U.S. patent application number 11/093814 was filed with the patent office on 2006-01-05 for cd39/ecto-adpase for treatment of thrombotic and ischemic disorders.
This patent application is currently assigned to The Trustees of Columbia University. Invention is credited to David J. Pinsky.
Application Number | 20060003930 11/093814 |
Document ID | / |
Family ID | 23477466 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003930 |
Kind Code |
A1 |
Pinsky; David J. |
January 5, 2006 |
CD39/ecto-adpase for treatment of thrombotic and ischemic
disorders
Abstract
The present invention relates to a method for treating an
ischemic disorder in a subject which comprises administering to the
subject a CD39 polypeptide (SEQ ID NO:2) or an active fragment
thereof which inhibits ADP or ATP mediated platelet aggregation or
leukocyte accumulation so as to treat the ischemic disorder in the
subject.
Inventors: |
Pinsky; David J.;
(Cresskill, NJ) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Assignee: |
The Trustees of Columbia
University
New York
NY
|
Family ID: |
23477466 |
Appl. No.: |
11/093814 |
Filed: |
March 30, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10049320 |
Aug 5, 2002 |
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PCT/US00/22060 |
Aug 11, 2000 |
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11093814 |
Mar 30, 2005 |
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09374586 |
Aug 13, 1999 |
6867177 |
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10049320 |
Aug 5, 2002 |
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Current U.S.
Class: |
800/3 ; 514/13.8;
514/14.9; 514/15.1; 514/16.4; 514/17.7 |
Current CPC
Class: |
C07K 14/70596 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/17 20060101
A61K038/17 |
Goverment Interests
[0002] The invention described herein was made in the course of
work done under Grant Nos. HL-47073, HL-46403, HL-07423 (AJM, MJB,
JHFD), HL-59488 and HL-55397 (DJP) and NS 02038 (ESC) Department of
Veterans Affairs and from National Institutes of Health. Therefore,
the United States Government has certain rights in this invention.
Claims
1-26. (canceled)
27. A method for treating an ischemic disorder in a subject which
comprises administering to the subject a CD39 polypeptide (SEQ ID
NO:2) or an active fragment thereof which inhibits ADP or ATP
mediated platelet aggregation or leukocyte accumulation so as to
treat the ischemic disorder in the subject.
28. The method of claim 27, wherein the leukocyte is a white blood
cell, a neutrophil, a monocyte or a platelet.
29. The method of claim 27, wherein the subject is a mammal.
30. The method of claim 27, wherein the mammal is a human.
31. The method of claim 29, wherein the ischemic disorder comprises
a peripheral vascular disorder, a pulmonary embolus, a venous
thrombosis, a myocardial, infarction, a transient ischemic attack,
unstable angina, a reversible ischemic neurological deficit, sickle
cell anemia or a stroke disorder.
32. The method of claim 27, wherein the subject is undergoing heart
surgery, lung surgery, spinal surgery, brain surgery, vascular
surgery, abdominal surgery, or organ transplantation surgery.
33. The method of claim 32, wherein the organ transplantation
surgery comprises heart, lung, pancreas or liver transplantation
surgery.
Description
[0001] This application is a continuation-in-part and claims
priority of U.S. Ser. No. 09/374,586, filed Aug. 13, 1999, the
contents of which are hereby incorporated by reference.
[0003] Throughout this application, various publications are
referenced by numbers. Full citations for these publications may be
found listed numerically at the end of the specification
immediately preceding the claims. The disclosures of these
publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the
state of the art as known to those skilled therein as of the date
of the invention described and claimed herein.
BACKGROUND OF THE INVENTION
[0004] Stroke is the third leading cause of death and the main
cause of permanent morbidity in the United States, 25 affecting
over 450,000 patients annually.sup.1. Recent studies in a murine
model of ischemic stroke demonstrated a pivotal role for platelets
in progressive microvascular thrombosis distal to the primary
obstruction o f a major cerebrovascular tributary.sup.2. This
progressive microvascular thrombosis is characterized by distal
platelet and fibrin accumulation, resulting in postischemic
hypoperfusion. ("no re-flow") and neuronal injury.sup.2. While
leukocyte adhesion receptors and recruited neutrophils contribute
to postischemic hypoperfusion, postischemic hypoperfusion cannot be
completely abrogated because even in the absence of neutrophils,
progressive microvascular thrombosis persists.sup.3,4. Two
thrombolytic agents, recombinant tissue-type plasminogen activator
(rtPA) and pro-urokinase, have been used for treatment of stroke.
However, their therapeutic utility is limited due to risk of
symptomatic and fatal intracranial hemorrhage.sup.5. In the United
States, less than 1% of patients presenting to community hospitals
with acute ischemic stroke receive rtPA.sup.6. Inhibition of the
final common pathway of platelet accumulation, via blockade of
glycoprotein Ilb/IIIa receptor-mediated platelet-platelet
interactions, does reduce microvascular thrombosis in experimental
stroked.sup.2. However, as with thrombolytic agents, small excesses
of a GPIIb/IIIa receptor blocker culminated in serious
intracerebral hemorrhage. It is therefore important to identify
novel strategies for inhibition of platelet function in acute
stroke that will reduce intravascular thrombosis without increasing
risk of intracerebral hemorrhage.
SUMMARY OF THE INVENTION
[0005] The present invention provides a method of treating or
preventing thrombotic or ischemic disorders in a subject which
comprises administering an agent to the subject, wherein the agent
inhibits platelet aggregation by increasing ADP catabolism. The
present invention also provides a method for determining whether a
compound inhibits platelet aggregation by increasing ADP catabolism
so as to treat or prevent thrombotic or ischemic disorders in a
subject, comprising: (a) inducing thrombotic or ischemic disorders
in an animal, which animal is an animal model for thrombotic or
ischemic disorders; (b) measuring the stroke outcome in said
animal, (c) measuring platelet deposition and/or fibrin deposition
in ischemic tissue, and (d) comparing the stroke outcome in step
(b) and the platelet deposition and/or fibrin deposition with that
of the animal model in the absence of the compound so as to
identify a compound capable of treating or preventing thrombotic or
ischemic disorders in a subject.
BRIEF DESCRIPTION OF THE FIGURES
[0006] FIGS. 1A-1F: Effects of saline (control), aspirin or CD39 on
the aggregation of murine platelets ion response to: (A) ADP 2.5
.mu.M; (B) collagen 2.5 .mu.g/mL; (C) ADP 10 .mu.M; (D) collagen 10
pg/mL; or (E) sodium arachidonate (0.1 mM). The % inhibition of
platelet aggregation is shown in (F) Agents were administered to
mice 45 minutes prior to harvest of blood, and preparation of
platelet-rich plasma for the indicated studies.
[0007] FIG. 2: Bleeding times in control, aspirin-treated,
CD39-treated, or CD39 null mice.
[0008] FIGS. 3A-3E: Effect of CD39 on stroke outcomes, and
comparison with aspirin. (A) Cerebral blood flow; (B) cerebral
infarction volume; (C) Neurological deficit stroke; (D) mortality;
(E) intracerebral hemorrhage.
