daqing W., Hrachovinova, Ingrid, Wagner, Denisa D..
Application Number | 20020031508 09/860618 |
Document ID | / |
Family ID | 22763420 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020031508 |
Kind Code |
A1 |
Wagner, Denisa D. ; et
al. |
March 14, 2002 |
Methods for diagnosing and treating hemostatic disorders by
modulating P-selectin activity
Abstract
The present invention identifies P-selectin as a modulator of
hemostasis. Accordingly, the present invention relates to methods
for the identification and use of modulators of P-selectin activity
as modulators of hemostasis. The invention also relates to methods
and compositions for the diagnosis and treatment of hemostatic
disorders, including, but not limited to, hemorrhagic disorders and
thrombotic disorders. The present invention describes methods for
the diagnostic evaluation and prognosis of various hemostatic
conditions, and for the identification of subjects exhibiting a
predisposition to such conditions. In addition, the present
invention provides methods for the diagnostic monitoring of
patients undergoing clinical evaluation for the treatment of a
hemostatic or vascular disorders, and for monitoring the efficacy
of compounds in clinical trials.
Inventors: |
Wagner, Denisa D.;
(Wellesley, MA) ; Andre, Patrick; (Jamaica Plain,
MA) ; Hartwell, Daqing W.; (Brookline, MA) ;
Hrachovinova, Ingrid; (Jamaica Plain, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
22763420 |
Appl. No.: |
09/860618 |
Filed: |
May 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60205734 |
May 19, 2000 |
|
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Current U.S.
Class: |
424/94.63 ;
424/145.1; 514/1.9; 514/13.5; 514/14.9; 514/19.1; 514/19.3 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
9/10 20180101; G01N 2333/70564 20130101; A61P 35/00 20180101; C07K
2319/30 20130101; G01N 33/86 20130101; A61P 7/00 20180101; A61P
43/00 20180101; A61K 48/00 20130101; A61P 7/02 20180101; A61P 7/04
20180101; A61K 38/178 20130101 |
Class at
Publication: |
424/94.63 ;
424/145.1; 514/12 |
International
Class: |
A61K 038/48; A61K
039/395; A61K 038/17 |
Claims
What is claimed:
1. A method for inducing hemostasis in a subject, comprising
administering to said subject an inducer of P-selectin activity,
such that hemostasis occurs.
2. The method of claim 1, wherein the inducer of P-selectin
activity increases the level of soluble P-selectin polypeptide in
the plasma of the subject.
3. The method of claim 2, wherein the inducer of P-selectin
activity increases the proteolytic cleavage of P-selectin from a
cell surface.
4. The method of claim 2, wherein the inducer of P-selectin
activity increases P-selectin gene expression.
5. The method of claim 1, wherein the inducer of P-selectin
activity binds to a P-selectin receptor or ligand and mimics the
activity of a P-selectin polypeptide.
6. The method of claim 5, wherein the inducer of P-selectin
activity is an antibody to a P-selectin receptor or ligand.
7. The method of claim 5, wherein the P-selectin ligand is
PSGL-1.
8. The method of claim 6, wherein the antibody is an antibody to
PSGL-1.
9. A method for inducing hemostasis in a subject, comprising
administering to said subject a soluble P-selectin polypeptide,
such that hemostasis occurs.
10. A method for inducing hemostasis in a subject, comprising
administering to said subject an isolated nucleic acid molecule
comprising a nucleotide sequence which encodes a soluble P-selectin
polypeptide, such that hemostasis occurs.
11. A method for inducing hemostasis in a subject, comprising
administering to said subject a recombinant cell expressing soluble
P-selectin polypeptide, such that hemostasis occurs.
12. A method for treating or preventing a disorder associated with
hypocoagulation in a subject, comprising administering to said
subject an inducer of P-selectin activity, such that the disorder
associated with hypocoagulation is treated or prevented.
13. The method of claim 12, wherein said disorder is a hemorrhagic
disorder.
14. The method of claim 12, wherein said disorder is
hemophilia.
15. The method of claim 12, wherein the inducer of P-selectin
activity increases the level of soluble P-selectin polypeptide in
the plasma of the subject.
16. A method for treating or preventing a disorder associated with
hypocoagulation in a subject, comprising administering to said
subject a soluble P-selectin polypeptide.
17. A method for treating a vasculature-associated disease in a
subject, comprising administering to said subject an inducer of
P-selectin activity, such that the vasculature-associated disease
is treated.
18. The method of claim 17, wherein said vasculature-associated
disease is a tumor.
19. The method of claim 18, wherein said subject is further treated
with a molecule effective to induce a procoagulant state in tumor
associated vasculature.
20. The method of claim 19, wherein said molecule comprises a first
binding region that binds to a component of a tumor cell or tumor
associated vasculature, operatively linked to a coagulation factor
or a second binding region that binds to a coagulation factor.
21. The method of claim 20, wherein said first binding region
comprises an antibody, or an antigen binding fragment thereof, that
binds to VCAM-1, operatively linked to tissue factor.
22. The method of claim 17, wherein the inducer of P-selectin
activity increases the level of soluble P-selectin polypeptide in
the plasma of the subject.
23. A method for treating a vasculature-associated disease in a
subject, comprising administering to said subject a soluble
P-selectin polypeptide.
24. A method for reducing hemostasis in a subject, comprising
administering to said subject an inhibitor of P-selectin activity,
such that procoagulant activity is reduced.
25. The method of claim 24, wherein the inhibitor of P-selectin
activity decreases the level of soluble P-selectin polypeptide in
the plasma of the subject.
26. The method of claim 25, wherein the inhibitor of P-selectin
activity decreases the proteolytic cleavage of P-selectin from the
cell surface.
27. The method of claim 26, wherein the inhibitor of P-selectin
activity decreases P-selectin gene expression.
28. The method of claim 24, wherein the inhibitor of P-selectin
activity is an anti-P-selectin antibody.
29. The method of claim 24, wherein the inhibitor of P-selectin
activity is recombinant soluble PSGL-1.
30. A method for reducing hemostasis in a subject, comprising
administering to said subject an isolated nucleic acid molecule
comprising a nucleotide sequence which is antisense to a nucleotide
sequence which encodes a P-selectin polypeptide, such that
hemostasis is reduced.
31. A method for treating or preventing a thrombotic disorder in a
subject, comprising administering to said subject an inhibitor of
P-selectin activity, such that the thrombotic disorder is treated
or prevented.
32. The method of claim 31, wherein said disorder is
arteriosclerosis.
33. The method of claim 31, wherein said disorder is deep vein
thrombosis.
34. The method of claim 31, wherein said disorder is angina.
35. The method of claim 31, wherein said thrombotic disorder is
restenosis following medical intervention.
36. The method of claim 31, wherein the inhibitor of P-selectin
activity decreases the level of soluble P-selectin polypeptide in
the plasma of the subject.
37. A method for modulating hemostatic potential in a subject,
comprising modulating P-selectin activity in said subject.
38. The method of claim 37, wherein said modulating step comprises
administering to the subject a modulator of P-selectin
activity.
39. The method of claim 38, wherein the modulator regulates the
level of soluble P-selectin in the plasma of said subject.
40. The method of claim 38, wherein the modulator is an inhibitor
of P-selectin activity.
41. The method of claim 38, wherein the modulator is an inducer of
P-selectin activity.
42. A method for diagnosing a procoagulant state in a subject,
comprising determining a P-selectin activity in a biological sample
of the subject, wherein an increased P-selectin activity in the
sample indicates a procoagulant state in the subject.
43. The method of claim 42, which comprises providing a test sample
of blood from a subject and comparing the level of soluble
P-selectin in the test sample to the level of soluble P-selectin in
a control blood sample from a subject with normal hemostatic
activity, wherein an increased level of soluble P-selectin in the
test sample as compared to the control sample is an indication of a
procoagulant state in the subject.
44. A method of identifying a subject having a thrombotic disorder,
or at risk for developing a thrombotic disorder, comprising
determining a P-selectin activity in a biological sample of the
subject, wherein an increased P-selectin activity in the sample
identifies a subject having a thrombotic disorder, or at risk for
developing a thrombotic disorder.
45. The method of claim 44 comprising: a) contacting a sample of
blood obtained from said subject with a P-selectin binding
substance; and b) detecting the presence of increased levels of
soluble P-selectin in said sample, thereby identifying a subject
having a thrombotic disorder, or at risk for developing a
thrombotic disorder.
46. A method for identifying a compound capable of modulating
hemostasis, comprising assaying the ability of the compound to
modulate a P-selectin activity, thereby identifying a compound
capable of modulating hemostasis.
47. The method of claim 46, wherein the P-selectin activity is the
expression of soluble P-selectin.
48. A pharmaceutical composition for modulating hemostasis
comprising a compound identified according to the method of claim
46.
49. A pharmaceutical composition for modulating hemostasis
containing at least one compound which is a modulator of P-selectin
activity.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of prior-filed
provisional patent application Ser. No. 60/205,734, filed May 19,
2000, entitled "Methods For Diagnosing and Treating Hemostatic
Disorders By Modulating P-Selectin Activity." The entire content of
the above-referenced application is incorporated herein by this
reference.
BACKGROUND OF THE INVENTION
[0002] The ability of cells to adhere to one another plays a
critical role in development, normal physiology, and disease
processes. This ability is mediated by adhesion molecules,
generally glycoproteins, expressed on the cell surface. Several
important classes of adhesion molecules include the integrins, the
selecting, and members of the immunoglobulin (Ig) superfamily.
Selectins play a central role in mediating leukocyte adhesion to
activated endothelium and platelets.
[0003] Blood clotting, along with inflammation and tissue repair,
are host defense mechanisms which function in parallel to preserve
the integrity of the vascular system after tissue injury. In
response to tissue injury, platelets, endothelial cells and
leukocytes are essential for the formation of a platelet plug,
deposition of leukocytes in injured tissue, initiation of
inflammation, and wound healing.
[0004] P-selectin, also known as CD62, granule membrane protein-140
(GMP-140), and platelet activation-dependent granule external
membrane protein (PADGEM), is an integral membrane glycoprotein
that is expressed on vascular endothelial cells and platelets, and
is involved in the recognition of various circulating cells. The
P-selectin molecule has an N-terminal lectin domain, a region with
homology to epidermal growth factor, a region with homology to
complement regulatory proteins, a transmembrane domain, and a short
cytoplasmic tail. The P-selectin ligand includes the Lex
carbohydrate structure, sialic acid, and the PSGL-1 protein (U.S.
Pat. No. 5,843,707).
[0005] P-selectin is constitutively stored in secretory granules
(e.g., a-granules and Weibel-Palade bodies) and is translocated to
the surface of platelets and endothelial cells in response to a
variety of stimuli, including cell activation, where it mediates
platelet-leukocyte and endothelium-leukocyte interactions. The cell
surface expression of P-selectin is tightly regulated, and
P-selectin is rapidly shed from the cell surface upon platelet
activation, appearing as a soluble fragment in the plasma (Berger,
G. et al. Blood (1998) 92:4446-4452). Soluble P-selectin may also
result from an alternatively spliced isoform of P-selectin lacking
the transmembrane domain (Ishiwata, N. et al. J. Biol Chem (1994)
269:23708). The plasma of healthy humans and mice contains little
soluble P-selectin, as detected by ELISA, and an increase in plasma
P-selectin concentration may indicate in vivo activation of and/or
damage to platelets and endothelial cells.
[0006] In addition to its role in leukocyte rolling and
extravasation in inflammation, P-selectin mediates
platelet-leukocyte adhesion within thrombi, and increases tissue
factor expression on monocytes, thereby promoting fibrin deposition
by leukocytes and thrombogenesis (Palabrica, T. et al. Nature
(1992) 359:848-851; Celi, A. et al. Proc Natl Acad Sci USA (1994)
91:8767-8771).
SUMMARY OF THE INVENTION
[0007] The present invention provides methods and compositions for
the regulation of hemostatic and thrombotic processes using
modulators of P-selectin activity (e.g., inducers and inhibitors of
P-selectin activity), as well as for the diagnosis and treatment of
hemostatic disorders.
[0008] In one aspect, the invention provides methods for inducing
hemostasis in a subject, comprising administering an inducer of
P-selectin activity to the subject. In one embodiment, the inducer
of P-selectin activity increases the level of circulating soluble
P-selectin in the subject. The inducer of P-selectin activity may
increase the level of soluble P-selectin polypeptide by increasing
the proteolytic cleavage of P-selectin from the cell surface, or by
increasing P-selectin gene expression. In another embodiment, the
inducer of P-selectin activity binds to a P-selectin ligand or
receptor (e.g., PSGL-1) and mimics the activity of a P-selectin
polypeptide, e.g., a soluble P-selectin polypeptide.
[0009] In an exemplary embodiment, the invention provides methods
for inducing hemostasis in a subject, comprising administering
soluble P-selectin polypeptide to the subject. In another
embodiment, an isolated nucleic acid molecule comprising a
nucleotide sequence which encodes a soluble P-selectin polypeptide
is administered to the subject to induce hemostasis. In a further
embodiment, hemostasis is induced in a subject by administering a
recombinant cell expressing soluble P-selectin polypeptide.
[0010] In another aspect, the invention provides methods for
treating or preventing a disorder associated with hypocoagulation,
e.g., a hemorrhagic disorder, in a subject, comprising
administering to the subject an inducer of P-selectin activity. In
one embodiment, a soluble P-selectin polypeptide is administered to
a subject to treat or prevent a disorder associated with
hypocoagulation.
[0011] In a further aspect, the invention provides methods for
treating a vasculature-associated disease in a subject, comprising
administering to the subject an inducer of P-selectin activity. In
a preferred embodiment, a soluble P-selectin polypeptide is
administered to a subject to treat or prevent a
vasculature-associated disease. In one embodiment, the
vasculature-associated disease is a tumor. In another embodiment,
the subject is further treated with a molecule effective to induce
a procoagulant state in tumor associated vasculature, e.g., a
molecule comprising a first binding region that binds to a
component of a tumor cell or tumor associated vasculature
operatively linked to a coagulation factor or a second binding
region that binds to a coagulation factor.
[0012] Another aspect of the invention provides methods for
reducing hemostasis in a subject, comprising administering to the
subject an inhibitor of P-selectin activity. In one embodiment, the
inhibitor of P-selectin activity decreases the level of soluble
P-selectin in plasma of the subject. The inhibitor of P-selectin
activity may decrease the level of the soluble P-selectin
polypeptide by decreasing the proteolytic cleavage of P-selectin
from the cell surface, or decreasing P-selectin gene expression. In
another embodiment, the inhibitor of P-selectin activity is an
anti-P-selectin antibody. In yet another embodiment, the inhibitor
of P-selectin activity is a recombinant soluble PSGL-1 polypeptide.
In a further embodiment, the invention provides a method for
reducing hemostasis in a subject, comprising administering an
isolated nucleic acid molecule comprising a nucleotide sequence
which is antisense to a nucleotide sequence which encodes a
P-selectin polypeptide, e.g., a soluble P-selectin polypeptide.
[0013] In another aspect, the invention provides methods for
treating or preventing a thrombotic disorder in a subject,
comprising administering to the subject an inhibitor of P-selectin
activity. Thrombotic disorders that may be treated or prevented
using the methods of the invention include arteriosclerosis, deep
vein thrombosis, angina, e.g., unstable angina, and restenosis
following medical intervention.
[0014] In a further aspect, the invention provides methods for
modulating hemostatic potential in a subject, comprising modulating
P-selectin activity in the subject. In one embodiment, a modulator
(e.g., an inducer or inhibitor) of P-selectin activity is
administered to a subject to modulate hemostatic potential. A
modulator of soluble P-selectin activity may act by regulating the
level of soluble P-selectin in the plasma of the subject.