[0009] FIG. 4. Covariate plot of cerebral infarct volume vs
intracerebral hemorrhage: Comparison of vehicle (saline) with
aspirin (ASA, 5 mg/kg prior to stroke), CD39 (4 and 8 mg/kg prior
to stroke), and CD39 (8 mg/kg, 3 hours following stroke
induction).
[0010] FIGS. 5A-5B: (A) Effect of CD39 on platelet deposition in
stroke; (B) Effect of CD39 on fibrin accumulation in stroke; A
positive fibrin control is shown in the leftmost lane.
Ipsilat.=ipsilateral (i.e., ischemic) hemisphere.
Contralat.=nonischemic left hemisphere.
[0011] FIGS. 6A-6E. Comparison of stroke outcomes: control
(C57BL/6J.times.129/J Fl) mice. (n=6), CD39-/- mice (n=5), and
CD39-/- mice "reconstituted" with CD39 (n=6). Cerebral blood flow
(A) cerebral infarct volume (B) neurological score (C) mortality,
and (D) intracerebral hemorrhage (E) (*p<0.05,
.dagger-dbl.p<0.01, .dagger.p<0.001).
[0012] FIG. 7. Ex vivo aggregation of murine platelets. Following
administration of vehicle (saline), solCD39 (4 mg/kg) or aspirin (5
mg/kg), platelets were stimulated with 10/ig/ml collagen (b), or
0.1 mM sodium arachidonate (c). Panel d indicates percent
inhibition of aggregation as compared to control with aggregation
quantified as described in the Methods section. SolCD39 treatment
yielded aggregation curves that returned to baseline following
stimulation with agonists in a, b, c. Aspirin treatment resulted in
this pattern only when arachidonate was the agonist.
[0013] FIG. 8. Inhibition of platelet (n=20) (a) and fibrin (n=3)
(b) deposition following induction of stroke in mice pretreated
with solCD3 9 (8mg/kg). Fibrin-positive control;
Ipsilat=ipsilateral (ie, ischemic hemisphere);
Contralat=nonischemic hemisphere.
[0014] FIG. 9. Comparative effects of vehicle, aspirin, and solCD39
on the various outcome after experimental stroke. (a) Relative
cerebral blood flows shown at occlusion, reperfusion, and sacrifice
at 24 hours for three groups (solCD39 4 mg/kg given preoperatively
(n=16) or postoperatively (n=9) and control (vehicle, given
postoperatively, n=10); (b) Relative cerebral blood flow at 24
hours for preoperative vehicle (n=24), aspirin (ASA, n=27)), or
solCD39 (n=ll,ll, and 16 for the 1 mg/kg, 2 mg/kg, and 4 mg/kg
doses, respectively.) Cerebral blood flow data for vehicle or
solCD39 given 3 hours after stroke, shown in the (a) panel, are
repeated here for comparison. At 24 hours, the following parameters
were also determined: (c) cerebral infarct volume; (d) neurological
deficit score (higher scores denote worse deficit (15)); (e)
mortality; and (f) intracerebral hemorrhage. (*p<0.05,
**p<0.01, ***p<0.001, .dagger.p<0.0001,
.dagger-dbl.p<0.00002).
[0015] FIG. 10. Covariate plot of cerebral infarct volume vs
intracerebral hemorrhage, as a function of genotype or treatment.
Comparison of vehicle (saline) with aspirin (ASA, 5 mg/kg) or
solCD39 (4 mg/kg) given to wild type mice (CD39+/+). Data are shown
for treatments given immediately prior to stroke. Standard errors
are shown in FIG. 3, but are omitted here for clarity.
[0016] FIG. 11. Generation of CD39-/- mice by homologous
recombination. A gene targeting vector, in which a 4.1 kb
Spel-BglII fragment containing exons 4-6 (encoding apyrase
conserved regions 2-4) (24) was replaced with a PGKneo cassette
(a), was introduced into 129-derived ES cells, and cells were
selected in G418 and gancyclovir. Nine ES clones with a disrupted
CD39 allele, identified by genomic Southern blot analyses of BglII
digested DNA (b), were injected into blastocysts and the resulting
chimeras crossed to C57BL/6 to produce heterozygotes (CD39.+-.).
CD39-/- mice, generated at the expected Mendelian frequency from
CD3 9.+-. intercrosses, were overtly normal and did not display
reproductive defects (not shown). The CD39-/- mice, generated at
the expected Mendelian frequency from CD39.+-. intercrosses, were
overtly normal and did not display reproductive defects (not
shown). The CD39-/- mice used represent random C57BL/6.times.129
hybrids. BglII (B), SpeI (S), Asp718 (A). (c) PCR analysis (25
cycles) of tail DNA, using the following amplicon sequences [primer
1 GAACAGAGTTGGCTAAGCCTC; Primer 2: GAATGTCCTTGGCCAGTTTCTGCC], used
to generate a 236 bp fragment of exon 6. Each lane is from a
different animal, with genotype as indicated above the lanes.
[0017] FIG. 12. Bleeding times in control, (n=15), aspirin-treated
(5 mg/kg, n=10), solCD39-treated (4, 8, and 20 mg/kg, n=25), and
solCD39-/- mice (n=10). (*p<0.05, ***p<0.001).
[0018] FIG. 13. Comparison of stroke outcomes in CD39+/+ control
(C57BL/6J.times.129/J Fl) mice (n=17), CD39-/- mice (n=21), and
CD39-/- mice "reconstituted" with solCD39 (n=18). Cerebral blood
flow (a)(*p<0.05 vs CD39-/-); cerebral infarct volume (b),
neurological score (c), mortality (d), and intracerebral hemorrhage
(e).
[0019] FIG. 14. This figure shows a 236 base pair PCR product,
corresponding to a region of the CD39 gene which was deleted.
Endothelial cells were purified based on immunomagnetic separation,
using CD31 as a sorting marker for endothelial cells. Staining for
von Willebrand Factor confirmed that the isolated cells were
endothelial cells. Note that both the CD39-/- mice, whose DNA was
prepared from tail clippings, and the CD39-/- endothelial cells,
lack the 236 bp product. Wild type (ie, CD39-gene containing) cells
and tails both show the 236 bp product in this PCR reaction.
Northern blot data (not shown) are similar to these blots.
[0020] FIG. 15. Effect of the CD39 gene (or its absence) on
survival and lung function after left lung ischemia. Left lung
ischemia was created as follows: Animals were initially
anesthetized intraperitoneally with 0.1 mg/mouse weight (g) of
ketamine and 0.01 mg/mouse weight (g) of xylazine, following by
intraperitoneal continuous infusion of one third of the initial
dose per hour using a syringe pump (model 100 series, KD Scientific
Inc. MA). After ensuring appropriate depth of anesthesia, mice were
intubated via tracheostomy and placed on a Harvard ventilator
(tidal volume=0.75 mL, respiratory rate=120/min) with room air,
followed by bilateral thoracotomy. The left hilum was cross-clamped
for a period of 1 hour after which the cross-clamp was released.
Reperfusion proceeded for 3 hours according to the following
groups: Then the contralateral (right) hilum was permanently
ligated, so that the animal's survival and gas exchange depended
solely upon the reperfused lung, and observation continued for 1
hour among the 4 groups. As the mouse continued to be ventilated;
death of the mouse was defined as a combination of (1) cessation of
regular cardiac activity; (2) the apparent collapse of the left
atrium; and (3) brief clonic activity indicating cessation of
cerebral blood flow.
[0021] Groups which were studied are indicated in the figure
(soluble CD39 was administered (where indicated) as an intravenous
4 mg/kg dose immediately before the procedure. Note that soluble
CD39 improves survival (a) and arterial oxygenation (b), as well as
decreases edema of the postischemic tissue [measured as Wet/Dry
weight ratio, panel (c)].
[0022] FIG. 16. Fibrin deposition, measured by immunoblotting and
densitometric quantification of immunoblots, revealed that CD39-/-
mice have increased fibrin formation, showing that the normal
expression of this molecule suppressed instravascular fibrin
formation under normal or stress conditions. Note that soluble CD39
significantly reduces fibrin formation in both wild type and
CD39-/- mice.
[0023] FIG. 17. Postschemic leukocyte accumulation is increased in
mice lacking the CD39 gene, and suppressed in mice given soluble
CD39. Note that this is true both of neutrophils, as quantified in
the (a) panel by measurements of myeloperoxidase activity, as well
as in mononuclear phagocytes (b), as quantified by specific
immunostaining and counting infiltrating mononuclear phagocytes per
high power field.
[0024] FIG. 18. Use of the endothelial cells derived from wild type
or CD39-/- (as shown in FIG. 1), for assessing altered endothelial
adhesivity for leukocytes under hypoxic conditions. The (a) panel
shows adhesion of polymorphonuclear leukocytes (PMNs), and the (b)
panel shows adhesion of either cell type under hypoxic conditions.
Note that ATP, which increases leukocyte adhesion and which can
also be catabolized by CD39 (as can be ADP), can have its effects
blocked by soluble CD39. These data suggest that CD39 may work by
suppressing ADP levels, ATP levels or both.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides a method for treating or
preventing stroke in a subject wherein the subject is susceptible
to intracranial hemorrhaging, comprising administering a CD39
polypeptide (SEQ ID NO:1) or an active fragment thereof which
inhibits adenosine diphosphate-mediated platelet aggregation by
increasing adenosine diphosphate catabolism to the subject.
[0026] In one embodiment of the method, the active fragment is CD39
polypeptide is a mutated or a truncated form of CD39
polypeptide.
[0027] In another embodiment of the method, the active fragment is
soluble CD39 (SEQ ID NO:2).
[0028] In another embodiment of the method, the CD39 polypeptide is
a recombinant CD39 polypeptide having IL-2 as its leader
sequence.
[0029] In another embodiment of the method, the recombinant CD39
polypeptide lacks a transmembrane domain.
[0030] In another embodiment of the method, the active fragment
comprises from amino acid number 1 to amino acid number 50 of SEQ
ID NO.:2.
[0031] In another embodiment of the method, the active fragment of
the CD39 polypeptide comprises about 20-80 amino acid residues of
SEQ ID NO:1 which mimics the active site of CD39.
[0032] In one embodiment of the method, the CD39 polypeptide or its
active fragment treats or prevents thrombotic or ischemic disorders
in a subject without increasing bleeding or intracerebral
hemorrhage.
[0033] As used herein, the term "ADP" means adenosine
diphosphate.
[0034] As used herein, "ischemic and thrombotic disorders"
encompass pulmonary embolism, lung ischemia, limb or gut ischemia,
myocardial ischemia, post surgical vasculopathy, postangioplasty
stenosis, shunt/fistula remodeling or thrombosis, cerebral
ischemia, or ischemia of other organs or tissues.
[0035] As used herein, the term "ischemic disorder" encompasses and
is not limited to a peripherial vascular disorder, a venous
thrombosis, a pulmonary embolus, a myocardial infarction, a
transient ischemic attack, lung ischemia, unstable angina, a
reversible ischemic neurological deficit, adjunct thromolytic
activity, excessive clotting conditions, reperfusion injury, sickle
cell anemia, a stroke disorder or an iatrogenically induced
ischemic period such as angioplasty.
[0036] As used herein, the term "thrombotic disorder" encompasses
disorders caused by the formation, development or presence of a
blood clot or a blood coagulation which is located inside of a
patient or inside of an extracorporeal circuit or system which
circulates blood of the patient. Thrombotic disorder also
encompasses disorders caused by the presence of a thrombus which
includes a blood clot partially or fully occluding a blood vessel
or formed in a heart cavity or by the activation of a plasmatic
coagulation system in a patient which includes the production of
fibrin, emeshed platelets, fibrin degradation product, protein C,
free protein S, coagulation factor II, immunoglobulin G or albumin
in the patient. Thrombotic disorder also encompasses disorders
cause by the formation of white thrombus which may be composed of
platelets and fibrin and is relatively poor in erythrocytes, a
disseminated fibrin deposit thrombus or a red thrombus which may be
composed of red cells and fibrin.
[0037] In another embodiment of the method, the CD39 polypeptide or
its active fragment can be replace by a peptide, an enzyme, a
pseudo enzyme, a catalyst, a peptidomimetic compound, a
glycosylated peptide, a small molecule, a mutated peptide or an
antibody.
[0038] As used herein, a polypeptide is an amino acid polymer of
amino acids linked together by peptide bonds; a nucleic acid is a
deoxyribonucleotide or ribonucleotide polymer of nucleotides linked
together by phosphodiester bonds; an antisense nucleic acid is a
nucleic acid that is the reverse complement of another nucleic acid
which may be capable of inhibiting transcription or translation of
the other nucleic acid.
[0039] In another embodiment of the method, the the CD39
polypeptide or its active fragment agent comprises a CD39
polypeptide (abbreviated as CD39) or a variant thereof.