[0015] Another aspect of the invention provides a method for
diagnosing a procoagulant state in a subject, comprising
determining an increased level of P-selectin activity in a
biological sample of the subject. In one embodiment, the level of
soluble P-selectin in a test sample of blood or plasma from a
subject is compared to the level of soluble P-selectin in a control
blood or plasma sample from a subject with normal hemostatic
activity, wherein an increased level of soluble P-selectin in the
test sample as compared to the control sample is an indication of a
procoagulant state in the subject.
[0016] In another aspect, the invention provides a method for
identifying a subject having a thrombotic disorder, or at risk for
developing a thrombotic disorder, comprising determining an
increased P-selectin activity in a biological sample of the
subject. In one embodiment, a sample of blood or plasma obtained
from a subject is contacted with a P-selectin binding substance,
and the detection of increased levels of soluble P-selectin
polypeptide in the sample identifies a subject having a thrombotic
disorder, or at risk for developing a thrombotic disorder.
[0017] Another aspect of the invention provides a method for
identifying a compound capable of modulating hemostasis, comprising
assaying the ability of the test compound to modulate a P-selectin
activity. In one embodiment, the P-selectin activity is the
expression of soluble P-selectin.
[0018] In a further aspect, the invention provides compositions for
modulating hemostasis comprising at least one modulator of
P-selectin activity.
[0019] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a photograph of en face examination of the
thrombotic deposits in wild-type mice (WT), P-selectin deficient
mice (PKO), and .DELTA.CT mice formed after a 2 minute
non-anticoagulated blood perfusion (blood flow, left to right). The
white arrow indicates platelet rich thrombus; the black arrow
indicates fibrin tail formed distally to the platelet thrombus.
[0021] FIG. 2 shows fibrin formation in a perfusion chamber of
non-anticoagulated blood from wild type mice (WT), P-selectin
deficient mice (P-sel -/-), and .DELTA.CT mice.
[0022] FIG. 3 shows macroscopic and microscopic grading of
hemorrhagic lesions formed in a local Shwartzman reaction in wild
type mice (WT) that were either untreated, perfused with human
IgG1, or perfused with soluble P-selectin-Ig (s-P-sel), and
.DELTA.CT mice.
[0023] FIG. 4 shows fibrin deposition in a local Shwartzman
reaction in wild type (WT) mice that were perfused with either
human IgG1 or soluble P-selectin-Ig (P-sel).
[0024] FIGS. 5A and B show the plasma clotting time of wild type
mice (WT), P-selectin deficient mice (P-sel -/-), and .DELTA.CT
mice that were either untreated or perfused with recombinant PSGL-1
or recombinant soluble P-selectin.
[0025] FIG. 6 shows the levels of microparticles in the circulation
of wild type mice (WT) that were either untreated, perfused with
human IgG1, or perfused with soluble P-selectin-Ig (s-P-sel), and
.DELTA.CT mice.
[0026] FIG. 7 shows the number of microparticles expressing tissue
factor in wild type (WT) and .DELTA.CT mice.
[0027] FIG. 8 shows the increased generation of procoagulant
microparticles in the circulation of von Willebrand factor
deficient mice (vWF -/-) that were perfused with soluble
P-selectin-Ig (sP-sel-Ig).
[0028] FIG. 9 shows the prothrombin clotting time of wild type mice
(WT), and von Willebrand factor deficient mice (vWF -/-) that were
either untreated, perfused with human IgG1, or perfused with
soluble P-selectin-Ig (sPselIg).
[0029] FIG. 10 shows the bleeding time in hemophilia A mice treated
with either human IgG1 or soluble P-selectin-Ig (P-sel-Ig).
[0030] FIG. 11A shows the reduction in the number of microparticles
after treatment of .DELTA.CT mice with soluble PSGL-Ig as compared
to control human Ig (*=p<0.05).
[0031] FIG. 11B shows the increase in clotting time after treatment
of .DELTA.CT mice with soluble PSGL-Ig as compared to control human
Ig (*=p<0.05).
[0032] FIG. 12A shows the generation of procoagulant microparticles
in human blood after incubation with either human IgG or soluble
P-selectin-Ig (P-sel-Ig). After 6 hrs. incubation with soluble
P-selectin-Ig, the numbers of microparticles significantly
increased by 30% (*=p<0.04).
[0033] FIG. 12B shows the generation of tissue factor positive
microparticles in human blood after incubation with either human
IgG or soluble P-selectin-Ig (P-sel-Ig). The number of tissue
factor positive evens was significantly increased at 6 hours by
incubation with P-selectin Ig, 30% (*=p<0.05).
[0034] FIG. 13A shows the clotting time of human whole blood after
incubation with human IgG or soluble P-selectin-Ig (P-sel-Ig). The
clotting time of whole blood incubated with soluble P-selectin-Ig
was shortened by about 20% after 2 hours (*=p<0.02) and by 60%
after 8 hours of incubation (**=p<0.004).
[0035] FIG. 13B shows the clotting time of human plasma after
incubation with human IgG or soluble P-selectin-Ig (P-sel-Ig). The
plasma clotting time of the soluble P-selectin treated blood was
shortened by 25% after 6 hours of incubation and by 40% after 8
hours (**p<0.004).
[0036] FIG. 14A shows activated partial thromboplastin time (APTT)
in factor VIII -/- mice (hemophilia A mice) treated with control Ig
or soluble P-selectin-Ig.
[0037] FIG. 14B shows plasma clotting time in factor VIII -/- mice
(hemophilia A mice) treated with control Ig or soluble
P-selectin-Ig.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention provides modulators (e.g., inducers,
inhibitors) of P-selectin activity as therapeutic and diagnostic
agents for the regulation of hemostasis. The present invention is
based on the discovery that soluble P-selectin induces a
procoagulant state in a mammal, for example a mouse or a human,
(e.g., by increasing the numbers of microparticles containing
tissue factor in the blood, reducing bleeding time, and/or reducing
clotting time).
[0039] As used herein, the term "modulator of P-selectin activity"
includes a compound or agent that is capable of modulating or
regulating at least one P-selectin activity, as described herein.
In a preferred embodiment, a modulator of P-selectin activity
modulates the expression of soluble P-selectin. A modulator of
P-selectin activity can be an inducer of P-selectin activity or an
inhibitor of P-selectin activity. As used herein, an "inducer of
P-selectin activity" stimulates, enhances, and/or mimics a
P-selectin activity. As used herein, an "inhibitor of P-selectin
activity" reduces, blocks or antagonizes a P-selectin activity.
[0040] As used interchangeably herein, a "P-selectin activity",
"biological activity of P-selectin" or "functional activity of
P-selectin" refers to an activity exerted by a P-selectin
polypeptide or nucleic acid molecule on a P-selectin responsive
cell (e.g., a hematopoietic cell or lymphocyte) or tissue, or on a
P-selectin ligand or receptor, as determined in vitro and in vivo,
according to standard techniques. In an exemplary embodiment, a
P-selectin activity is the ability to modulate hemostasis. In one
embodiment, a P-selectin activity is a procoagulant activity. In
another embodiment, a P-selectin activity is the ability to
increase the number of microparticles containing tissue factor. In
yet another embodiment, a P-selectin activity is the ability to
bind a P-selectin ligand, e.g., PSGL-1.
[0041] Accordingly, the invention provides a method for regulating
hemostasis in a subject, at least in part, by increasing or
decreasing P-selectin activity in the subject (e.g., by increasing
or decreasing levels of circulating soluble P-selectin). As used
interchangeably herein, the terms "hemostasis", "hemostatic
activity", or "hemostatic potential" refer to the control of
bleeding, including the physiological properties of
vasoconstriction and coagulation. Blood coagulation assists in
maintaining the integrity of mammalian circulation after injury,
inflammation, disease, congenital defect, dysfunction or other
disruption. After initiation of clotting, blood coagulation
proceeds through the sequential activation of certain plasma
proenzymes to their enzyme forms (see, for example, Coleman, R. W.
et al. (eds.) Hemostasis and Thrombosis, Second Edition, (1987)).
These plasma glycoproteins, including Factor XII, Factor XI, Factor
IX, Factor X, Factor VII, and prothrombin, are zymogens of serine
proteases. Most of these blood clotting enzymes are effective on a
physiological scale only when assembled in complexes on membrane
surfaces with protein cofactors such as Factor VIII and Factor V.
Other blood factors modulate and localize clot formation, or
dissolve blood clots. Activated protein C is a specific enzyme that
inactivates procoagulant components. Calcium ions are involved in
many of the component reactions. Blood coagulation follows either
the intrinsic pathway, where all of the protein components are
present in blood, or the extrinsic pathway, where the cell-membrane
protein tissue factor plays a critical role. Clot formation occurs
when fibrinogen is cleaved by thrombin to form fibrin. Blood clots
are composed of activated platelets and fibrin.
[0042] As used herein, the term "procoagulant state" includes
physiological conditions that are conducive to and/or promote blood
clotting, hemostasis, and/or thrombosis. Hemostatic potential,
e.g., the potential for blood coagulation under the appropriate
physiological conditions, or hemostatic activity can be assessed
using well established laboratory tests including prothrombin time
(PT), activated partial thromboplastin time (APTT), bleeding time,
and thrombin time. As used interchangeably herein, "modulating or
modulation of hemostasis" and "regulating or regulation of
hemostasis" includes the induction (e.g., stimulation, increase) of
hemostasis, as well as the inhibition (e.g., reduction, decrease)
of hemostasis.
[0043] In one aspect of the invention, hemostasis is induced in a
subject by administering an inducer of P-selectin activity. In an
exemplary embodiment, an inducer of P-selectin activity increases
the plasma level of soluble P-selectin polypeptide. In this
respect, an inducer of P-selectin activity may act to stimulate the
translocation of P-selectin from a cellular storage pool to the
cell surface, or to increase the proteolytic cleavage and release
of soluble P-selectin from the surface of a cell expressing
P-selectin, e.g., an endothelial cell or a platelet. In another
embodiment, an inducer of P-selectin activity increases P-selectin
gene expression by stimulating either gene transcription or
translation. In a preferred embodiment, an inducer of P-selectin
activity will preferentially stimulate the expression of an
alternatively spliced isoform of the P-selectin gene encoding a
soluble P-selectin polypeptide lacking the transmembrane domain. In
yet another embodiment, an inducer of P-selectin activity binds to
a P-selectin ligand or receptor (e.g., PSGL-1) and mimics the
activity of a P-selectin polypeptide on a P-selectin responsive
cell. The inducer of P-selectin activity can thereby elicit a
biological response of P-selectin, e.g., the release of
microparticles containing tissue factor. Accordingly, in one
embodiment, an inducer of P-selectin activity is an antibody, e.g.,
an anti-PSGL-1 antibody.
[0044] In another embodiment of the invention, a soluble P-selectin
polypeptide is administered to a subject to induce hemostasis. As
used herein, a "soluble P-selectin polypeptide" includes a
P-selectin polypeptide comprising amino acid sequences
corresponding to the extracellular domain of a P-selectin protein,
or a fragment thereof. The nucleic acid and amino acid sequences of
P-selectin proteins have been described (see, for example, Sanders,
W. E. et al. (1992) Blood 80:795-800; and GenBank Accession Numbers
NM.sub.--003005 and M25322 (human); GenBank Accession Numbers
NM.sub.--013114 and L23088 (rat); GenBank Accession Numbers
NM.sub.--011347 and M87861 (mouse); and GenBank Accession Number
L12041 (bovine)). In another embodiment, a soluble P-selectin
polypeptide comprises at least a lectin domain, an EGF-like repeat,
and at least two complement-binding domains of a P-selectin
protein. In yet another embodiment, a soluble P-selectin
polypeptide binds to a P-selectin ligand, e.g., PSGL-1. In a
preferred embodiment, a soluble P-selectin polypeptide of the
invention is a soluble P-selectin fusion protein. In one
embodiment, the P-selectin fusion protein is a P-selectin-Ig fusion
protein comprising a signal sequence, a lectin domain, an EGF-like
repeat, and at least two complement-binding domains of a P-selectin
protein operatively linked to the Fc region (hinge, C1 and C2) of
an immunoglobulin, e.g., human IgG1.
[0045] In a further embodiment of the invention, hemostasis is
induced in a subject by administering an isolated nucleic acid
molecule comprising a nucleotide sequence which encodes a soluble
P-selectin polypeptide. In yet another embodiment, a recombinant
cell expressing a soluble P-selectin polypeptide is administered to
a subject to induce hemostasis.
[0046] Another embodiment of the invention provides methods for
inducing hemostasis in a subject presenting insufficient hemostatic
function, such as a subject having, or at risk of developing a
disorder associated with hypocoagulation. As used herein, the term
"hypocoagulation" refers to a decreased ability or inability to
form blood clots. Such disorders include hemorrhagic disorders,
e.g., hemophilia (e.g., hemophilia A or B), and disorders resulting
from a deficiency in clotting factors or platelet ligands, e.g., a
deficiency in von Willebrand's factor resulting in von Willebrand
disease. The induction of a procoagulant state would prevent or
stop spontaneous bleeding and would also be beneficial preceding
surgical intervention in a patient, or to promote wound
healing.
[0047] The methods of the present invention are also useful for the
treatment of a vasculature-associated disease. As used herein, a
"vasculature-associated disease" is a disease having a pathology
that is dependent on a vascular blood supply. Thus, it is
contemplated that achieving coagulation in the vasculature of the
disease site, e.g., in the intratumoral vasculature of a solid
tumor, would prove beneficial. Such vasculature-associated diseases
include benign and malignant tumors or growths, such as BPH,
diabetic retinopathy, vascular restenosis, arteriovenous
malformations (AVM), meningioma, hemangioma, neovascular glaucoma
and psoriasis. Also included within this group are synovitis,
dermatitis, endometriosis, angiofibroma, rheumatoid arthritis,
atherosclerotic plaques, corneal graft neovascularization,
hemophilic joints, hypertrophic scars, osler-weber syndrome,
pyogenic granuloma retrolental fibroplasia, scleroderma, trachoma,
and vascular adhesions.
[0048] In one embodiment, an inducer of P-selectin activity, e.g.,
soluble P-selectin, is administered in addition to therapies
designed to induce thrombosis of tumor blood vessels, in order to
potentiate tumor necrosis. Such therapies utilize strategies for
targeting coagulation factors to the tumor vasculature, for
example, as described in U.S. Pat. No. 5,877,289. Markers of tumor
vasculature or stroma may be specifically induced and then targeted
using a binding ligand, such as an antibody. Exemplary inducible
antigens include E-selectin, P-selectin, MHC Class II antigens,
VCAM-1, ICAM-1, endoglin, ligands reactive with LAM-1, vascular
addressins and other adhesion molecules.
[0049] Moreover, the present invention provides a method for
reducing hemostasis in a subject by administering an inhibitor of
P-selectin activity. The inhibition of hemostasis, e.g., clot
formation, is desirable in situations where vessel patency is of
importance.
[0050] In an exemplary embodiment, an inhibitor of P-selectin
activity decreases the level of circulating soluble P-selectin in
the subject. The inhibitor of P-selectin activity may act to
decrease the translocation of P-selectin from a cellular storage
pool to the cell surface, or to decrease the proteolytic cleavage
and release of soluble P-selectin from the surface of a cell
expressing P-selectin, e.g., an endothelial cell or a platelet. In
another embodiment, an inhibitor of P-selectin activity decreases
P-selectin gene expression by reducing either gene transcription or
translation. In a preferred embodiment, an inhibitor of P-selectin
activity will preferentially reduce the expression of an
alternatively spliced isoform of the P-selectin gene encoding a
soluble P-selectin polypeptide lacking the transmembrane domain. In
yet another embodiment, an inhibitor of P-selectin activity acts as
an antagonist, wherein it binds to a P-selectin ligand or receptor
(e.g., PSGL-1) and blocks the activity of a P-selectin polypeptide
on a P-selectin responsive cell. In one embodiment of the
invention, an inhibitor of P-selectin activity is an
anti-P-selectin antibody. In another embodiment, an inhibitor of
P-selectin activity is a soluble PSGL-1 polypeptide. PSGL-1 nucleic
acids, polypeptides, and soluble forms thereof are disclosed in
U.S. Pat. No. 5,843,707.