[0040] Variants in amino acid sequence of CD39 are produced when
one or more amino acids in naturally occurring CD39 is substituted
with a different natural amino acid, an amino acid derivative, a
synthetic amino acid, an amino acid analog or a non-native amino
acid. Particularly preferred variants include homologous CD39 of
humans or of different species of animals. Variants of a CD39 may
include biologically active fragments of naturally occurring CD39,
wherein sequences of the variant differ from the wild type CD39
sequence by one or more conservative amino acid substitutions. Such
substitutions typically would have minimal influence on the
secondary structure and hydrophobic nature of the CD39. The amino
acid sequences of CD39 and one variant of CD39 have been previously
determined and are the following: TABLE-US-00001
MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPENVKYGIVLDAGS (SEQ ID
NO:1) SHTSLYIYKWPAEKENDTGVVHQVEECRVKGPGISKFVQKVNEIGIYLTDCMERARE
VIPRSQHQETPVYLGATAGMRLLRMESEELADRVLDVVERSLSNYPFDFQGARIITG
QEEGAYGWITINYLLGKFSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVPQNQ
TIESPDNALQFRLYGKDYNVYTHSFLCYGKDQALWQKLAKDIQVASNEILRDPCFHP
GYKKVVNVSDLYKTPCTKRFEMTLPFQQFEIQGIGNYQQCHQSILELFNTSYCPYSQ
CAFNGIFLPPLQGDFGAFSAFYFVMKFLNLTSEKVSQEKVTEMMKKFCAQPWEEIKT
SYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADSWEHIHFIGKIQGSDAGWTLGYML
NLTNMIPAEQPLSTPLSHSTYVFLMVLFSLVLFTVAIIGLLIFHKPSYFWKDMV
TQNKALPENVKYGIVLDAGSSHTSLYIYKWPAEKENDTGVVHQVEECRVKGPGISKF (SEQ ID
NO:2) VQKVNEIGIYLTDCMERAREVIPRSQHQETPVYLGATAGMRLLRMESEELADRVLDV
VERSLSNYPFDFQGARIITGQEEGAYGWITINYLLGKFSQKTRWFSIVPYETNNQET
FGALDLGGASTQVTFVPQNQTIESPDNALQFRLYGKDYNVYTHSFLCYGKDQALWQK
LAKDIQVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLPFQQFEIQGIGNY
QQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFGAFSAFYFVMKFLNLTSEKVSQ
EKVTEMMKKFCAQPWEEIKTSYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADSWEH
IHFIGKIQGSDAGWTLGYMLNLTNMIPAEQPLSTPLSHST
[0041] Variants may also have sequences which differ by one or more
non-conservative amino acid substitutions, deletions or insertions
which do not abolish the biological activity associated with CD39.
Conservative substitutions (substituents) typically include the
substitution of one amino acid for another with similar
characteristics such as substitutions within the following groups:
valine, glycine; glycine, alanine; valine, isoleucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine. The non-polar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine,.proline, phenylalanine, tryptophan and methionine. The
polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine and glutamine. The positively
charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid
and glutamic acid.
[0042] A CD39 variant of this invention includes a CD39 varied by
changes such as insertions, deletions and substitutions, either
conservative or nonconservative where such changes might provide
for certain advantages in their use such as increased potency,
bioavailability, stability or decreased toxicity or degradation
under physiological conditions.
[0043] One embodiment of the present invention is a truncated
variant of the CD39 which variant is capable of increasing
adenosine diphosphate catabolism or having improved availability or
decreased immunogenicity or increased activity.
[0044] In other embodiments, variants with amino acid substitutions
which are less conservative may also result in desired derivatives
of CD39, e.g., by causing desirable changes in charge, conformation
and other biological properties. Such substitutions would include
for example, substitution of hydrophilic residue for a hydrophobic
residue, substitution of a cysteine or proline for another residue,
substitution of a residue having a small side chain for a residue
having a bulky side chain or substitution of a residue having a net
positive charge for a residue having a net negative charge. When
the result of a given substitution cannot be predicted with
certainty, the derivatives may be readily assayed according to the
methods disclosed herein to determine the presence or absence of
the desired characteristics.
[0045] Just as it is possible to replace substituents of the
scaffold (i.e., amino acids which make up the CD39), it is also
possible to substitute functional groups which decorate the
scaffold with groups characterized by similar features (i.e.,
R-groups which are part of each amino acid). These substitutions
will initially be conservative, i.e., the replacement group will
have approximately the same size, shape, hydrophobicity and charge
as the original group. Non-sequence modifications may include, for
example, in vivo or in vitro chemical derivatization of portions of
naturally occurring CD39, as well as changes in acetylation,
methylation, phosphorylation, carboxylation or glycosylation.
[0046] In a further embodiment the CD39 is modified by chemical
modifications in which activity is preserved. For example, the CD39
may be aminated, sulfated, singly or multiply halogenated,
alkylated, carboxylated, or phosphorylated. The CD39 may also be
singly or multiply acylated, such as with an acetyl group, with a
farnesyl moiety, or with a fatty acid, which may be saturated,
monounsaturated or polyunsaturated. The fatty acid may also be
singly or multiply fluorinated. The invention also includes
methionine analogs of CD39, for example the methionine sulfone and
methionine sulfoxide analogs. The invention also includes salts of
CD39, such as ammonium salts, including alkyl or aryl ammonium
salts, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate,
dihydrogen phosphate, thiosulfate, carbonate, bicarbonate,
benzoate, sulfonate, thiosulfonate, mesylate, ethyl sulfonate and
benzensulfonate salts.
[0047] Variants of CD39 may also include peptidomimetic compounds
of CD39. Such compounds are well known to those of skill in the art
and are produced through the substitution of certain R groups or
amino acids in the protein with non-natural replacements. Such
substitutions may increase the stability, bioavailability, or
activity of such CD39 compound.
[0048] In another embodiment of the method, the CD39 polypeptide is
a recombinant CD39 polypeptide having IL-2 as its leader sequence.
A different leader sequence may be used to drive the secretion of
the protein.
[0049] In another embodiment of the method, the recombinant CD39
polypeptide lacks a transmembrane domain.
[0050] In another embodiment of the method, the agent comprises a
biologically active fragment of the CD39 polypeptide or its
variants thereof or a non-protein compound that augments ADP
catabolism.
[0051] In another embodiment of the method, the active fragment of
the CD39 polypeptide has 20-80 amino acid residues which mimics the
active site of CD39 or its variants thereof.
[0052] In another embodiment of the method, the CD39 polypeptide or
its active fragment can be replaced by a nucleic acid encoding CD39
or its variants or a biologically active fragment thereof.
[0053] In another embodiment of the method, the stroke is
associated with pulmonary embolism, post surgical vasculopathy,
postangioplasty stenosis, and shunt/fistula remodeling or
thrombosis.
[0054] In another embodiment of the method, the stroke is
associated with lung ischemia, limb ischemia, gut ischemia,
myocardial ischemia.
[0055] In another embodiment, the time of administration comprises
from about 5 days before surgery or onset of the disorder to about
5 days after surgery or the onset of the disorder. In another
embodiment, the period of time comprises from about 1 hour before
surgery or the onset of the disorder to about 12 hours after
surgery or the onset of the disorder. In another embodiment, the
period of time comprises from about 12 hours before surgery or the
onset of the disorder to about 1 hour after surgery or the onset of
the disorder. In another embodiment, the period of time comprises
from about 1 hour before surgery or the onset of the disorder to
about 1 hour after surgery or the onset of the disorder.
[0056] In one embodiment, the subject is a mammal. In another
embodiment, the mammal is a human. In another embodiment, the
amount of CD39 polypeptide or its active fragment administered
comprises from about 75 .mu.g/kg to about 550 .mu.g/kg. In another
embodiment, the amount comprises 300 .mu.g/kg.
[0057] In another embodiment of the method, the administration of
the CD39 polypeptide or its active fragment occurs at the onset of
stroke in a subject.
[0058] In another embodiment of the method, the administration of
the CD39 polypeptide or its active fragment is prior to stroke
onset in a subject.
[0059] In another embodiment of the method, the administration of
the CD39 polypeptide or its active fragment occurs after the stroke
onset in a subject.
[0060] In another embodiment of the method, the CD39 polypeptide or
its active fragment is administered in a dosage of 1-20 mg/kg of
the subject's body weight.
[0061] In another embodiment of the method, the CD39 polypeptide or
its active fragment is administered in a dosage of 4-8 mg/kg of the
subject's body weight.
[0062] In another embodiment of the method, the subject is a mouse,
a rat, a dog, a primate or a human.
[0063] In a further embodiment of the method, the CD39 polypeptide
or its active fragment is administered with a pharmaceutically
acceptable carrier.
[0064] The present invention also provides a method for determining
whether a compound inhibits platelet aggregation by increasing ADP
catabolism so as to treat or prevent thrombotic or ischemic
disorders in a subject, comprising: (a) inducing thrombotic or
ischemic disorders in an animal, which animal is an animal model
for thrombotic or ischemic disorders; (b) measuring the stroke
outcome in said animal, (c) measuring platelet deposition and/or
fibrin deposition in ischemic tissue, and (d) comparing the stroke
outcome in step (b) and the platelet deposition and/or fibrin
deposition with that of the animal model in the absence of the
compound so as to identify a compound capable of treating or
preventing thrombotic or ischemic disorders in a subject.
[0065] In one embodiment of the method, the animal model comprises
CD39-deficient mice; wherein the thrombotic or ischemic disorders
are induced by administering an agonist to said mice.
[0066] In another embodiment of the method, the compound treat or
prevent thrombotic or ischemic disorders in a subject without
increasing intracerebral hemorrhage or bleeding.
[0067] In another embodiment of the method, the stroke outcome is
determined from the measurements of platelet deposition, bleeding
time and infarction volume.
[0068] In another embodiment of the method, the compound can be
administered orally or by injection.
[0069] In another embodiment of the method, the compound is
identified by the method.
[0070] In another embodiment of the method, the administration of
the compound is prior to stroke onset in the animal.
[0071] In yet another embodiment of the method, the administration
of the compound occurs at the onset of stroke in the animal.
[0072] In a further embodiment of the method, the administration of
the compound occurs after the stroke onset in the animal.
[0073] The present invention further provides a pharmaceutical
composition comprising the compound of identified by the methods
and a pharmaceutically acceptable carrier as an agent to treat
thrombotic or ischemic disorders in a subject.
[0074] In one embodiment of the pharmaceutical composition, the
composition comprises CD39 and a pharmaceutically acceptable
carrier
[0075] As used herein, the term "pharmaceutically acceptable
carrier" encompasses any of the standard pharmaceutically
acceptable carriers, such as phosphate buffered saline solution,
water, emulsions such as oil/water emulsion or a triglyceride
emulsion, various types of wetting agents, tablets, coated tablets
and capsules. Typically, such carriers contain excipients such as
starch, milk, sugar, certain types of clay, gelatin, stearic acid,
talc, vegetable fats or oils, gums, glycols, or other known
excipients. Such carriers may also include flavor and color
additives or other ingredients.
[0076] By means of well-known techniques such as titration and by
taking into account the observed pharmacokinetic characteristics of
the agent in the individual subject, a skilled artisan can
determine the appropriate dosages for treatment methods of the
present invention.
[0077] Mutants or fragments of CD39 can be produced by known
genetic engineering techniques, using as the starting material
recombinant cDNA encoding CD39 in an appropriate cloning vector or
using direct chemical synthesis.
[0078] This invention will be better understood from the
Experimental Details which follow. These sections are set forth to
aid in an understanding of the invention but are not intended to,
and should not be construed to, limit in any way the invention as
set forth in the claims which follow thereafter.
Experimental Details
EXAMPLE 1
CD39/ecto-ADPase Inhibits Thrombosis and Limits Ischemic Cerebral
Injury in Wild Type and Reconstituted CD39 Null Mice
[0079] Initial studies demonstrated a significant contribution of
leukocyte adhesion receptors and recruited neutrophils to
postischemic no-reflow, but even in the absence of neutrophils,
postischemic no-reflow was not completely abrogated as progressive
microvascular thrombosis ensued.sup.3,4. More recent studies in a
murine model of ischemic stroke demonstrated the cardinal role of
platelets in progressive microvascular thrombosis distal to the
site of primary obstruction of a major cerebrovascular
tributary..sup.2 Progressive microvascular thrombosis,
characterized by accumulation of platelets and fibrin at downstream
sites, contributes to post-ischemic hypoperfusion (no reflow) and
cerebral tissue damage.sup.2. Recent research has indicated t-hat
inhibition of the final common pathway of platelet accumulation,
via blockage of glycoprotein IIb/IIIa receptor-mediated
platelet-platelet interactions, could reduce microvascular
thrombosis in stroke.sup.2. However, the therapeutic window for GP
IIb/IIIa receptor blockade, similar to thrombolytic agents is
narrow, with even small excesses dosing culminating devastating
intracerebral hemorrhage.
[0080] When the integrity of the blood vessel wall is compromised,
platelets adhere to collagen in the subendothelium, leading to
platelet activation and the release of additional agonist:
adenosine diphosphate (ADP), thromboxane (TXA.sub.2), and
serotonin. Of these, ADP is the most important platelet agonist and
recruiting agent present in the microenvironment of the
thrombus.sup.7. There are three primary mechanisms by which
endothelial cells, under homeostatic conditions, maintain blood
fluidity at the blood/vessel wall interface. These include the
local generation of nitric oxide, release of eicosanoids, and
expression of ectoADPase (CD39), a highly conserved enzyme which
promotes ADP catabolism, thus potently inhibiting platelet
aggregation.sup.8. Endothelial cells express CD39 constitutively.
When recombinant CD39 was transfected into COS cells, they acquired
the ability to inhibit ADP-induced platelet aggregation,
establishing CD39 as a prime thromboregulatory enzyme.sup.9.
Recently, CD39 was prepared in soluble form by deletion of two
transmembrane domains and inclusion of a new leader sequence, and
expressed in CHO cells.sup.11. This soluble CD39 (CD39) also
blocked ADP-induced platelet aggregation in vitro.sup.11. The
present studies were designed to elucidate the role of CD39 in the
microvascular thrombosis of stroke, driven by the hypothesis that
augmenting endogenous CD39 should inhibit ADP-mediated
autoamplification of platelet recruitment in distal microvessels
and thereby reduce accretion of thrombus stroke. The studies
presented herein provide an improved method to use CD39 to inhibit
microvascular thrombosis confer cerebroprotection in stroke without
intracerebral hemorrhage.
[0081] Abbreviations: rtPA, recombinant tissue-type plasminogen
activator; CBF, cerebral blood flow; ADP, adenosine diphosphate;
TXA2, thromboxane A2; ICH, intracerebral hemorrhage.