[0051] Alternatively, the invention provides a method for reducing
hemostasis in a subject by administering an isolated nucleic acid
molecule comprising a nucleotide sequence which is antisense, e.g.,
complementary to, to a nucleotide sequence encoding a P-selectin
polypeptide.
[0052] Thus, the methods of the invention are useful for the
treatment or prevention of thrombotic disorders. As used herein,
the term "thrombotic disorder" includes any disorder or condition
characterized by excessive or unwanted coagulation or hemostatic
activity, or a hypercoagulable state. Thrombotic disorders include
disorders diseases involving platelet adhesion and thrombus
formation, and may manifest as an increased propensity to form
thromboses, e.g., an increased number of thromboses, thrombosis at
an early age, a familial tendency towards thrombosis, and
thrombosis at unusual sites. Examples of thrombotic disorders
include, but are not limited to, thromboembolism, deep vein
thrombosis, pulmonary embolism, stroke, myocardial infarction,
miscarriage, thrombophilia associated with anti-thrombin III
deficiency, protein C deficiency, protein S deficiency, resistance
to activated protein C, dysfibrinogenemia, fibrinolytic disorders,
homocystinuria, pregnancy, inflammatory disorders,
myeloproliferative disorders, arteriosclerosis, angina, e.g.,
unstable angina, disseminated intravascular coagulation, thrombotic
thrombocytopenic purpura, cancer metastasis, sickle cell disease,
and glomerular nephritis. In addition, inhibitors of soluble
P-selectin expression or activity are administered to prevent
thrombotic events or to prevent re-occlusion during or after
therapeutic clot lysis or procedures such as angioplasty or
surgery.
[0053] Furthermore, measuring the level P-selectin activity in a
biological sample, e.g., in blood, would provide diagnostic
information of a procoagulant state, e.g., the likelihood of a
thrombotic or clotting event. Accordingly, in one embodiment, the
invention provides a method for diagnosing a procoagulant state in
a subject by detecting an increased level of circulating soluble
P-selectin as compared with the levels of soluble P-selectin in the
blood of individual with clinically established normal levels of
hemostatic activity. In another embodiment, the invention provides
a method of identifying a subject having a thrombotic disorder, or
at risk for developing a thrombotic disorder, by detecting the
presence of increased levels of P-selectin activity (e.g.,
increased levels of circulating soluble P-selectin).
[0054] As used herein, a "hemostatic disorder" includes a disorder
or condition characterized by aberrant or unwanted hemostatic
activity. A hemostatic disorder may result from excessive coagulant
activity, e.g., a thrombotic disorder, or it may result from
insufficient coagulant activity, e.g., a hemorrhagic disorder.
[0055] Furthermore, another aspect of the invention provides a
method for identifying a compound capable of modulating hemostasis
by assaying the ability of the compound to modulate a P-selectin
activity, e.g., the expression of soluble P-selectin.
[0056] Various aspects of the invention are described in further
detail in the following subsections.
[0057] I. Isolated P-selectin Proteins and Anti-P-selectin
Antibodies
[0058] The methods of the invention include the use of isolated
P-selectin polypeptides, and biologically active portions thereof.
As used herein, a "P-selectin protein" or "P-selectin polypeptide"
includes a soluble P-selectin polypeptide and a soluble P-selectin
fusion protein.
[0059] The genomic organization and coding sequence for human
P-selectin have been determined, and the cDNA has been cloned and
sequenced (see, for example, GenBank Accession Numbers
NM.sub.--003005 and M25322). In addition, the sequences encoding
rat (GenBank Accession Numbers NM.sub.--013114 and L23088), mouse
(GenBank Accession Numbers NM.sub.--011347 and M87861), and bovine
(GenBank Accession Number L12041) P-selectin have been disclosed.
Furthermore, a comparison of the amino acid sequences and
structural domains of human and mouse P-selectin is disclosed in
Sanders, WE et al. (1992) Blood 80:795-800.
[0060] Isolated soluble P-selectin proteins for use in the methods
of the present invention preferably have an amino acid sequence
that is sufficiently identical to the amino acid sequence of a
native P-selectin protein. As used herein, the term "sufficiently
identical" refers to an amino acid (or nucleotide) sequence which
contains a sufficient or minimum number of identical or equivalent
(e.g., an amino acid residue that has a similar side chain) amino
acid residues (or nucleotides) to a P-selectin amino acid (or
nucleotide) sequence such that the polypeptide shares common
structural domains or motifs, and/or a common functional activity
with a native P-selectin protein. For example, amino acid or
nucleotide sequences which share common structural domains have at
least 30%, 40%, or 50% identity, preferably 60% identity, more
preferably 70%-80%, and even more preferably 90-95% identity across
the amino acid sequences of the domains and contain at least one,
and more preferably two or more structural domains or motifs, are
defined herein as sufficiently identical. For example, a soluble
P-selectin polypeptide may comprise at least one or more of the
following domains: a signal peptide, a lectin domain, an EGF-like
repeat, a complement binding domain, and a cytoplasmic domain.
Furthermore, amino acid or nucleotide sequences which share at
least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or
90-95% identity and share a common functional activity (e.g., a
soluble P-selectin activity as described herein) are defined herein
as sufficiently identical. A P-selectin polypeptide may differ in
amino acid sequence from the P-selectin polypeptides disclosed
herein due to natural allelic variation or mutagenesis.
Accordingly, isolated soluble P-selectin polypeptides having a
P-selectin activity can be used in the methods of the
invention.
[0061] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-identical
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence. The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position (as used herein amino acid or nucleic acid "identity" is
equivalent to amino acid or nucleic acid "homology"). The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences, taking into account
the number of gaps, and the length of each gap, which need to be
introduced for optimal alignment of the two sequences.
[0062] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the
percent identity between two amino acid or nucleotide sequences is
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4.
[0063] As used herein, a "biologically active portion" of a
P-selectin polypeptide (e.g., a soluble P-selectin polypeptide)
includes a fragment of a P-selectin polypeptide which retains a
P-selectin polypeptide activity. Typically, a biologically active
portion of a P-selectin polypeptide comprises at least one domain
or motif with at least one activity of the P-selectin polypeptide,
e.g., modulating hemostatic activity. Biologically active portions
of a P-selectin polypeptide include polypeptides comprising amino
acid sequences sufficiently identical to or derived from the amino
acid sequence of a P-selectin protein, which include less amino
acids than the full length P-selectin polypeptide, and exhibit at
least one activity of a soluble P-selectin polypeptide.
Biologically active portions of a P-selectin polypeptide can be
used as targets for developing agents which modulate a P-selectin
polypeptide activity, e.g., a hemostatic activity. A biologically
active portion of a P-selectin polypeptide comprises a polypeptide
which can be prepared by recombinant techniques and evaluated for
one or more of the functional activities of a P-selectin
polypeptide.
[0064] In one embodiment, P-selectin polypeptides can be isolated
from cells or tissue sources by an appropriate purification scheme
using standard protein purification techniques. For example, a
soluble P-selectin polypeptide can be isolated from the culture
medium of cells, e.g., activated endothelial cells, that have been
induced to shed P-selectin from the cell surface. In another
embodiment, P-selectin polypeptides are produced by recombinant DNA
techniques. For example, a soluble P-selectin polypeptide can be
isolated from a host cell transfected with a polynucleotide
sequence encoding a soluble isoform of P-selectin (e.g., an isoform
of P-selectin lacking a transmembrane domain) or a soluble
P-selectin fusion protein. Alternative to recombinant expression, a
soluble P-selectin polypeptide can be synthesized chemically using
standard peptide synthesis techniques.
[0065] An "isolated" or "purified" polypeptide or protein, or
biologically active portion thereof is substantially free of
cellular material or other contaminating proteins from the cell or
tissue source from which the P-selectin polypeptide is derived, or
substantially free from chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of
cellular material" includes preparations of P-selectin polypeptide
in which the protein is separated from cellular components of the
cells from which it is isolated or recombinantly produced. In one
embodiment, the language "substantially free of cellular material"
includes preparations of P-selectin protein having less than about
30% (by dry weight) of non-P-selectin protein (also referred to
herein as a "contaminating protein"), more preferably less than
about 20% of non-P-selectin protein, still more preferably less
than about 10% of non-P-selectin protein, and most preferably less
than about 5% non-P-selectin protein. When the P-selectin
polypeptide or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, more
preferably less than about 10%, and most preferably less than about
5% of the volume of the protein preparation.
[0066] The language "substantially free of chemical precursors or
other chemicals" includes preparations of P-selectin polypeptide in
which the protein is separated from chemical precursors or other
chemicals which are involved in the synthesis of the protein. In
one embodiment, the language "substantially free of chemical
precursors or other chemicals" includes preparations of P-selectin
polypeptide having less than about 30% (by dry weight) of chemical
precursors or non-P-selectin chemicals, more preferably less than
about 20% chemical precursors or non-P-selectin chemicals, still
more preferably less than about 10% chemical precursors or
non-P-selectin chemicals, and most preferably less than about 5%
chemical precursors or non-P-selectin chemicals.
[0067] The methods of the invention may also use soluble P-selectin
polypeptides that are chimeric or fusion proteins. As used herein,
a soluble P-selectin "chimeric protein" or "fusion protein"
comprises a soluble P-selectin polypeptide operatively linked to a
non-soluble P-selectin polypeptide. A "soluble P-selectin
polypeptide" includes a P-selectin polypeptide that comprises amino
acid sequences corresponding to the extracellular domain of a
P-selectin protein, or a biologically active portion thereof,
whereas a "non-soluble P-selectin polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous to a P-selectin
polypeptide, e.g., a protein which is different from the soluble
P-selectin polypeptide and which is derived from the same or a
different organism. Within a soluble P-selectin fusion protein the
soluble P-selectin polypeptide may include, for example, all or a
portion of the extracellular domain of a P-selectin protein. In a
preferred embodiment, a soluble P-selectin fusion protein comprises
at least a signal sequence, a lectin domain, an EGF-like repeat,
and at least two complement-binding domains of a P-selectin
protein. Within the fusion protein, the term "operatively linked"
is intended to indicate that the soluble P-selectin polypeptide and
the non-soluble P-selectin polypeptide are fused in-frame to each
other. The non-soluble P-selectin polypeptide can be fused to the
N-terminus or C-terminus of the soluble P-selectin polypeptide.
[0068] For example, in a preferred embodiment, the fusion protein
is a soluble P-selectin-immunoglobulin fusion protein in which the
Fc region, e.g., the hinge, C1 and C2 sequences, of an
immunoglobulin, (e.g., human IgG1) is fused to the C-terminus of
the soluble P-selectin sequences. Selectin immunoglobulin chimeras
can be constructed essentially as described in WO 91/08298. Such
fusion proteins can facilitate the purification of recombinant
soluble P-selectin polypeptides. In another embodiment, the fusion
protein is a soluble P-selectin polypeptide containing a
heterologous signal sequence at its N-terminus. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
soluble P-selectin can be increased through use of a heterologous
signal sequence.
[0069] The soluble P-selectin polypeptides and fusion proteins of
the invention can be incorporated into pharmaceutical compositions
and administered to a subject in vivo. In an exemplary embodiment,
a soluble P-selectin polypeptide or fusion protein may be used to
modulate hemostasis in a subject (e.g., induce a procoagulant
state). In another embodiment, a soluble P-selectin polypeptide or
fusion protein may be used to treat a hemostatic disorder, e.g., a
hemorrhagic disorder. In another embodiment, a soluble P-selectin
polypeptide or fusion protein may be used to treat a
vasculature-associated disease. Use of soluble P-selectin
polypeptides and fusion proteins may also be useful therapeutically
for the treatment of disorders caused by, for example, (i) aberrant
modification or mutation of a gene encoding a P-selectin protein;
(ii) mis-regulation of a P-selectin gene; and (iii) aberrant
post-translational modification of a P-selectin protein. In
addition, the soluble P-selectin polypeptides and fusion proteins
can be used to affect the bioavailability of a P-selectin ligand,
e.g., PSGL-1.
[0070] Moreover, the soluble P-selectin polypeptides and fusion
proteins of the invention can be used as immunogens to produce
anti-P-selectin antibodies in a subject, to purify P-selectin
ligands, and in screening assays to identify molecules which
modulate P-selectin activity, and/or modulate the interaction of a
P-selectin polypeptide with a P-selectin ligand or receptor.
[0071] Preferably, a soluble P-selectin fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A soluble P-selectin-encoding nucleic acid can be
cloned into such an expression vector such that the fusion moiety
is linked in-frame to the soluble P-selectin polypeptide.
[0072] The methods of the present invention may also include the
use of variants of a P-selectin polypeptide which function as
either P-selectin agonists (mimetics) or as P-selectin antagonists.
Variants of the P-selectin polypeptide can be generated by
mutagenesis, e.g., discrete point mutation or truncation of a
P-selectin protein. An agonist of a P-selectin polypeptide can
retain substantially the same, or a subset, of the biological
activities of the naturally occurring form of a P-selectin
polypeptide. An antagonist of a P-selectin polypeptide can inhibit
one or more of the activities of a native form of the P-selectin
polypeptide by, for example, competitively modulating a P-selectin
activity (e.g., a hemostatic activity) of a P-selectin polypeptide.
Thus, specific biological effects can be elicited by treatment with
a variant of limited function. In one embodiment, treatment of a
subject with a variant having a subset of the biological activities
of the naturally occurring form of the protein has fewer side
effects in a subject relative to treatment with the naturally
occurring form of the P-selectin polypeptide.
[0073] In one embodiment, variants of a soluble P-selectin
polypeptide which function as either soluble P-selectin agonists
(mimetics) or as soluble P-selectin antagonists can be identified
by screening mutants, e.g., truncation mutants, of a soluble
P-selectin polypeptide for soluble P-selectin polypeptide agonist
or antagonist activity. The activity of a variant soluble
P-selectin polypeptide, e.g., the ability to modulate hemostatic
activity, can be assessed in an animal model such as the animal
models described and exemplified herein, e.g., a P-selectin
deficient mouse, or a von Willebrand factor deficient mouse.
[0074] An isolated P-selectin polypeptide, or a portion or fragment
thereof, can be used as an immunogen to generate antibodies that
bind P-selectin using standard techniques for polyclonal and
monoclonal antibody preparation (see, generally R. H. Kenneth, in
Monoclonal Antibodies: A New Dimension In Biological Analyses,
Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981)
Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic
Cell Genet. 3:231-36). Moreover, the ordinarily skilled artisan
will appreciate that there are many variations of such methods
which also would be useful.
[0075] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-P-selectin antibody can be identified
and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with P-selectin to thereby isolate immunoglobulin library members
that bind P-selectin. Kits for generating and screening phage
display libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZAP.TM. Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, Ladner et al. U.S. Pat. No.
5,223,409; Kang et al. PCT International Publication No. WO
92/18619; Dower et al. PCT International Publication No. WO
91/17271; Winter et al. PCT International Publication WO 92/20791;
Markland et al. PCT International Publication No. WO 92/15679;
Breitling et al. PCT International Publication WO 93/01288;
McCafferty et al. PCT International Publication No. WO 92/01047;
Garrard et al. PCT International Publication No. WO 92/09690;
Ladner et al. PCT International Publication No. WO 90/02809; Fuchs
et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.
Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins
et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991)
Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA
89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377;
Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al.
(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et
al. Nature (1990) 348:552-554.
[0076] Additionally, recombinant anti-P-selectin antibodies, such
as chimeric and humanized monoclonal antibodies, comprising both
human and non-human portions, which can be made using standard
recombinant DNA techniques, can also be used in the methods of the
present invention. Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in Robinson et al.
International Application No. PCTIUS86/02269; Akira, et al.
European Patent Application 184,187; Taniguchi, M., European Patent
Application 171,496; Morrison et al. European Patent Application
173,494; Neuberger et al. PCT International Publication No. WO
86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.
European Patent Application 125,023; Better et al. (1988) Science
240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA
84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et
al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.
80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et
al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539;
Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0077] An anti-P-selectin antibody (e.g., a monoclonal antibody)
can be used in the methods of the invention to modulate the
expression and/or activity of a soluble P-selectin polypeptide.
Alternatively, an antibody against a P-selectin ligand or receptor,
e.g., PSGL-1, may be useful in the methods of the invention. For
example, an anti-PSGL-1 antibody may be used to mimic the activity
of soluble P-selectin. In one embodiment an activating anti-PSGL-1
antibody induces the release of microparticles containing tissue
factor.
[0078] An anti-P-selectin antibody can also be used to isolate
soluble P-selectin polypeptides or fusion proteins by standard
techniques, such as affinity chromatography or immunoprecipitation.
An anti-P-selectin antibody can facilitate the purification of
natural soluble P-selectin from cell cultures and of recombinantly
produced soluble P-selectin expressed in host cells. Moreover, an
anti-P-selectin antibody can be used to detect soluble P-selectin
polypeptide (e.g., in a blood sample) in order to evaluate the
abundance and pattern of expression of the soluble P-selectin
polypeptide. Anti-P-selectin antibodies can be used diagnostically
to monitor protein levels in blood as part of a clinical testing
procedure, e.g., to, for example, determine hemostatic activity,
i.e., a procoagulant state. Detection can be facilitated by
coupling (i.e., physically linking) the antibody to a detectable
substance. Examples of detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, 131I, .sup.35S or .sup.3H.
[0079] II. Isolated Nucleic Acid Molecules
[0080] The methods of the invention include the use of isolated
nucleic acid molecules that encode P-selectin polypeptides (e.g., a
soluble P-selectin polypeptide) or biologically active portions
thereof. The nucleotide sequences encoding human (GenBank Accession
Numbers NM.sub.--003005 and M25322), rat (GenBank Accession Numbers
NM.sub.--013114 and L23088), mouse (GenBank Accession Numbers
NM.sub.--011347 and M87861), and bovine (GenBank Accession Number
L12041) P-selectin have been disclosed.
[0081] As used herein, the term "nucleic acid molecule" is intended
to include DNA molecules (e.g., cDNA or genomic DNA) and RNA
molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0082] The term "isolated nucleic acid molecule" includes nucleic
acid molecules which are separated from other nucleic acid
molecules which are present in the natural source of the nucleic
acid. For example, with regards to genomic DNA, the term "isolated"
includes nucleic acid molecules which are separated from the
chromosome with which the genomic DNA is naturally associated.
Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank the nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. For example, in various
embodiments, the isolated nucleic acid molecule encoding soluble
P-selectin can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1
kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank
the nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived. Moreover, an "isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of
other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
[0083] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule encoding soluble P-selectin, a soluble
P-selectin fusion protein, or a portion thereof, can be isolated
using standard molecular biology techniques (e.g., as described in
Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0084] A nucleic acid of the invention can be amplified using cDNA,
mRNA or alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to P-selectin
nucleotide sequences can be prepared by standard synthetic
techniques, e.g., using an automated DNA synthesizer.
[0085] A nucleic acid fragment encoding a "biologically active
portion" of a P-selectin polypeptide can be prepared by isolating a
portion of the nucleotide sequence of a P-selectin gene having a
P-selectin biological activity (the biological activities, e.g.,
the hemostatic activity, of soluble P-selectin are described
herein), expressing the encoded portion of the P-selectin
polypeptide (e.g., by recombinant expression in vitro) and
assessing the activity of the encoded portion of the P-selectin
polypeptide.
[0086] The skilled artisan will further appreciate that changes can
be introduced by mutation into the nucleotide sequence encoding a
P-selectin polypeptide, thereby leading to changes in the amino
acid sequence of the encoded P-selectin polypeptide, without
altering the functional ability of the P-selectin polypeptide. For
example, nucleotide substitutions leading to amino acid
substitutions at "non-essential" amino acid residues can be made in
the sequence of a P-selectin gene. A "non-essential" amino acid
residue is a residue that can be altered from the wild-type
sequence of a P-selectin polypeptide without altering the
biological activity, whereas an "essential" amino acid residue is
required for biological activity. For example, amino acid residues
that are conserved among the P-selectin proteins from different
species are predicted to be particularly unamenable to
alteration.
[0087] Accordingly, the methods of the invention may include the
use of nucleic acid molecules encoding P-selectin polypeptides that
contain changes in amino acid residues that are not essential for
activity.
[0088] An isolated nucleic acid molecule encoding a P-selectin
polypeptide can be created by introducing one or more nucleotide
substitutions, additions or deletions into the nucleotide sequence
of a P-selectin gene such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into a nucleic acid
sequence by standard techniques, such as site-directed mutagenesis
and PCR-mediated mutagenesis. Preferably, conservative amino acid
substitutions are made at one or more predicted non-essential amino
acid residues. A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a soluble P-selectin
polypeptide is preferably replaced with another amino acid residue
from the same side chain family. Alternatively, in another
embodiment, mutations can be introduced randomly along all or part
of a P-selectin coding sequence, such as by saturation mutagenesis,
and the resultant mutants can be expressed recombinantly and
screened for biological activity to identify mutants that retain
activity, e.g., in an animal model described herein. In a preferred
embodiment, a mutant soluble P-selectin polypeptide protein can be
assayed for the ability to modulate hemostatic activity.
[0089] In addition to the nucleic acid molecules encoding
P-selectin polypeptides described herein, another aspect of the
invention pertains to isolated nucleic acid molecules which are
antisense thereto. An "antisense" nucleic acid comprises a
nucleotide sequence which is complementary to a "sense" nucleic
acid encoding a protein, e.g., complementary to the coding strand
of a double-stranded cDNA molecule or complementary to an mRNA
sequence. Accordingly, an antisense nucleic acid can hydrogen bond
to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire P-selectin coding strand, or to only a
portion thereof. In one embodiment, an antisense nucleic acid
molecule is antisense to a "coding region" of the coding strand of
a nucleotide sequence encoding P-selectin. The term "coding region"
refers to the region of the nucleotide sequence comprising codons
which are translated into amino acid residues. In another
embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide sequence
encoding P-selectin. The term "noncoding region" refers to 5' and
3' sequences which flank the coding region that are not translated
into amino acids.
[0090] Given the coding strand sequences encoding P-selectin,
antisense nucleic acids of the invention can be designed according
to the rules of Watson and Crick base pairing. The antisense
nucleic acid molecule can be complementary to the entire coding
region of P-selectin mRNA, but more preferably is an
oligonucleotide which is antisense to only a portion of the coding
or noncoding region of P-selectin mRNA. For example, the antisense
oligonucleotide can be complementary to the region surrounding the
translation start site of P-selectin mRNA. An antisense
oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30,
35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid
of the invention can be constructed using chemical synthesis and
enzymatic ligation reactions using procedures known in the art. For
example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted nucleotides can be used. Examples of modified
nucleotides which can be used to generate the antisense nucleic
acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomet-
hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
[0091] In yet another embodiment, the P-selectin nucleic acid
molecules of the present invention can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the
stability, hybridization, or solubility of the molecule. For
example, the deoxyribose phosphate backbone of the nucleic acid
molecules can be modified to generate peptide nucleic acids (see
Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1):
5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics, e.g., DNA mimics, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup B. et al. (1996) supra; Perry-OKeefe et al. Proc. Natl. Acad.
Sci. 93: 14670-675.
[0092] PNAs of P-selectin nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of P-selectin nucleic acid molecules can also be used in the
analysis of single base pair mutations in a gene, (e.g., by
PNA-directed PCR clamping); as `artificial restriction enzymes`
when used in combination with other enzymes, (e.g., SI nucleases
(Hyrup B. (1996) supra)); or as probes or primers for DNA
sequencing or hybridization (Hyrup B. et al. (1996) supra;
Perry-O'Keefe supra).
[0093] In another embodiment, PNAs of P-selectin can be modified,
(e.g., to enhance their stability or cellular uptake), by attaching
lipophilic or other helper groups to PNAs, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of
P-selectin nucleic acid molecules can be generated which may
combine the advantageous properties of PNA and DNA. Such chimeras
allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases),
to interact with the DNA portion while the PNA portion would
provide high binding affinity and specificity. PNA-DNA chimeras can
be linked using linkers of appropriate lengths selected in terms of
base stacking, number of bonds between the nucleobases, and
orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA
chimeras can be performed as described in Hyrup B. (1996) supra and
Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For
example, a DNA chain can be synthesized on a solid support using
standard phosphoramidite coupling chemistry and modified nucleoside
analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thy- midine
phosphoramidite, can be used as a between the PNA and the 5' end of
DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA
monomers are then coupled in a stepwise manner to produce a
chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn
P. J. et al. (1996) supra). Alternatively, chimeric molecules can
be synthesized with a 5'DNA segment and a 3'PNA segment (Peterser,
K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
[0094] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (See, e.g., Krol et al.
(1988) Bio-Techniques 6:958-976) or intercalating agents. (See,
e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the
oligonucleotide may be conjugated to another molecule, (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
[0095] III. Recombinant Expression Vectors and Host Cells
[0096] The methods of the invention include the use of vectors,
preferably expression vectors, containing a nucleic acid encoding a
P-selectin polypeptide (or a portion thereof, e.g., a soluble
P-selectin polypeptide). As used herein, the term "vector" refers
to a nucleic acid molecule capable of transporting another nucleic
acid to which it has been linked. One type of vector is a
"plasmid", which refers to a circular double stranded DNA loop into
which additional DNA segments can be ligated. Another type of
vector is a viral vector, wherein additional DNA segments can be
ligated into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the methods of the invention may include other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0097] The recombinant expression vectors used in the methods of
the invention comprise a nucleic acid of the invention in a form
suitable for expression of the nucleic acid in a host cell, which
means that the recombinant expression vectors include one or more
regulatory sequences, selected on the basis of the host cells to be
used for expression, which is operatively linked to the nucleic
acid sequence to be expressed. Within a recombinant expression
vector, "operably linked" is intended to mean that the nucleotide
sequence of interest is linked to the regulatory sequence(s) in a
manner which allows for expression of the nucleotide sequence
(e.g., in an in vitro transcription/translation system or in a host
cell when the vector is introduced into the host cell). The term
"regulatory sequence" is intended to include promoters, enhancers
and other expression control elements (e.g., polyadenylation
signals). Such regulatory sequences are described, for example, in
Goeddel; Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990). Regulatory sequences
include those which direct constitutive expression of a nucleotide
sequence in many types of host cells and those which direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, and the like. The expression vectors used in the methods
of the invention can be introduced into host cells to thereby
produce proteins or peptides, including fusion proteins or
peptides, encoded by nucleic acids as described herein (e.g.,
soluble P-selectin polypeptides, fusion proteins, and the
like).
[0098] The recombinant expression vectors used in the methods of
the invention can be designed for expression of P-selectin
polypeptides or fusion proteins in prokaryotic or eukaryotic cells,
e.g., for use in the methods of the invention. For example, soluble
P-selectin polypeptides or fusion proteins can be expressed in
bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0099] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility and/or stability of the recombinant protein; and 3) to
aid in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin
and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRITS
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein. Purified P-selectin fusion proteins
(e.g., soluble P-selectin-Ig) can be utilized to modulate
hemostatic potential, as described and exemplified herein. In one
embodiment, a soluble P-selectin fusion protein expressed in a
retroviral expression vector of the present invention can be
utilized to infect cells, e.g., hematopoietic cells, which are
subsequently transplanted into recipients. The hemostatic activity
of the subject recipient is then examined after sufficient time has
passed (e.g., six (6) weeks).
[0100] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
prophage harboring a T7 gn1 gene under the transcriptional control
of the lacUV 5 promoter.
[0101] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid
to be inserted into an expression vector so that the individual
codons for each amino acid are those preferentially utilized in E.
coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[0102] In another embodiment, the P-selectin expression vector is a
yeast expression vector. Examples of vectors for expression in
yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO
J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell
30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2
(Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen
Corp, San Diego, Calif.).
[0103] Alternatively, P-selectin polypeptides can be expressed in
insect cells using baculovirus expression vectors. Baculovirus
vectors available for expression of proteins in cultured insect
cells (e.g., Sf 9 cells) include the pAc series (Smith et al.
(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and
Summers (1989) Virology 170:31-39).
[0104] In yet another embodiment, a nucleic acid used in the
methods of the invention is expressed in mammalian cells using a
mammalian expression vector. Examples of mammalian expression
vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian
cells, the expression vector's control functions are often provided
by viral regulatory elements. For example, commonly used promoters
are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian
Virus 40. For other suitable expression systems for both
prokaryotic and eukaryotic cells see chapters 16 and 17 of
Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0105] In another embodiment, the recombinant mammalian expression
vector used in the methods of the invention is capable of directing
expression of the nucleic acid preferentially in a particular cell
type (e.g., tissue-specific regulatory elements are used to express
the nucleic acid). Tissue-specific regulatory elements are known in
the art. Non-limiting examples of suitable tissue-specific
promoters include the albumin promoter (liver-specific; Pinkert et
al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters
(Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular
promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
8:729-733) and immunoglobulins (Banerji et al. (1983) Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748),
neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),
endothelial cell-specific promoters (e.g., KDR/flk promoter; U.S.
Pat. No. 5,888,765), pancreas-specific promoters (Edlund et al.
(1985) Science 230:912-916), and mammary gland-specific promoters
(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). Developmentally-regulated
promoters are also encompassed, for example the murine hox
promoters (Kessel and Gruss (1990) Science 249:374-379) and the
.alpha.-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.
3:537-546).
[0106] The expression characteristics of an endogenous P-selectin
gene within a cell line or microorganism may be modified by
inserting a heterologous DNA regulatory element into the genome of
a stable cell line or cloned microorganism such that the inserted
regulatory element is operatively linked with the endogenous
P-selectin gene. For example, an endogenous P-selectin gene which
is normally "transcriptionally silent", i.e., a P-selectin gene
which is normally not expressed, or is expressed only at very low
levels in a cell line or microorganism, may be activated by
inserting a regulatory element which is capable of promoting the
expression of a normally expressed gene product in that cell line
or microorganism. Alternatively, a transcriptionally silent,
endogenous P-selectin gene may be activated by insertion of a
promiscuous regulatory element that works across cell types.
[0107] A heterologous regulatory element may be inserted into a
stable cell line or cloned microorganism, such that it is
operatively linked with an endogenous P-selectin gene, using
techniques, such as targeted homologous recombination, which are
well known to those of skill in the art, and described, e.g., in
Chappel, U.S. Pat. No. 5,272,071; PCT publication No. WO 91/06667,
published May 16, 1991.
[0108] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to P-selectin mRNA.