[0082] Mice: Mice lacking CD39 were generated by homologous
recombination in ES cells. Briefly, a gene targeting vector was
created in which exons 3-5, containing Apyrase Conserved Regions
1-3 (ACR 1-3) were replaced with a PGKneo cassette. The resulting
targeting vector was introduced into 129 derived ES cells. ES
clones carrying a CD39 allele disrupted by homologous recombination
were identified by genomic Southern blot analyses and injected into
blasdtocysts. The resulting chimeras were bred to C57BL/6 to
generate mice heterozygous for the CD39 mutation (CD39.+-.), which
were subsequently intercrossed to generate mice deficient in CD39
(CD39-/- mice were born at the expected Mendelian frequency mice
used throughout these experiments represent were on a background of
50% 129J; other mice used for experiments as indicated were CD39+/+
C57BL/6 mice or control C57BL/6/129J CD39+/+ mice (designated F1
controls: for clarity). Animals were 7-9 weeks of age and weighed
between 22 and 26 g.
[0083] Materials: Specific materials were acquired form the
following sources. ADP, heparin (Sigma, St. Louis, Mo.) collagen
(Hormon Chemie, Munchen, Germany); sodium arachidonate (Nu-Check
Prep, Elysian, Minn.).
[0084] Transient Middle Cerebral Artery Occlusion: Mice were
anesthetized (0.3 ml if 10 mg/mL ketamine and 0.5 mg/mL xylazine,
IP) and positioned supine on a rectal temperature controlled
operating surface (yellow Springs Instruments, Inc). Animal core
temperature was maintained at 37.+-.2.degree. C. during surgery and
for 90 minutes after surgery. A midline neck incision was created
to expose the right carotid sheath under the operating microscope
(.times.6 to .times.40 zoom, Leica). The common carotid artery was
isolated with 4-o silk, and the occipital, pterygopalatine, and
external carotid Artemis were each isolated and divided. MCAO was
accomplished by advancing a 13-mm, heat-blunt tipped 6-0 nylon
suture via an arteriotomy made in the external carotid stump. After
placement of the occluding suture, the external carotid artery
stump was cauterized to prevent bleeding through the arteriotomy,
and arterial flow was established. The duration of carotid
occlusion never exceeded 3 minutes. After 45 minutes, the occluding
suture an cautery was once again employed to prevent bleeding S
through the arteriotomy. The wound was closed with surgical
staples. These procedures have previously described in
detail.sup.25.
[0085] Measurement of Cerebral Cortical Blood Flow: Transcranial
measurements of cerebral blood flow were made using laser Doppler
(Permed, Inc. Piscataway, N.J.) as previously described.sup.25.
Using a 0.7 mm straight laser Doppler probe (model PF 303, Perimed)
and previously published landmarks (2 mm posterior to the bregma, 6
mm to each side of the midline).sup.25, relative cerebral blood
flow measurement were made as follows; after anesthesia,
immediately after occlusion, pre-reperfusion, immediate
post-reperfusion, and at sacrifice. Data are expressed as the ratio
of the Doppler signal intensity of the ischemic compared with the
nonischemic hemisphere. The surgical procedure was considered to be
technically adequate if a .gtoreq.70% reduction in cerebral blood
flow was observed immediately after placement of the intraluminal
occluding suture.
[0086] Neurological Exam: Twenty-four hours after surgery, mice
were examined for neurological deficit using a modified four-tiered
grading system published by Hata.sup.26. A score of 1 was given if
the animal demonstrated spontaneous movements and extended both
forelimbs when rolling supine (primitive Moro reflex); a score of
two was given if the animal spontaneously circled clockwise when
viewed from above; a score of three was given if the animal
exhibited marginal activity and leaned to one side or had
incomplete extension of the contralateral forelimb when rolled
supine. A score of four was given if the animal exhibited no
spontaneous movements.
[0087] Calculation of Infarct Volume: After neurological
examination, mice were anesthetized, and final cerebral blood flow
measurements obtained. Mice were sacrificed and brains were removed
and placed in a mouse brain matrix (Activational Systems Inc.) For
1 mm sectioning. Section were immersed in 2% TTC (Sigma) in 0.9%
saline, incubated for 12 minutes at 37.degree. C. Infarcted brain
was stained as an area of unstained tissue. Infarct volumes were
calculated from digital images of 1 mm sections and expressed as
the percentage of infarct in the ipsilateral hemisphere. This
method of calculating infarct volumes has been used previously and
has been correlate.
[0088] Preparation of CD39: Recombinant CD39 was prepared as
described.sup.11. In brief, the CD39 cDNA insert, containing the
CD39 sequence and IL-2 leader, was stably transfected into CHO
cells, affinity purified from conditioned medium, followed by
removal of N-linked sugars. Biochemical purity was assured as
described.sup.11. Doses used are indicated in the test.
[0089] Measurement of Bleeding Time: Bleeding times were measured
in mice which were not subjected to experimental manipulation other
than by receiving either vehicle (saline) or CD39 prepared in
physiological saline and administered intravenously 5 minutes prior
to the experiment. Following anesthesia, a standardized incision
was made on the central dorsal tail vein, and the tail was then
immersed in physiological saline at 37.5.degree. C. Time was
recorded from the moment blood was observed to emerge from the
wound until cessation of blood flow.sup.29.
[0090] Measurements of cerebral thrombosis: .sup.111Indium-platelet
accumulation: Platelet accumulation was determined using
.sup.111Indium labeled platelets, collected and prepared as
previously described.sup.27,30. In brief, pooled blood was
collected from control mice in 3.8% sodium citrate for
anticoagulation (10 mL total). Platelets were isolated by
differential centrifugation, first at 300.times.g for 5 minutes to
obtain platelet rich plasma, which was then washed three times at
2000.times.g for 15 minutes in 10 ml of acid/citrate/dextrose
anticoagulant (ACD-A, containing 38 mmol/L citric acid, 75 mmol/L
sodium citrate, and 135 mmol/L glucose). The pellet was suspended
in 5 mL of ACD-A and centrifuged at 100.times.g for 5 minutes to
remove contaminating red blood cells, and the supernatant was
collected. .sup.111In-oxyquinoline (70 .mu.L of 1 mCi/mL, Amersham
Mediphysics) was added, and the suspension was shaken gently for 30
minutes at room temperature. The radiolabeled platelets were washed
three times in ACD-A and resuspended in PBS, and the platelet
number was adjusted to 5.times.10.sup.6 mL (1.times.10.sup.6 counts
were given to each animal). Immediately prior to insertion of the
occluding suture, 0.2 mL of .sup.111In-labeled platelet suspension
was injected intravenously into the penile vein; at 24 hours of
reperfusion, brain tissue was harvested and platelet accumulation
was quantified as the ipsilateral/contralateral cpm ratio.
[0091] Detection of Intracerebral Fibrin: Mice were first subjected
to focal cerebral ischemia and reperfusion as describe above. In
order to detect fibrin by immunoblotting, mice were heparinized
(1000 U/ml, 0.2 mL given intravenously) about 1 minute prior to
sacrifice) in order to minimize postmortem thrombosis. Following
separation into right and left hemispheres and plasmin digestion to
solubilize fibrin, immunoblotting for fibrin was performed as
described previously.sup.31 using a monoclonal anti-fibrin IgG1
(Biodesign International, ME) that had been prepared with human
fibrin-like beta peptide as immunogen. This antibody was shown to
react with murine fibrin but not murine fibrinogen in preliminary
experiments.