Regulatory sequences operatively linked to a nucleic acid cloned in
the antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0109] Another aspect of the invention pertains to use of host
cells into which a P-selectin nucleic acid molecule of the
invention is introduced, e.g., a P-selectin nucleic acid molecule
within a recombinant expression vector or a P-selectin nucleic acid
molecule containing sequences which allow it to homologously
recombine into a specific site of the host cell's genome. The terms
"host cell" and "recombinant host cell" are used interchangeably
herein. It is understood that such terms refer not only to the
particular subject cell but to the progeny or potential progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0110] A host cell can be any prokaryotic or eukaryotic cell. For
example, a P-selectin polypeptide or fusion protein can be
expressed in bacterial cells such as E. coli, insect cells, yeast
or mammalian cells (such as hematopoietic cells, leukocytes, human
umbilical vein endothelial cells (HUVEC), human microvascular
endothelial cells (HMVEC), Chinese hamster ovary cells (CHO) or COS
cells). Other suitable host cells are known to those skilled in the
art.
[0111] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0112] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding a soluble P-selectin polypeptide or can be introduced
on a separate vector. Cells stably transfected with the introduced
nucleic acid can be identified by drug selection (e.g., cells that
have incorporated the selectable marker gene will survive, while
the other cells die).
[0113] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) a P-selectin polypeptide or fusion protein for use in the
methods of the invention. In one embodiment, a host cell (into
which a recombinant expression vector encoding a soluble P-selectin
polypeptide or fusion protein has been introduced) is cultured in a
suitable medium such that a soluble P-selectin polypeptide or
fusion protein is produced. In another embodiment, a soluble
P-selectin polypeptide or fusion protein is isolated from the
medium or the host cell. A recombinant cell expressing soluble
P-selectin or a soluble P-selectin fusion protein can also be
administered to a subject to modulate hemostasis.
[0114] IV. Methods of Treatment
[0115] The present invention discloses methods for modulating
hemostatic potential by modulating P-selectin activity (e.g., the
levels of soluble P-selectin). Accordingly, the present invention
provides for both prophylactic and therapeutic methods of treating
a subject at risk of (or susceptible to) or having a hemostatic
disorder, e.g., a disorder associated with aberrant or unwanted
hemostatic activity, or a vasculature-associated disease. With
regards to both prophylactic and therapeutic methods of treatment,
such treatments may be specifically tailored or modified, based on
knowledge obtained from the field of pharmacogenomics.
"Pharmacogenomics", as used herein, refers to the application of
genomics technologies such as gene sequencing, statistical
genetics, and gene expression analysis to drugs in clinical
development and on the market. More specifically, the term refers
the study of how a patient's genes determine his or her response to
a drug (e.g., a patient's "drug response phenotype", or "drug
response genotype".) Thus, another aspect of the invention provides
methods for tailoring an individual's prophylactic or therapeutic
treatment with either soluble P-selectin or modulators of
P-selectin activity according to that individual's drug response
genotype. Pharmacogenomics allows a clinician or physician to
target prophylactic or therapeutic treatments to patients who will
most benefit from the treatment and to avoid treatment of patients
who will experience toxic drug-related side effects.
[0116] A. Prophylactic Methods
[0117] The assessment of P-selectin activity can used as a measure
of hemostatic activity. Accordingly, in one aspect, the invention
provides a method for preventing in a subject, a hemostatic
disorder, e.g., a disorder associated with an aberrant or unwanted
hemostatic activity, or a vasculature-associated disease by
administering to the subject a modulator of P-selectin activity, or
a soluble P-selectin polypeptide. Subjects at risk for a hemostatic
disorder or a vasculature-associated disease can be identified by,
for example, any or a combination of diagnostic or prognostic
assays as described herein, e.g., by assessing P-selectin activity
in a biological sample (i.e., plasma levels of soluble P-selectin).
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the hemostatic
disorder, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
disorder, for example, a soluble P-selectin polypeptide, or a
modulator of P-selectin activity, e.g., a P-selectin agonist or
antagonist, can be used for treating the subject. The appropriate
agent can be determined based on screening assays described
herein.
[0118] B. Therapeutic Methods
[0119] Described herein are methods and compositions whereby
hemostatic disorders, vasculature-associated diseases, and symptoms
thereof, may be ameliorated. Certain hemostatic disorders, e.g., a
hypocoagulable state or a hemorrhagic disorder, are brought about,
at least in part, by the absence or reduction of hemostatic
activity. As such, an increase in hemostatic activity would bring
about the amelioration of disease symptoms. In addition, certain
vasculature-associated diseases are supported by a blood supply to
the disease site, for example, to provide oxygen and nutrients.
Similarly, the induction of a procoagulant state in the vasculature
supplying such disease sites would provide a beneficial effect.
[0120] Alternatively, certain other hemostatic diseases, e.g., a
thrombotic disorder, are brought about, at least in part, by the
presence or increase in hemostatic activity. As such, an reduction
in hemostatic activity would bring about the amelioration of
disease symptoms.
[0121] Techniques for the modulating hemostasis using modulators of
P-selectin activity are discussed herein. Accordingly, another
aspect of the invention pertains to methods of modulating
hemostasis or hemostatic potential for therapeutic purposes.
[0122] In an exemplary embodiment, the modulatory methods of the
invention involve administering a modulator of P-selectin activity,
or a soluble P-selectin polypeptide. A modulator of P-selectin
activity includes an agent that modulates (e.g., induces or
inhibits) one or more activities of P-selectin, or an agent that
modulates soluble P-selectin expression. A modulator of P-selectin
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring target molecule of a
soluble P-selectin polypeptide (e.g., a P-selectin ligand), an
anti-P-selectin antibody, a soluble P-selectin agonist or
antagonist, a peptidomimetic of a soluble P-selectin agonist or
antagonist, or other small molecule. In one embodiment, the agent
is an inducer of P-selectin activity. Examples of such inducers
include active soluble P-selectin polypeptides, a nucleic acid
molecule encoding a soluble P-selectin polypeptide, and a soluble
P-selectin mimetic, e.g., an activating anti-PSGL-1 antibody. In
another embodiment, the agent is an inhibitor of P-selectin
activity. Examples of such inhibitors include antisense soluble
P-selectin nucleic acid molecules, anti-P-selectin antibodies, and
soluble P-selectin inhibitors, e.g., soluble PSGL-1. As such, the
present invention provides methods of treating an individual
afflicted with a disease or disorder characterized by aberrant or
unwanted hemostatic activity. In one embodiment, the method
involves administering a modulator of P-selectin activity. In
another embodiment, the method involves administering a soluble
P-selectin polypeptide or a nucleic acid encoding a soluble
P-selectin polypeptide to induce hemostasis and/or a procoagulant
state.
[0123] (i) Methods for Inhibiting Soluble P-selectin Expression,
Synthesis, or Activity
[0124] As discussed above, certain hemostatic disorders, e.g.,
thrombotic disorders, may result from an increased or excessive
level of hemostatic activity. In such circumstances, hemostatic
activity, e.g., thrombosis, may have a causative or exacerbating
effect on the disease state. In such cases, a reduction in
hemostasis or hemostatic activity may be achieved by reducing
circulating levels of soluble P-selectin. As such, an inhibitor of
P-selectin activity may be used in accordance with the invention to
reduce hemostasis. Such compounds may include, but are not limited
to, small organic molecules, peptides, antibodies, and the
like.
[0125] For example, compounds can be administered that compete with
endogenous ligand for a soluble P-selectin polypeptide. The
resulting reduction in the amount of ligand-bound soluble
P-selectin polypeptide will modulate hemostatic activity. Compounds
that can be particularly useful for this purpose include, for
example, soluble proteins or peptides, such as peptides comprising
one or more of the extracellular domains, or portions and/or
analogs thereof, of the P-selectin ligand, PSGL-1, including, for
example, soluble fusion proteins such as Ig-tailed fusion proteins.
(For a discussion of the production of Ig-tailed fusion proteins,
see, for example, U.S. Pat. No. 5,116,964).
[0126] In one embodiment, an inhibitor of P-selectin activity which
reduces or inhibits the translocation of P-selectin from cellular
storage pools to the cell surface, or which reduce or inhibit the
proteolytic cleavage of cell surface P-selectin, can be effective
in reducing circulating soluble P-selectin levels, and thus
modulating hemostatic activity. Alternatively, an inhibitor of
P-selectin activity which reduces P-selectin gene expression (e.g.,
P-selectin gene transcription or translation), or the expression of
an alternatively spliced isoform of P-selectin lacking the
transmembrane domain, can be used to reduce hemostasis.
[0127] Further, antisense and ribozyme molecules which inhibit
expression of the P-selectin gene may also be used in accordance
with the invention to inhibit hemostasis. Still further, triple
helix molecules may be utilized in inhibiting soluble P-selectin
activity.
[0128] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a P-selectin protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention include direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies which
bind to cell surface receptors or antigens. The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are preferred.
[0129] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0130] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave P-selectin mRNA transcripts to
thereby inhibit translation of P-selectin mRNA. A ribozyme having
specificity for a P-selectin-encoding nucleic acid can be designed
based upon the nucleotide sequence of a P-selectin cDNA. For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in a
P-selectin-encoding mRNA (see, for example, Cech et al. U.S. Pat.
No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).
Alternatively, P-selectin mRNA can be used to select a catalytic
RNA having a specific ribonuclease activity from a pool of RNA
molecules (see, for example, Bartel, D. and Szostak, J. W. (1993)
Science 261:1411-1418).
[0131] P-selectin gene expression can also be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the P-selectin gene (e.g., the P-selectin promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the P-selectin gene in target cells (see, for
example, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;
Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher,
L. J. (1992) Bioassays 14(12):807-15).
[0132] Antibodies that are both specific for the P-selectin protein
and interfere with its activity may also be used to modulate or
inhibit P-selectin activity. Such antibodies may be generated,
using standard techniques, against the P-selectin protein itself or
against peptides corresponding to portions of the protein. Such
antibodies include but are not limited to polyclonal, monoclonal,
Fab fragments, single chain antibodies, or chimeric antibodies.
[0133] In instances where the target gene protein is intracellular,
e.g., localized in storage granules, and whole antibodies are used,
internalizing antibodies may be preferred. Lipofectin liposomes may
be used to deliver the antibody or a fragment of the Fab region
which binds to the target epitope into cells. Where fragments of
the antibody are used, the smallest inhibitory fragment which binds
to the target protein's binding domain is preferred. For example,
peptides having an amino acid sequence corresponding to the domain
of the variable region of the antibody that binds to the target
gene protein may be used. Such peptides may be synthesized
chemically or produced via recombinant DNA technology using methods
well known in the art (described in, for example, Creighton (1983),
supra; and Sambrook et al. (1989) supra). Single chain neutralizing
antibodies which bind to intracellular target gene epitopes may
also be administered. Such single chain antibodies may be
administered, for example, by expressing nucleotide sequences
encoding single-chain antibodies within the target cell population
by utilizing, for example, techniques such as those described in
Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).
[0134] In certain embodiments, antibodies that are specific for the
extracellular domain of the P-selectin protein, for example, and
that interfere with its activity, are particularly useful in
modulating hemostasis. Such antibodies are especially efficient
because they can access the target domains directly from the
bloodstream. Any of the administration techniques described below
which are appropriate for peptide administration may be utilized to
effectively administer inhibitory P-selectin antibodies to their
site of action.
[0135] Antibodies for the modulation of P-selectin function are
disclosed in U.S. Pat. Nos. 6,033,667; 5,800,815; and
5,622,701.
[0136] The inhibitors of P-selectin, as described herein, may be
administered alone or in conjunction with other agents, compounds,
or compositions which are useful in reducing hemostasis or
thrombosis, including, but not limited to, heparin, aspirin, and
other anti-coagul ants such as warfarin (Coumadin.TM.), nicoumalone
(Sintrom.TM.), or anti-platelet aggregation agents such as
inhibitors of .alpha.IIb.beta.3.
[0137] (ii) Methods for Restoring or Increasing P-selectin
Polypeptide Activity
[0138] Certain hemostatic disorders, e.g., hemorrhagic disorders,
may result from an reduced level of hemostatic activity. Moreover,
the progression of some vasculature-associated disorders is
dependent on a blood supply to the disease site. In such
circumstances, a reduction in or insufficient hemostatic activity,
may have a causative or exacerbating effect on the disease state.
In such cases, an increase in hemostasis or induction of a
procoagulant state may be achieved by using an inducer of
P-selectin activity to increase P-selectin activity, preferably by
increasing circulating levels of soluble P-selectin.
[0139] Described in this section are methods whereby the level of
soluble P-selectin activity may be increased to levels wherein the
symptoms of hypocoagulation disorders or vasculature-associated
diseases are ameliorated. The level of soluble P-selectin
polypeptide activity may be increased, for example, by either
increasing the level of P-selectin gene expression, e.g., an
alternatively spliced isoform of P-selectin lacking the
transmembrane domain, or by increasing the plasma level of active
soluble P-selectin protein which is present.
[0140] For example, a soluble P-selectin polypeptide or fusion
protein, at a level sufficient to ameliorate disease symptoms may
be administered to a patient exhibiting such symptoms. Any of the
techniques discussed herein may be used for such administration.
One of skill in the art will readily know how to determine the
concentration of effective, non-toxic doses of the soluble
P-selectin polypeptide, utilizing techniques such as those
described herein.
[0141] Additionally, RNA sequences encoding a soluble P-selectin
polypeptide may be directly administered to a patient exhibiting
disease symptoms, at a concentration sufficient to produce a level
of soluble P-selectin polypeptide such that disease symptoms are
ameliorated. Any of the techniques discussed below, which achieve
intracellular administration of compounds, such as, for example,
liposome administration, may be used for the administration of such
RNA molecules. The RNA molecules may be produced, for example, by
recombinant techniques such as those described herein.
[0142] Further, subjects may be treated by gene replacement
therapy. One or more copies of a gene encoding soluble P-selectin,
or a soluble P-selectin fusion protein, that directs the production
of a functional soluble P-selectin polypeptide or fusion protein,
may be inserted into cells using vectors which include, but are not
limited to adenovirus, adeno-associated virus, and retrovirus
vectors, in addition to other particles that introduce DNA into
cells, such as liposomes. Additionally, techniques such as those
described above may be used for the introduction of soluble
P-selectin gene sequences into human cells.
[0143] Cells, preferably, autologous cells, containing soluble
P-selectin expressing gene sequences may then be introduced or
reintroduced into the subject at positions which allow for the
amelioration of disease symptoms.
[0144] In one embodiment, inducers of P-selectin activity which
increase or enhance the translocation of P-selectin from cellular
storage pools to the cell surface, or which increase or enhance the
proteolytic cleavage of cell surface P-selectin, can be effective
in increasing circulating soluble P-selectin levels, and thus
modulating hemostatic activity. Alternatively, compounds which
stimulate P-selectin gene expression (e.g., P-selectin gene
transcription or translation), or the expression of an
alternatively spliced isoform of P-selectin lacking the
transmembrane domain, can be used to induce hemostasis.
Furthermore, inducers of P-selectin activity which enhance
P-selectin activity, e.g., a soluble P-selectin agonist, may be
used in accordance with the invention to induce hemostasis. In
another embodiment, inducers of P-selectin activity which mimic
P-selectin activity may be used to modulate hemostatic activity.
For example, an inducer of P-selectin activity, e.g., an antibody,
which binds to and activates a P-selectin ligand or receptor on a
cell can be used to modulate hemostasis. In one embodiment, an
antibody against PSGL-1, preferably an activating antibody, binds
to PSGL-1 on a cell and modulates hemostatic activity. In another
embodiment, an inducer of P-selectin activity binds to a P-selectin
ligand or receptor on a cell induces release of microparticles
containing tissue factor.
[0145] Such inducers of P-selectin activity may include, but are
not limited to, small organic molecules, peptides, antibodies, and
the like.
[0146] Inducers of P-selectin activity, as described herein, may be
administered alone or in conjunction with other anti-hemorrhagic or
pro-coagulant agents, compounds or compositions, including, but not
limited to Factor VIII, von Willebrand factor, platelets, the
absorption analogue DDAVP, and fibrin, e.g., fibrin glue. In one
embodiment, inducers of P-selectin activity as described herein may
be administered to a patient suffering from, for example,
hemophilia A or von Willebrand disease where antibodies to Factor
VIII have been developed by the patient, thereby reducing the
effectiveness of Factor VIII replacement therapy alone.
[0147] C. Pharmacogenomics
[0148] A modulators of P-selectin activity, for example, as
identified by a screening assay described herein, or a soluble
P-selectin polypeptide, can be administered to individuals to treat
(prophylactically or therapeutically) hemostatic disorders
associated with aberrant or unwanted hemostatic activity. In
conjunction with such treatment, pharmacogenomics (i.e., the study
of the relationship between an individual's genotype and that
individual's response to a foreign compound or drug) may be
considered. Differences in metabolism of therapeutics can lead to
severe toxicity or therapeutic failure by altering the relation
between dose and blood concentration of the pharmacologically
active drug. Thus, a physician or clinician may consider applying
knowledge obtained in relevant pharmacogenomics studies in
determining whether to administer a modulator of P-selectin
activity or a soluble P-selectin polypeptide, as well as tailoring
the dosage and/or therapeutic regimen of treatment with a modulator
of P-selectin activity, or a soluble P-selectin polypeptide.
[0149] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):254-266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic
defects or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0150] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0151] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drugs
target is known (e.g., P-selectin), all common variants of that
gene can be fairly easily identified in the population and it can
be determined if having one version of the gene versus another is
associated with a particular drug response.
[0152] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0153] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a soluble P-selectin polypeptide, or modulator
thereof, of the present invention) can give an indication whether
gene pathways related to toxicity have been turned on.
[0154] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment an individual. This knowledge, when applied to dosing or
drug selection, can avoid adverse reactions or therapeutic failure
and thus enhance therapeutic or prophylactic efficiency when
treating a subject with a soluble P-selectin polypeptide or soluble
P-selectin modulator.
[0155] VI. Screening Assays
[0156] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules (organic or inorganic) or other drugs) which modulate
P-selectin activity, and which may thus be used to modulate
hemostatic potential.
[0157] These assays are designed to identify compounds, for
example, that bind to a P-selectin polypeptide, e.g., a soluble
P-selectin polypeptide, bind to other proteins that interact with a
P-selectin polypeptide, and modulate the interaction of a
P-selectin polypeptide with other proteins, e.g., a P-selectin
ligand, and thus modulate P-selectin activity. Screening assays can
also be used to identify modulators of P-selectin activity, for
example, that regulate P-selectin gene expression, the alternative
splicing of the P-selectin gene encoding a soluble P-selectin
isoform, the translocation of P-selection from cellular storage
pools to the cell surface, and the proteolytic cleavage of
P-selection on the cell surface resulting in the release of soluble
P-selectin. Moreover, screening assays can be used to identify
inducers of P-selectin activity, for example, that mimic the
activity of a P-selectin polypeptide, e.g., the binding of
P-selectin to a P-selectin ligand or receptor, or the activity of
P-selectin towards a P-selectin responsive cell. Such compounds may
include, but are not limited to, peptides, antibodies, or small
organic or inorganic compounds.
[0158] Compounds identified via assays such as those described
herein may be useful, for example, for modulating hemostasis, and
for treating hemostatic disorders and/or vasculature-associated
diseases. In instances whereby a hemostatic disorder or a
vasculature-associated disease results from an overall lower level
of coagulation, useful compounds would bring about an effective
increase in the level of P-selectin activity, e.g., an inducer of
P-selectin activity. In other instances wherein a hemostatic
disorder results from an overall increased level of coagulation or
thrombosis, compounds that reduce the level of P-selectin activity
would be beneficial, e.g., an inhibitor of P-selectin activity.
Cell and animal models for testing the effectiveness of compounds
identified by techniques such as those described in this section
are discussed herein.
[0159] The test compounds can be obtained using any of the numerous
approaches in combinatorial library methods known in the art,
including: biological libraries; spatially addressable parallel
solid phase or solution phase libraries; synthetic library methods
requiring deconvolution; the `one-bead one-compound` library
method; and synthetic library methods using affinity chromatography
selection. The biological library approach is limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).
[0160] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0161] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner USP 5,223,409), spores (Ladner USP '409), plasmids
(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on
phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990)
Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.
87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner
supra.).
[0162] In one embodiment, an assay is a cell-based assay comprising
contacting a cell with a test compound and determining the ability
of the test compound to modulate (e.g., induce or inhibit)
P-selectin activity. For example, a cell expressing a P-selectin
ligand or receptor, e.g., a leukocyte, is contacted with soluble
P-selectin polypeptide either alone or in the presence of a test
compound, and the ability of the test compound to modulate soluble
P-selectin induced release of microparticles containing tissue
factor is determined, as described herein. A similar cell-based
assay could be used to identify a compound which mimics soluble
P-selectin hemostatic activity, for example, by assaying the test
compound for the ability to induce the release of microparticles
containing tissue factor.
[0163] Furthermore, in another embodiment, a cell based assay can
be used to determine the ability of the test compound to modulate
the translocation of P-selectin to the cell surface, or to modulate
the proteolytic cleavage of P-selectin from the cell surface. The
presence of P-selection on the surface of a cell can be assessed by
standard techniques, such as flow cytometry. The cleavage of
P-selectin and concurrent release of soluble P-selectin can be
assessed by measuring the level of membrane-associated P-selectin
as compared to the level of soluble P-selectin in the culture
medium.
[0164] In a further embodiment, modulators of P-selectin activity
are identified in a method wherein a cell is contacted with a
candidate compound and the expression of soluble P-selectin mRNA or
protein in the cell culture is determined by standard techniques.
The level of expression of soluble P-selectin mRNA or protein in
the presence of the candidate compound is compared to the level of
expression of soluble P-selectin mRNA or protein in the absence of
the candidate compound. The candidate compound can then be
identified as a modulator of soluble P-selectin activity based on
this comparison. For example, when expression of soluble P-selectin
mRNA or protein is greater (statistically significantly greater) in
the presence of the candidate compound than in its absence, the
candidate compound is identified as a inducer of P-selectin
activity. Alternatively, when expression of soluble P-selectin mRNA
or protein is less (statistically significantly less) in the
presence of the candidate compound than in its absence, the
candidate compound is identified as an inhibitor of P-selectin
activity.
[0165] In another embodiment, the ability of a test compound to
modulate soluble P-selectin binding to a receptor or ligand can
also be determined, for example by coupling soluble P-selectin with
a radioisotope or enzymatic label such that the binding of the
soluble P-selectin can be determined by detecting labeled soluble
P-selectin in a complex. For example, compounds (e.g., P-selectin
polypeptides, P-selectin ligands) can be labeled with .sup.125I,
.sup.35S, .sup.14C, or .sup.3H, either directly or indirectly, and
the radioisotope detected by direct counting of radioemmission or
by scintillation counting. Compounds can further be enzymatically
labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or luciferase, and the enzymatic label detected by
determination of conversion of an appropriate substrate to
product.
[0166] Animal-based systems which act as models for hemostatic
function or disease, such as the animal models described and
exemplified herein, e.g., P-selectin deficient mice and vWF
deficient mice, include, but are not limited to, non-recombinant
and engineered transgenic animals. Models for studying
vasculature-associated disease in vivo include animal models of
tumori genesis, tumor metastasis, and arteriosclerosis. Models for
studying thrombotic disorders in vivo include animal models of
thrombosis such as those described in, at least, for example,
Leadley et al. (2000) J Phannacol Toxicol Methods 43: 101, and
Dorffler-Melly, et al. (2000) Basic Res Cardiol 95:503.
[0167] The animal-based model systems may be used in a variety of
applications, for example, as part of screening strategies designed
to identify compounds which are modulators of P-selectin activity.
Thus, the animal-based models may be used to identify drugs,
pharmaceuticals, therapies and interventions which may be effective
in modulating hemostasis and treating hemostatic disorders and
vasculature-associated diseases. For example, animal models may be
exposed to a compound, suspected of exhibiting an ability to
modulate P-selectin activity, and the response of the animals to
the exposure may be monitored by assessing hemostatic activity
before and after treatment. Hemostatic activity can be assessed
using a clinically established test, e.g., a test of plasma
clotting time, or using a method exemplified herein, e.g., fibrin
formation in a perfusion chamber, plasma levels of soluble
P-selectin and fibrinogen, hemorrhagic lesions in a local
Schwartzman reaction, tissue factor activity.
[0168] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulator of P-selectin activity can be identified using a
cell-based assay, and the ability of the agent to modulate
P-selectin activity can be confirmed in vivo, e.g., in an animal
such as an animal model for hemostasis or a hemostatic
disorder.
[0169] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further test a modulator of
P-selectin activity as described herein in an appropriate animal
model for the ability to hemostatic potential. For example, an
inducer or inhibitor of P-selectin activity can be used in an
animal model to determine the LD50 and the ED50 in animal subjects,
and such data can be used to determine the in vivo efficacy,
toxicity, or side effects of treatment with such a potential
modulator of hemostatic activity.
[0170] With regard to intervention, any treatments which modulate
P-selectin activity and/or hemostatic potential should be
considered as candidates for human therapeutic intervention.
Dosages of test agents may be determined by deriving dose-response
curves. Furthermore, this invention pertains to uses of newly
identified modulators of P-selectin activity for modulating
hemostasis, as described herein.
[0171] Additionally, gene expression patterns may be utilized to
assess the ability of a compound, e.g., a modulator of P-selectin
activity, to modulate hemostasis. For example, the expression
pattern of one or more genes may form part of a "gene expression
profile" or "transcriptional profile" which may be then be used in
such an assessment. "Gene expression profile" or "transcriptional
profile", as used herein, includes the pattern of mRNA expression
obtained for a given tissue or cell type under a given set of
conditions. Such conditions may include, but are not limited to,
hemostatic disorders and/or vasculature-associated disease,
including any of the control or experimental conditions described
herein, for example, in a local Schwartzman reaction, or in an
animal model of P-selectin deficiency or vWF deficiency. Gene
expression profiles may be generated, for example, by utilizing a
differential display procedure, Northern analysis and/or RT-PCR. In
one embodiment, P-selectin gene sequences may be used as probes
and/or PCR primers for the generation and corroboration of such
gene expression profiles.
[0172] Gene expression profiles may be characterized for known
states, either hemostatic disease or normal, e.g., within the
animal-based model systems described herein. Subsequently, these
known gene expression profiles may be compared to ascertain the
effect a test compound has to modify such gene expression profiles,
and to cause the profile to more closely resemble that of a more
desirable profile.
[0173] For example, administration of a compound may cause the gene
expression profile of a hemostatic disorder model system to more
closely resemble the control system.
[0174] VI. Predictive Medicine
[0175] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining P-selectin activity, e.g.,
soluble P-selectin expression in the context of a biological sample
(e.g., blood, serum, cells, tissue) to thereby determine hemostatic
activity, and to determine whether an individual is afflicted with
a hemostatic disorder, or is at risk of developing a hemostatic
disorder. The invention also provides for prognostic (or
predictive) assays for determining whether an individual is
manifesting a procoagulant state. Such assays can be used for
prognostic or predictive purpose to modulate hemostasis, and
thereby prophylactically treat an individual prior to the onset of
a hemostatic disorder.
[0176] Another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs, compounds) on hemostatic activity
or procoagulant state in clinical trials.
[0177] These and other agents are described in further detail in
the following sections.
[0178] A. Diagnostic Assays
[0179] The present invention encompasses methods for diagnostic and
prognostic evaluation of hemostatic disease conditions, and for the
identification of subjects exhibiting a predisposition to such
conditions.
[0180] An exemplary method for detecting the presence or absence
hemostatic activity in a biological sample involves obtaining a
biological sample from a test subject and contacting the biological
sample, e.g., a blood sample, with a compound or an agent capable
of detecting P-selectin activity, e.g., a P-selectin binding
substance that detects soluble P-selectin protein, such that the
presence of P-selectin activity is detected in the biological
sample.
[0181] A preferred agent for detecting soluble P-selectin protein
is an antibody capable of binding to soluble P-selectin protein,
preferably an antibody with a detectable label. Antibodies can be
polyclonal, or more preferably, monoclonal. An intact antibody, or
a fragment thereof (e.g., Fab or F(ab')2) can be used. The term
"labeled", with regard to the probe or antibody, is intended to
encompass direct labeling of the probe or antibody by coupling
(i.e., physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently labeled streptavidin.
[0182] The term "biological sample" is intended to include tissues,
cells and biological fluids isolated from a subject, as well as
tissues, cells and fluids present within a subject. That is, the
detection method of the invention can be used to detect P-selectin
activity in a biological sample in vitro as well as in vivo. In
vitro techniques for detection of P-selectin protein include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. For a detailed
explanation of methods for carrying out Western blot analysis, see
Sambrook et al, 1989, supra, at Chapter 18. The protein detection
and isolation methods employed herein may also be such as those
described in Harlow and Lane, for example, (Harlow, E. and Lane,
D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated
herein by reference in its entirety.
[0183] Detection of P-selectin activity can be accomplished, for
example, by immunofluorescence techniques employing a fluorescently
labeled antibody (see below) coupled with light microscopic, flow
cytometric, or fluorimetric detection.
[0184] Often a solid phase support or carrier is used as a support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0185] One means for labeling an anti-P-selectin polypeptide
specific antibody is via linkage to an enzyme and use in an enzyme
immunoassay (EIA) (Voller, "The Enzyme Linked Immunosorbent Assay
(ELISA)", Diagnostic Horizons 2:1-7, 1978, Microbiological
Associates Quarterly Publication, Walkersville, Md.; Voller, et
al., J. Clin. Pathol. 31:507-520 (1978); Butler, Meth. Enzymol.
73:482-523 (1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press,
Boca Raton, Fla., 1980; Ishikawa, et al., (eds.) Enzyme
Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is bound
to the antibody will react with an appropriate substrate,
preferably a chromogenic substrate, in such a manner as to produce
a chemical moiety which can be detected, for example, by
spectrophotometric, fluorimetric or by visual means. Enzymes which
can be used to detectably label the antibody include, but are not
limited to, malate dehydrogenase, staphylococcal nuclease,
delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0186] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect
fingerprint gene wild type or mutant peptides through the use of a
radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles
of Radioimmunoassays, Seventh Training Course on Radioligand Assay
Techniques, The Endocrine Society, March, 1986, which is
incorporated by reference herein). The radioactive isotope can be
detected by such means as the use of a gamma counter or a
scintillation counter or by autoradiography.
[0187] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0188] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0189] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0190] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0191] Furthermore, in vivo techniques for detection of P-selectin
protein include introducing into a subject a labeled
anti-P-selectin antibody. For example, the antibody can be labeled
with a radioactive marker whose presence and location in a subject
can be detected by standard imaging techniques.
[0192] In one embodiment, the biological sample contains protein
molecules from the test subject. A preferred biological sample is a
blood sample isolated by conventional means from a subject (e.g.,
venipuncture).
[0193] Moreover, it will be understood that any of the above
methods for detecting soluble P-selectin can be used to monitor the
course of treatment or therapy.
[0194] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of P-selectin
activity, e.g., soluble P-selectin, such that the presence of
P-selectin activity is detected in the biological sample, and
comparing the presence of P-selectin activity in the control sample
with the presence of P-selectin activity in the test sample, to
thereby assess hemostatic activity.
[0195] In one embodiment, an increased level of P-selectin activity
is indicative of increased hemostatic activity, e.g., a
procoagulant state. In another embodiment, a decreased level of
P-selectin activity is indicative of decreased hemostatic activity,
e.g., a hypocoagulable state.