[0092] Measurement of intracerebral hemorrhage: ICH was quantified
using a spectrophotometric assay for hemoglobin which has been
recently developed and validated for use in a murine model of
stroke.sup.32. In brief, mouse brains were homogenized, sonicated,
centrifuged, and hemoglobin in the supernatants was converted (with
Drabkin's regent) to cyanomethemoglobin, whose concentration was
assessed by measuring O.D. at 550 nm against a standard curve
generated with known amounts of hemoglobin.
[0093] Preparation of Murine Platelets: Mice (untreated, treated
with 4 mg/kg CD39, or treated with 4 mg/kg aspirin) were
anesthetized and heparinized (10 U/g), prior to blood collection
via cardiac puncture performed with 22 gauge, 1 cc syringe.
Immediately following collection, 80 .mu.l 3.8% trisodium citrate
was added to each mL of blood. Blood from 6-8 mice was pooled in a
15 mL tube centrifuge tube (Flacon, polypropylene). Platelet-rich
plasma (PRP) was prepared with an initial whole blood
centrifugation (900 g, 3 min, 20.degree. C., no brake), and a
second centrifugation of PRP (100 g, 2 min) to eliminate residual
erythrocytes and leukocytes. The stock suspension of PRP was
maintained at room temperature. PRP platelet counts were
400-700.times.10.sup.3 platelets per .mu.l.
[0094] Platelet aggregation studies: PRP (200 .mu.l) was
pre-incubated (3 min, 37.degree. C.) with 100 .mu.l Tris-buffered
saline (TSG) buffer (15 mM Tris, 134 mM NaCl, 5 mM glucose, pH 7.4)
in an aggregometer cuvette (Lumiaggregometer; Chrono-Log,
Havertown, Pa.). After the 3 min, 37.degree. C. preincubation=5.0 C
10.sup.5 platelets, platelet agonist (ADP, collagen, sodium
arachidonate) were added at the concentration indicated. Total
volumes were adjusted to 300 .mu.l with TSG buffer, and the
aggregation response recorded for 2-4 min. Agonists were prepared
as 100.times. solution: ADP was in TSG buffer, collagen in
acidified isotonic glucose (Gormon Chemie, Munchen, Germany), and
sodium arachidonate in 0.85% saline. All aggregation studies were
completed within two hours of blood collection.
[0095] Pharmacokinetic analysis: C57/6J BL mice (6-8 weeks of age;
maintained under specific pathogen free conditions; Jackson
Laboratory, Bar Harbor, Me.) were intravenously injected with 200
.mu.g recombinant CD39. At the indicated times after injection (1
h, 6 h, 12 h, and 24 h) mice were bled by cardiac puncture, PRP was
prepared as described above, and % platelet inhibition was
documented at the respective timepoints. Aggregometry performed on
the PRP samples was performed in triplicate.
[0096] Quantitation of Platelet Aggregation: Area under the curve
was estimated by multiplying the height of the curve with its width
at half height. The former was measured form pre-simulation
baseline to the middle of the highest point of the curve. However,
when curves were less than 1/3 of maximal height, the lowest point
of the shape change portion of the curve was considered baseline.
Width at half height was extrapolated if the curve did not
re-approach baseline, but was always considered 6 min (=cm) or
less. These criteria underestimate large aggregation responses, and
overestimate small ones. Thus we knowingly underestimated the
effects of compounds with great inhibitory capacity. This approach
was preferred over one that could have overemphasized inhibitory
potential.
[0097] Murine platelet aggregation: C57/6J BL mice (6-8 weeks) were
obtained from Jackson Laboratories, Bar Harbor, Me.). Untreated
mice, and mice treated with 4 mg/kg CD39, with 5 mg/kg aspirin or
phosphate buffered saline, were anesthetized and heparinized (10
U/g), prior to blood collection via cardiac puncture; 80 .mu.L 3.8%
trisodium citrate was added to each mL of blood. Samples from 6-8
mice were pooled and platelet-rich plasma (PRP) prepared by
centrifugation (900 g, 3 min, 20.degree. C., followed by 100 g, 2
min to eliminate residual erythrocytes and leukocytes). PRP
contained 400-700.times.10.sup.3 platelets per .mu.L. PRP (200
.mu.L) was preincubated (3 min, 37.degree. C.) with 100 .mu.L
Tris-buffered saline (TSG) buffer (15 mM Tris, 134 mM NaCl, 5 mM
glucose, pH 7.4).sup.11,23,24 in an aggregometer cuvette
(Lumiaggregometer; Chrono-Log, Havertown, Pa.). Platelet agonists
(ADP, collagen (Hormon Chemie, Munchen, Germany), or sodium
arachidonate (Nu-Check Prep, Elysian, Minn.)) were added at the
final concentrations indicated. Aggregation responses were recorded
for 2-4 min, and expressed as area under the curve (height times
width at 1/2 height). All experiments were completed within two
hours of blood collection.
EXAMPLE 2
Murine Stroke Model
[0098] A previously validated murine model of stroke injury was
employed.sup.2,3,25. Anesthetized mice were maintained at
37.+-.2.degree. C. during and 90 min following surgery. A midline
neck incision was made and the right carotid artery exposed. Middle
cerebral artery occlusion was accomplished by advancing a 13-mm
heat-blunt tipped 6-0 nylon suture via an arteriotomy in the
external carotid stump. The external carotid artery was cauterized
to secure hemostasis, and arterial flow re-established. Carotid
artery occlusion never exceeded 3 min. The occluding suture was
removed after 45 min and cautery was again locally applied to
prevent bleeding at the arteriotomy site. Surgical staples were
used for wound closure. Procedures for Doppler measurement of
cerebral cortical blood flow, neurological score.sup.26,
calculation of infarct volume, measurement of cerebral thrombosis
using .sup.111In-labeled platelets.sup.2,27, detection of
intracerebral fibrin.sup.2, and measurement of intracerebral
hemorrhage.sup.2,28 have been described in earlier section of this
application.
EXAMPLE 3
[0099] Recent studies have demonstrated that CD39 inhibits
ADP-mediated platelet aggregation.sup.11. To ascertain the relative
potency of CD39 and another agent thought to improve outcomes
following transient cerebral attacks in human (aspirin.sup.12),
aggregometry studies were performed using murine platelets treated
with saline (control), CD39, or aspirin. Control and
aspirin-treated platelets strongly aggregated in response to
challenge with either ADP (FIGS. 1A & 1C) or collagen (FIGS. 1B
& 1D). In sharp contrast, CD9 completely abrogated the platelet
aggregation response to ADP addition, and attenuated the
aggregation response to collagen (inhibition was greater with a
lower of collagen). Dose-response data showed an increase in the
response to sodium arachidonate, the precursor of thromboxane
A.sub.2 was somewhat different. As expected, aspirin abrogated the
platelet response to arachidonate (FIG. 1E). CD39, on the other
hand, did not inhibit the initial activation phase of platelet
aggregation to arachidonate, but rapidly disaggregated platelets
during the initial recruitment phase. The data are quantified to
show percent inhibition of platelet aggregation in response to
aspirin or CD39 treatment (FIG. 1F).