[0196] B. Prognostic Assays
[0197] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
hemostatic disorder e.g., a disorder associated with aberrant or
unwanted hemostatic activity (i.e., a thrombotic disorder, a
hemorrhagic disorder). As used herein, the term "aberrant" includes
a level of hemostatic activity which deviates from clinically
established normal levels of hemostatic activity under defined
physiological conditions. Aberrant hemostatic activity includes
increased or decreased hemostatic activity. As used herein, the
term "unwanted" includes an unwanted phenomenon involved in a
biological response such as hemorrhage or thrombosis. For example,
the term unwanted includes hemostatic activity which is undesirable
in a subject.
[0198] The assays described herein, such as the preceding
diagnostic assays or the following assays, can be utilized to
identify a subject having or at risk of developing a hemostatic
disorder. Thus, the present invention provides a method for
identifying a hemostatic disorder associated with aberrant or
unwanted hemostatic activity in which a test sample is obtained
from a subject and P-selectin activity is detected, wherein the
presence of aberrant or unwanted P-selectin activity is diagnostic
for a subject having or at risk of developing a hemostatic
disorder. As used herein, a "test sample" refers to a biological
sample obtained from a subject of interest. For example, a test
sample can be a biological fluid (e.g., serum), cell sample, or
tissue.
[0199] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
hemostatic disorder. For example, such methods can be used to
determine whether a subject can be effectively treated with an
agent for a hemorrhagic disorder or a thrombotic disorder. Thus,
the present invention provides methods for determining whether a
subject can be effectively treated with an agent for a hemostatic
disorder, e.g., a disorder associated with aberrant or unwanted
hemostatic activity, in which a test sample is obtained and
P-selectin activity is detected (e.g., wherein the level of
P-selectin activity is diagnostic for a subject that can be
administered the agent to treat a hemostatic disorder).
[0200] Furthermore, any cell type or tissue in which P-selectin
activity is expressed may be utilized in the prognostic assays
described herein.
[0201] C. Monitoring of Effects During Clinical Trials
[0202] The present invention provides methods for evaluating the
efficacy of drugs and monitoring the progress of patients involved
in clinical trials for the treatment of hemostatic disorders.
[0203] Monitoring the influence of agents (e.g., drugs) on
P-selectin activity can be applied not only in basic drug
screening, but also in clinical trials. For example, the
effectiveness of an agent determined by a screening assay as
described herein to induce P-selectin activity can be monitored in
clinical trials of subjects exhibiting decreased or insufficient
hemostatic activity. Alternatively, the effectiveness of an agent
determined by a screening assay to inhibit P-selectin activity can
be monitored in clinical trials of subjects exhibiting increased
hemostatic activity, e.g., thrombosis or a procoagulant state. In
such clinical trials, P-selectin activity can be used as a "read
out" or marker of hemostatic activity. In addition, the level of
P-selectin activity may be used as a read out of a particular drug
or agent's effect on a hemostatic activity.
[0204] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an modulator of P-selectin activity (e.g., an agonist,
antagonist, peptidomimetic, protein, peptide, nucleic acid, small
molecule, or other drug candidate identified by the screening
assays described herein) including the steps of (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of P-selectin activity in the
preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of P-selectin in the post-administration samples; (v)
comparing the level of P-selectin activity in the
pre-administration sample with that in the post administration
sample or samples; and (vi) altering the administration of the
agent to the subject accordingly. For example, increased
administration of an inducer of P-selectin activity may be
desirable to increase P-selectin activity to higher levels than
detected, i.e., to increase the effectiveness of the agent to
promote hemostasis. Alternatively, increased administration an
inhibitor of P-selectin activity may be desirable to lower
P-selectin activity to lower levels than detected, i.e. to increase
the effectiveness of the agent to downregulate hemostasis.
According to such an embodiment, P-selectin activity may be used as
an indicator of the effectiveness of an agent, even in the absence
of an observable phenotypic response.
[0205] VII. Pharmaceutical Compositions
[0206] Active compounds for use in the methods of the invention can
be incorporated into pharmaceutical compositions suitable for
administration. As used herein, the language "active compounds"
includes nucleic acid molecules encoding soluble P-selectin,
soluble P-selectin proteins, and active fragments thereof, and
anti-P-selectin antibodies. Active compounds also include
modulators of soluble P-selectin activity, e.g., inducers and
inhibitors, identified compounds that modulate P-selectin gene
expression, synthesis, and/or activity, or compounds that mimic
P-selectin activity, e.g., an anti-PSGL-1 antibody. Such
compositions typically comprise the compound, nucleic acid
molecule, protein, or antibody and a pharmaceutically acceptable
carrier. As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0207] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, ophthalmic, and rectal
administration, including direct installation into a disease site.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0208] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0209] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a inducer or inhibitor of
P-selectin activity, a soluble P-selectin fusion protein) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0210] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0211] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0212] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0213] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0214] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0215] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals. In one embodiment, a "therapeutically effective dose"
refers to that amount of an active compound sufficient to result in
modulation of hemostasis or hemostatic potential. In another
embodiment, a therapeutically effective dose refers to an amount of
an active compound sufficient to result in amelioration of symptoms
of a hemostatic disorder or a vasculature-associated disease. In
yet another embodiment, a therapeutically effective dose refers to
that amount of an active compound sufficient to modulate the level
and/or activity of soluble P-selectin.
[0216] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0217] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0218] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments.
[0219] In a preferred example, a subject is treated with antibody,
protein, or polypeptide in the range of between about 0.1 to 20
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. It will also be appreciated that the effective dosage of
antibody, protein, or polypeptide used for treatment may increase
or decrease over the course of a particular treatment. Changes in
dosage may result and become apparent from the results of
diagnostic assays as described herein.
[0220] The present invention encompasses active agents which
modulate soluble P-selectin expression or activity. An agent may,
for example, be a small molecule. For example, such small molecules
include, but are not limited to, peptides, peptidomimetics, amino
acids, amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, organic or inorganic compounds
(i.e., including heteroorganic and organometallic compounds) having
a molecular weight less than about 10,000 grams per mole, organic
or inorganic compounds having a molecular weight less than about
5,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 1,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 500
grams per mole, and salts, esters, and other pharmaceutically
acceptable forms of such compounds. It is understood that
appropriate doses of small molecule agents depends upon a number of
factors within the ken of the ordinarily skilled physician,
veterinarian, or researcher. The dose(s) of the small molecule will
vary, for example, depending upon the identity, size, and condition
of the subject or sample being treated, further depending upon the
route by which the composition is to be administered, if
applicable, and the effect which the practitioner desires the small
molecule to have upon the nucleic acid or polypeptide of the
invention.
[0221] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate expression or activity of a polypeptide or nucleic acid of
the invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0222] In certain embodiments of the invention, a modulator of
P-selectin activity is administered in combination with other
agents (e.g., a small molecule), or in conjunction with another,
complementary treatment regime. For example, in one embodiment, an
inducer of P-selectin activity is used to treat a
vasculature-associated disease. In the instance where the
vasculature-associated disease is a tumor, the subject may be
treated with an inducer of P-selectin activity, and further treated
with a molecule effective to induce a procoagulant state in tumor
associated vasculature, e.g., a molecule comprising a first binding
region that binds to a component of a tumor cell or tumor
associated vasculature (e.g., VCAM-1) operatively linked to a
coagulation factor or a second binding region that binds to a
coagulation factor, thereby increasing effectiveness of the
treatment at the disease site. The vessels at the disease site in
other vasculature-associated diseases may be similarly targeted
with a coagulation factor or pro-coagulant agent, such that the
specificity and effectiveness of the inducer of P-selectin activity
is enhanced. In another embodiment, an inhibitor of P-selectin
activity may be used in conjunction with anti-coagulant agents
(e.g., integrin inhibitors, aspirin, heparin) in the treatment of
thrombotic disorders, such as restenosis following medical
intervention.
[0223] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. The conjugates of the invention can be
used for modifying a given biological response, and the drug moiety
is not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a coagulation factor such as tissue
factor; a protein such as vascular endothelial growth factor
("VEGF"), platelet derived growth factor, and tissue plasminogen
activator; biological response modifiers such as, for example,
lymphokines, cytokines and growth factors; or a toxin.
[0224] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2.sup.nd Ed.),
Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);
Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
[0225] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0226] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0227] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Figures, are
incorporated herein by reference.
EXAMPLES
Example 1
Hemostatic Potential in Animals with Increased Levels of Soluble
P-selectin
[0228] Transgenic mice that express P-selectin lacking the
cytoplasmic domain (.DELTA.CT mice) have been generated by gene
replacement through homologous recombination in embryonic stem
cells (Hartwell, D. W. et al. J Cell Biol (1998) 143:1129-1141).
These mutant animals display an elevated level of soluble
P-selectin in the plasma.
[0229] This example describes studies of the hemostatic potential
in .DELTA.CT mice as compared to wild type controls.
[0230] A. Fibrin Formation in a Perfusion Chamber
[0231] Fibrin formation of non-anticoagulated blood from wild type
(WT), .DELTA.CT mice, and P-selectin deficient (P-sel -/-) mice
(Mayadas, T. N. et al. Cell (1993) 74:541-554) was compared ex vivo
in a perfusion chamber. Leukocyte rolling and neutrophil
extravasation, as well as hemostasis are compromised in these mice
(Subramaniam, M. et al. Blood (1996) 87:1238-1242).
[0232] Briefly, glass capillary tubes (0.56 mm inner diameter) were
coated with 1 mg/ml human fibrillar type III collagen (Sigma, St.
Louis) as previously described (Andre, P. et al. Arterioscler
Thromb Vasc Biol (1996) 16:56-63). Mice were anesthetized with 2.5%
tribromoethanol (0.15 ml/10 g). Non-anticoagulated blood was
collected directly from the vena cava of the mice using a butterfly
25 G, and perfused through the collagen coated perfusion chamber
using silastic tubing. A flow rate of 220 .mu.l/minute was
established for 2 minutes by a syringe pump (Harvard Apparatus)
mounted distal to the chamber, resulting in a shear rate of 212
s.sup.-1, according to the equation: .gamma.=4Q/.pi.r.sup.3.
Immediately after the blood perfusion, the thrombotic deposits
formed onto the collagen surface were rinsed for 20 seconds with
PBS and fixed in an ice cold 2.5% cacodylate buffered
glutaraldehyde (pH 7.4) at the same shear rate. The perfusion
chamber was then removed from the flow system and fixed in a
freshly prepared fixative buffer for 24 hours at 4.degree. C. En
face visualization of the thrombotic deposits was performed under
light microscopy after epon embedding.
[0233] FIG. 1 is a photograph of en face examination of the
thrombotic deposits formed after a 2 minute non-anticoagulated
blood perfusion (blood flow, left to right). The white arrow
indicates platelet rich thrombus; the black arrow indicates fibrin
tail formed distally the platelet thrombus. As shown in FIG. 2, in
4 out of 11 perfusion chambers performed with wild type animals
(one perfusion chamber per animal), a fibrin tail was found
distally to the platelet aggregate. In 8 out of 9 perfusion
chambers performed in .DELTA.CT mice, a fibrin tail was present. In
addition, the fibrin tail from the .DELTA.CT mice was significantly
longer than that observed in the wild type mice. None of the
perfusion chambers performed with P-selectin deficient blood
exhibited a fibrin tail. The statistical comparison between fibrin
formation in the 3 genotypes was performed using the Log rank test.
A Student's t test was used to compare the length of the fibrin
tail.
[0234] B. Levels of Soluble P-selectin and Fibrinogen in Plasma
[0235] The level of soluble P-selectin in plasma was measured using
a modified sandwich ELISA procedure as previously described
(Hartwell, D. W. et al. J Cell Biol (1998) 143:1129-1141). Briefly,
plasma samples of wild type (WT) and .DELTA.CT mice were incubated
for 2 hours at 37.degree. C. with monoclonal anti-mouse P-selectin
antibody (RB 40.34, Pharmingen Corp., San Diego, Calif.)-coated
plates. After washing, a biotinylated rabbit anti-P-selectin
antibody (Pharmingen Corp., San Diego, Calif.) was added to the
wells and incubated for 2 hours. ExtrAvidin-conjugated alkaline
phosphatase was added and the activity was revealed with
p-nitrophenyl phosphate (Sigma Chemical Co., St Louis, Mo.). Plates
were read at 405 nm in an Epson LX-300 ELISA reader (Dynatech
Laboratories, Chantilly, Va.). The plasma level of fibrinogen was
measured according to the Sigma Diagnostics Procedure No. 886 (St.
Louis, Mo.) and expressed in mg/dL.
[0236] As shown in Table 1 below, a 3-fold increase in the level of
soluble P-selectin was found in the plasma of .DELTA.CT mice
compared with wild type mice. In contrast, no significant
difference was observed in the plasma fibrinogen levels in these
animals.
1 TABLE 1 Soluble P-selectin in plasma Fibrinogen level in plasma
(.mu.g/ml) n (mg/dl) N WT 0.34 4 WT 367 .+-. 24 13 .DELTA.CT 1.05 4
.DELTA.CT 344 .+-. 14 13
[0237] C. Hemorrhagic Lesions in a Local Shwartzman Reaction
[0238] Local Shwartzman reaction is a hemorrhagic and necrotic
lesion induced by endotoxin and cytokines, and is a prototypic
model for the interrelation between the inflammatory and hemostatic
systems. Briefly, 12 to 14 week old age-matched male wild type (WT)
and .DELTA.CT mice were primed on day 0 by a subcutaneous injection
of Escherichia coli LPS 055:B5 (Difco Laboratories, Detroit, Mich.)
at 100 .mu.g/mouse in 0.1 ml of sterile phosphate buffered saline
(PBS). Twenty four hours later (day 1), recombinant TNF-A (Genzyme,
Cambridge, Mass.) at 0.3 .mu.g/mouse was injected at the same skin
site, as described (Subramaniam, M et al. Blood (1996)
87:1238-1242). On day 2, the hemorrhagic lesions were examined and
scored on a scale of 0 to 4 without knowledge of the mouse
genotypes. Hematoxylin-eosin stained paraffin sections were
prepared from the lesion site and the degree of inflammatory cell
infiltration as well as hemorrhage were scored microscopically, on
a scale of 0 to 4.
[0239] Macroscopic and microscopic evaluation of the injection
sites revealed that after 48 hours, the average size of the
hemorrhagic lesions in .DELTA.CT mice was about 50% of that in the
wild type (see FIG. 3). A highly significant reduction of the
hemorrhage was also observed in wild type animals perfused with
soluble P-selectin-Ig (1 .mu.g/g; Pharmingen Corp., San Diego, CA)
injected 1 hour prior to TNFa challenge as compared to those
injected with human IgG1 (Sigma Chemical Co., St Louis, Mo.).
[0240] D. Fibrin Deposition in a Local Shwartzman Reaction
[0241] Paraffin sections from the Shwartzman lesion site of wild
type mice injected with human IgG1 or soluble P-selectin, as
described above, were de-paraffinized, sequentially blocked with
avidin D solution and biotin blocking solution (Vector, Burlingame,
Calif.), and then stained with a rabbit anti-human fibrinogen
antibody (1:1000 dilution; A0080, Dako, Carpinteria, Calif.) which
cross-reacts with mouse fibrin/fibrinogen. Sections were then
sequentially treated with a biotinylated goat anti-rabbit secondary
antibody (Zymed Laboratories Inc., South San Francisco, Calif.),
and an ABC mix solution (Vector, Burlingame, Calif.). Development
was done by treating the sections with an AEC substrate kit for
horseradish peroxidase (Vector, Burlingame, Calif.). Sections were
counterstained with hematoxylin for 5 minutes.
[0242] All vessels which presented fibrin staining outside of the
vessel wall were classified as "leakage". Vessels which presented
fibrin staining on the luminal surface of the endothelial cells
without fibrin outside the vessel wall were classified as "ring".
The results are shown in FIG. 4. Wild type animals injected with
soluble P-selectin exhibited a significant decrease in the
percentage of "leakage" vessels, and an increase in the percentage
of "ring" vessels, as compared with animals perfused with human
IgG1.
[0243] E. Plasma Clotting Time
[0244] The plasma clotting time of wild type mice, either
untreated, or infused with either human IgG1 (control) or soluble
P-selectin (s-P-sel), P-selectin deficient, and .DELTA.CT mice,
either untreated or infused with human recombinant PSGL-1
(r-PSGL-1), was determined as follows. Briefly, 1 ml of blood was
drawn from the retro-orbital venous plexus using plain
microhematocrit capillary tubes and collected into polypropylene
tubes containing 10% final volume of acid-citrate-dextrose (ACD: 38
mM citric acid, 75 mM trisodium citrate, 100 mM dextrose). Platelet
poor plasma was prepared by centrifugation at 1,500 g for 25
minutes, followed by centrifugation at 15,000 g for 2 minutes to
remove any contaminating cells from the plasma. Plasma clotting
time was induced under stirring conditions (800 rpm) at 37.degree.
C. in an aggregometer by adding an equal volume of pre-warmed 20 mM
CaCl.sub.2 solution to the plasma in a siliconized tube.
[0245] As shown in FIG. 5, .DELTA.CT mice presented a significant
reduction of the clotting time compared with wild type mice. In
addition, a significant increase of the clotting time was observed
on day 4 in .DELTA.CT mice injected intravenously (on days 0 and 2)
with human recombinant PSGL-1 IgG (10 mg/kg). In contrast,
injection of soluble P-selectin in wild type mice significantly
reduced the clotting time compared with the IgG treated control
group.
[0246] F. Microparticles in Mouse Plasma
[0247] The levels of microparticles circulating in vivo in wild
type mice, untreated, or infused with either human IgG1 (control)
or soluble P-selectin (s-P-sel), and in .DELTA.CT mice was
determined as follows. Briefly, platelet poor plasma was prepared
as described above. Subsequently, 300 .mu.l of platelet poor plasma
was collected per mouse, and three samples of platelet poor plasma
from mice of the same genotype were pooled together, diluted 1:3
with buffer (10 mmol/L HEPES, 5 mmol/L KCl, 1 mmol/L MgCl.sub.2,
136 mmol/L NaCl, pH 7.4), and centrifuged for 1.5 hours at 100,000
g. The supernatant was discarded and the pellet of microparticles
was resuspended in a fixed volume (120 .mu.l) of the same
buffer.
[0248] Flow cytometric analysis was performed on a Becton-Dickinson
FACSCalibur (Franklin Lakes, N.J.) with CellQuest software
(Becton-Dickinson, San Jose, Calif.). The light scatters and
fluorescent channels were set at logarithmic gain (forward scatter
was EOO with a threshold of 12 and sideward scatter was 300). To
count the total population of microparticles, 30 .mu.l aliquots
were incubated for 15 minutes in the dark with calcein AM (0.25
.mu.g/ml; Molecular Probes, Eugene, Oreg.). The total number of
events were counted for a set interval of 10 seconds.
[0249] FIG. 6 shows that the number of microparticles was increased
by 1.9-fold in .DELTA.CT mice compared with wild type animals.
Furthermore, a 2.7-fold increase in microparticles was obtained
when wild type mice were injected intravenously with soluble
P-selectin-Ig, as compared to human IgG1.
[0250] To identify the origin of the procoagulant activity,
microparticle samples were stained for 20 minutes at room
temperature with a sheep anti-rabbit tissue factor IgG (American
Diagnostica Inc., Greenwich, Conn.) which recognizes mouse tissue
factor (5 .mu.g/ml final concentration). A FITC-conjugated rabbit
anti-sheep IgG (1:1000 dilution; Zymed Laboratories Inc., South San
Francisco, Calif.) was used as a secondary antibody. As controls,
an identical concentration of control IgG antibodies were used (rat
IgG, Sigma Chemical Co., St. Louis, Mo.; FITC-conjugated sheep IgG,
Caltag Laboratories, Burlingame, Calif.). The microparticles were
analyzed by flow cytometry.
[0251] FIG. 7 shows that there are an increased number of
microparticles expressing tissue factor in the plasma of .DELTA.CT
mice.
[0252] G. Treatment of .DELTA.CT Mice with Soluble PSGL-Ig
[0253] Soluble PSGL-Ig infusion decreases the pro-coagulant
phenotype of .DELTA.CT mice as shown by a significant decrease in
the number of microparticles and a prolonged clotting time of
plasma. Infusion of control Ig had no such effect.
[0254] Plasma clotting time was determined as described above. For
analysis of microparticles in plasma of .DELTA.CT mice treated with
PSGL-Ig, 200 .mu.l of blood was collected by retro-orbital puncture
on day 0. Platelet-poor plasma was obtained, and 40 l was diluted
in 260 .mu.l PBS and immediately analyzed for microparticle number
by FACS. Mice were then infused i.v. (days 0 and 2) with 10 mg/kg
PSGL-Ig or control Ig. On day 4, 200 .mu.l of blood was collected
from the other eye, and the number of microparticles was
determined.
[0255] FIG. 11A shows the number of microparticles present in 40
.mu.l of .DELTA.CT plasma, before (open bars) and after (filled
bars) two infusions of PSGL-Ig and control Ig in .DELTA.CT mice
(*=p<0.05).
[0256] FIG. 11B shows that the clotting time at the end of the
experiment (e.g., after 4 days) was significantly longer in mice
treated with soluble PSGL-Ig (filled bar) than in control Ig
treated group (open bar) (*=p<0.05). These data show that
inhibition of soluble P-selectin decreases the pro-coagulant state
in vivo.
Example 2
Activity of Soluble P-selectin in von Willebrand Factor Deficient
Mice and Mice with Hemophilia A
[0257] von Willebrand factor (vWF) deficient mice have only about
20% of the wild type level of factor VIII (anti-hemophilia factor),
and thus have difficulty making fibrin clots (Denis, C. et al. Proc
Natl Acad Sci USA (1998) 95:9524-9529). Mice with hemophilia A are
lacking factor VIII completely (Bi, L. et al. (1995) Nature
Genetics 10:119-121. This example describes the hemostatic activity
of soluble P-selectin in these animals.
[0258] A. Tissue Factor Activity in Platelet Poor Plasma
[0259] Platelet poor plasma was prepared from pooled plasma of vWF
deficient mice (vWF -/-) infused with soluble P-selectin-Ig (n=2)
or IgG1 (control; n=3). Microparticles were prepared by repeated
centrifugation of platelet poor plasma. Briefly, the first
centrifugation step at 12,000 g for 2 minutes was performed to
remove any contaminating cells. The supernatant was then diluted in
a 20 mM HEPES, 1 mM EDTA, pH7.2 solution and ultracentrifuged at
200,000 g for 90 minutes. The supernatant was discarded, and the
pelleted microparticles were resuspended (1/2 of the initial
volume) in a 10 mM HEPES, 136 mM NaCl, pH7.4 solution.
Determination of tissue factor activity of the microparticle
solution was measured through its ability to promote the activation
of factor X (150 nM) by factor VIla (5 nM) in the presence of 1 mM
CaCl.sub.2. The reaction was allowed to proceed for 20 minutes at
37.degree. C. and was stopped by the addition of an excess of EDTA
(5 mM final concentration). A chromogenic substrate of factor Xa,
Spectrozyme.RTM. fXa, was added at a final concentration of 0.3 mM.
The change in absorbance at 405 nm versus time was immediately
recorded using a plate reader equipped with kinetics software
(DYNEX Technologies, Inc.). The linear changes in absorbance
directly correlate with the concentration of factor Xa generated in
the assay.
[0260] As shown in Table 2 below, the tissue factor activity of the
solution of microparticles from vWF deficient mice infused with
soluble P-selectin-Ig was 2.1 fold higher than that of control mice
infused with IgG1.
2TABLE 2 Tissue Factor (Xa) Activity in OD/minute vWF -/- + IgG1
vWF -/- + soluble P-selectin-Ig 2.54 5.26
[0261] B. Procoagulant Microparticle Generation by Infusion of
Soluble P-selectin-Ig
[0262] The levels of microparticles circulating in vivo in vWF
deficient mice, infused with either human IgG1 (control) or soluble
P-selectin-Ig (sP-sel-Ig) was determined as described above. FIG. 8
shows that the number of microparticles was increased when vWF
deficient mice were injected intravenously with soluble
P-selectin-Ig, as compared to human IgG1 (control).
[0263] C. Prothrombin Clotting Time
[0264] Prothrombin clotting time (PT) is a global coagulation
screening test. It involves extrinsic pathway of coagulation
starting with activation of TF-VII(a) complex. PT time is measured
in prewarmed (37.degree. C.) platelet poor plasma after adding
thromboplastin as a source of tissue factor, and Ca.sup.2+.
[0265] Diluted prothrombin time was measured when pooled platelet
poor plasma sample (0.1 ml) was mixed with 0.2 ml of diluted rabbit
brain thromboplastin (IL TEST PT). Clotting time was determined
using photometry detection of the first fibrin threads formed. FIG.
9 shows a prolonged prothrombin clotting time in vWF deficient
plasma (vWF 4-) compared with wild type (wt) when the
thromboplastin concentration decreased. This can be explained by
the 20% of normal level of factor VIII found in the vWF deficient
mice.
[0266] Clotting time of vWF deficient mice infused with either
soluble P-selectin-Ig or IgG1 (control) was tested at the high
dilution of thromboplastin (1:20,000) because it is known that at
that dilution, prothrombin clotting time is preferentially tissue
factor dependent. The infusion of soluble P-selectin-Ig in vWF
deficient mice shortened the prothrombin clotting time by 28% when
compared with vWF deficient mice infused with IgG1.
[0267] D. Bleeding Time
[0268] Bleeding time was measured as described by Dejana, et al.
(1979) Thromb. Res. 15:199-201. Briefly, factor VIII-deficient mice
were injected with 1.2 .mu.g soluble P-selectin-Ig (P-sel-Ig) or
human IgG1 control per gram of mouse. Six hours later mice were put
in a restrainer, and a distal 3-mm segment of the tail was severed
with a razor blade. The tail was immediately immersed in 0.9%
isotonic saline at 37.degree. C. with the tip of tail 5 cm below
the body. The bleeding time was defined as the time required for
the stream of blood to cease. The infusion of soluble P-selectin
reduced bleeding time in hemophilia A mice (factor VIII-deficient
mice).
[0269] As shown in FIG. 10, bleeding time was significantly
decreased for hemophilia A mice treated with soluble P-selectin-Ig
as compared to hemophilia A mice treated with human IgG1.
[0270] E. Activated Partial Thromboplastin Time (APTT)
[0271] Activated partial thromboplastin time (APTT) is a global
coagulation screening test. It involves the intrinsic pathway of
coagulation.
[0272] The effect on soluble P-selectin on activated partial
thromboplastin time and plasma clotting time in factor
VIII-deficient mice (hemophilia A mice) was determined as follows.
Briefly, hemophilia A mice were treated with 1.2 .mu.g/g body
weight P-selectin-Ig or human IgG1. Mice were bled into ACD six
hours after perfusion. Platelet poor plasma was prepared as
described above. Activated partial thromboplastin time (APTT) was
determined with APTT reagent and clotting was initiated by addition
of calcium ions. APTT and plasma clotting time are reduced in
soluble P-selectin-Ig treated hemophilia A mice.
[0273] As shown in FIG. 14, APTT in soluble P-selectin-Ig treated
hemophilia A mice was shorter as compared to mice treated with
human IgG1 (p<0.0013, determined by unpaired t test).
Recalcified clotting time of plasma of hemophilia A mice treated
with soluble P-selectin-Ig was significantly reduced (p<0.0058,
determined by unpaired t test) as compared to mice treated with
control IgG1.
[0274] The foregoing Examples demonstrate the hemostatic activity
of soluble P-selectin. The infusion of soluble P-selectin into a
mouse induces a procoagulant state in the animal. When such an
animal is wounded, fibrin is deposited more rapidly at the site of
the vessel injury thus reducing leakage from the blood vessels. The
plasma of the animal infused with soluble P-selectin clots faster.
Transgenic animals expressing higher levels of soluble P-selectin
(.DELTA.CT mice) also form fibrin more readily than wild-type
animals and are protected from excessive leakage in hemorrhagic
injury. In contrast, animals lacking all forms of P-selectin have
an increased hemorrhagic response and slightly longer bleeding time
than wild type. These data indicate that the level of soluble
P-selectin is a predictor of coagulation potential in a mammal.
[0275] Moreover, we have observed that infusion of soluble
P-selectin into a mouse increases the numbers of microparticles
containing tissue factor in the blood. Similarly, transgenic mice
expressing higher than normal levels of soluble P-selectin have
more tissue factor-containing microparticles in circulation.
Infusion of soluble PSGL-1 (a ligand/inhibitor of P-selectin)
reduces the numbers of tissue factor-containing microparticles and
prolongs clotting time of the plasma in these mice. Thus,
modulating P-selectin activity by, for example, modulating levels
of soluble P-selectin can either increase or decrease hemostatic
potential in a subject, and thus is useful for the diagnosis and
treatment of hemostatic disorders.
Example 3
Soluble P-selectin Generates Microparticles in Human Blood
[0276] An in vitro system was developed to further demonstrate how
soluble P-selectin induces pro-coagulant activity. Generation of
microparticles after the addition of 15 .mu.g/ml of human
P-selectin-Ig chimera or control human IgG1 was determined as
described herein. Human blood was collected in ACD. The blood
samples from four donors, each treated separately, were incubated
at 37.degree. C. Samples were handled under aseptic conditions to
avoid LPS contamination. The generation of microparticles was
analyzed by flow cytometry in platelet poor plasma diluted in PBS.
Forward scatter and sideward scatter plot was used for the quadrant
analysis to quantify the newly formed large procoagulatnt
microparticles. Tissue factor positive microparticles were analyzed
by flow cytometry. The microparticles were stained with a
FITC-conjugated mouse anti-human tissue factor (American
Diagnostica.TM.).
[0277] As shown in FIG. 12A, after 6 hours incubation with soluble
P-selectin, the numbers of procoagulant microparticles were
increased by 30% as compared to human IgG control
(*=p<0.04).
[0278] As shown in FIG. 12B, the number of tissue factor positive
evens was significantly increased by incubation with soluble
P-selectin-Ig in 6 hours by 30% (*=p<0.05).
Example 4
Soluble P-selectin Shortens Whole Blood and Plasma Clotting Time in
Human Blood
[0279] Whole blood recalcified clotting time and plasma recalcified
clotting time in human blood after the addition of 15 .mu.g/ml of
human P-selectin-Ig chimera or control human IgG1 was determined as
follows. The human blood was collected in ACD. The blood samples
from four donors, each treated separately, were incubated at
37.degree. C. Samples were handled under aseptic conditions to
avoid LPS contamination. The whole blood clotting time was measured
in siliconized tubes in a Soloclot Coagulation and Platelet
Analyzer (Sienco.TM.).
[0280] As shown in FIG. 13A, the whole blood clotting time of human
blood incubated with soluble P-selectin was shortened by about 20%
after 2 hours (*=p<0.02) and by 60% after 8 hours of incubation
(**=p<0.004) as compared to blood treated with IgG.
[0281] As shown in FIG. 13B, the plasma clotting time of the
soluble P-selectin blood was shortened by 25% after 6 hours of
incubation and by 40% after 8 hours of incubation. (**p<0.004)
as compared to control IgG and untreated plasma.
[0282] Equivalents
[0283] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *
References