[0100] Reduction in sequelae of stroke by CD39: Experiments were
performed to demonstrate the therapeutic utility of intravenously
injected CD39 in stroke. CD39 inhibited platelet as well as fibrin
accumulation in the ipsilateral cerebral hemisphere following
induction of stroke (FIGS. 3A & 3B). As postulated, the ability
of CD39 to reduce thrombosis was accompanied by improved
postischemic cerebral perfusion (FIG. 3A). In contrast, aspirin,
when administered at a clinically relevant dose that inhibited the
ex vivo response of platelets to arachidonate, did not improve
postischemic cerebral blood flow (FIG. 3A). Preoperatively
administered CD39 conferred a dose-dependent diminution of cerebral
infarct volume (FIG. 3B). In contrast, although aspirin showed a
tendency to decrease cerebral infarct volume, the effect was not
statistically significant. CD39 treatment (either prior to, or up
to 3 hours following stroke) reduced both neurological deficit
(FIG. 3C) and mortality (FIG. 3D).
EXAMPLE 4
[0101] CD39 and aspirin were examined with regard to development of
intracerebral hemorrhage following stroke (FIG. 3E) Whereas aspirin
increased intracerebral hemorrhage significantly, there was no
statistically significant increase in intracerebral hemorrhage at
any dose of CD39 tested (FIG. 3E). At these doses CD39 inhibited
both platelet and fibrin accumulation and promoted an increase in
postischemic blood flow (FIGS. 3A, 5A & 5B). A covariate plot
of cerebral infarct volume vs. intracerebral hemorrhage for each
treatment indicates that aspirin is less capable of reducing
infarct volume and preventing intracerebral hemorrhage than are
several regimens of CD39 treatment (FIG. 4).
EXAMPLE 5
[0102] To further characterize the role of endogenous CD39 in
regulation of hemostasis, CD39 null mice were generated by a gene
targeting vector in which exons 3-5, containing Apyrase Conserved
Regions 1-3 (ACR 1-3), were replaced with a PGKneo cassette.
Homozygous null CD39 mice did not display an obvious phenotype in
the unperturbed state, including normal bleeding times (FIG. 2).
These bleeding times can be contrasted with unperturbed state,
including normal bleeding times (FIG. 2). These bleeding times can
be contrasted with the marked increase in bleeding time induced by
aspirin treatment, and the dose-dependent increase in bleeding time
evoked by CD39. Hematologic profiles, including platelet counts,
hemoglobin levels, white blood cell counts and differentials (Table
1), and PT/PTT (not shown) were normal in these mice. To test the
hypothesis that a latent prothrombotic phenotype could be induced
in a clinically relevant platelet-dependent model (Stroke.sup.2),
mice were subjected to focal cerebral ischemia. CD39 null mice
exhibited diminished blood flow following reperfusion compared with
their genetically matched controls (FIG. 6A). When CD39 (200
.mu.g/mL was given to the CZ39 null mice, these "reconstituted
mice" exhibited postischemic flows which were similar to untreated
control mice. When these null mice were sacrifices at 24 hours,
there were increased cerebral infarction volumes compared with
genotype-matched control mice. CD39 null mice reconstituted with
CD39 were protected in stroke, as shown by their markedly
diminished infarct volumes at 24 hours (FIG. 6B). Other parameters
which were measured but which did not differ between groups
included neurological deficit scores, overall mortality, and
intracerebral hemorrhage, measured in a spectrophotometric
hemoglobin assay :which we have recently validated for use in
stroke.sup.32 (FIGS. 6C, 6D, & 6E).
EXAMPLE 6
[0103] Reconstitution of CD39-/- mice with CD39: To further
characterize the contributions of endogenous CD39 to hemostasis and
thrombosis, CD39-/- mice were generated by a gene targeting vector
in which exons 4-6, encoding apyrase conserved regions
2-4.sup.13-16, were replaced with a PGKneo cassette. Homozygous
CD39-/- mice did not display an obvious phenotype in the
unperturbed state. Hematological profiles were normal, including
erythrocyte parameters, platelet counts, leukocyte counts and
differentials, and coagulation screening tests. Bleeding times of
CD39-/- mice were normal, in contrast to the markedly increased
bleeding time following aspirin treatment, and a dose-dependent
increase in bleeding time induced by CD39 (FIG. 2).
EXAMPLE 7
[0104] The next group of experiments were performed to demonstrate
the utility of CD39 as a therapeutic agent in stroke. First, the
antithrombotic action of CD39 was established by its ability to
inhibit platelet accumulation in stroke (FIG. 5A) as well as to
inhibit fibrin accumulation in the ipsilateral cerebral hemisphere
(established by fibrin immunoblot (FIG. 5B). As expected, the
ability of CD39 to diminish thrombosis in stroke was accompanied by
improved postischemic cerebral perfusion (FIG. 3A). In contrast,
aspirin, even when used at a clinically relevant dose which
inhibited the response of platelets to arachidonate ex vivo, did
not improve postischemic cerebral blood flow (FIG. 3A). In terms of
cerebral infarction volume, CD39 administered preoperatively
conferred a dose-dependent diminution of cerebral infarct volumes,
in contrast to aspirin, which only tended to decrease cerebral
infarct volumes (this reduction was not statistically significant)
(FIG. 3B). Similarly, CD39 reduced both neurological deficit (FIG.
3C) and mortality (FIG. 3D). Of especial importance were data in
which the effects of aspirin and CD39 were examined in terms of
intracerebral hemorrhage. Although aspirin did increase
intracerebral hemorrhage significantly, there was not statistically
significant increase postischemic blood flow. The relationship
between the ability of either aspirin, CD39, or vehicle to diminish
cerebral infarction volume and their propensity to increase
intracerebral hemorrhage are shown in FIG. 3.
EXAMPLE 8
[0105] Other forms of ischemia were also studied. Because of the
integral role of platelets in other forms of coagulation,
thrombosis, vascular remodeling (and disorders such as pulmonary
embolism, lung ischemia, limb or gut ischemia, myocardial ischemia,
post surgical vasculopathy, postangioplasty stenosis, shunt/fistula
remodeling or thrombosis), I have tested the effect of CD39 in
another ischemic disorder involving a different vascular bed than
that in the brain. For these additional studies, I have used a
mouse model of lung ischemia and reperfusion to show that CD39
confers significant postischemic protection to the lung tissues and
blood vessels.
[0106] In these studies, murine ischemic reperfusion model was
used. In the mouse model of lung ischemia, mice were initially
anesthetized intraperitoneal with 0.1 mg/mouse weight (g) of
ketamine and 0.01 mg/mouse weight (g) of xylazine, following by
intraperitoneal continuous infusion of one third of the initial
dose per hour using a syringe pump (model 100 series, KD Scientific
Inc. MA). After ensuring appropriate depth of anesthesia, mice were
intubated via tracheostomy and placed on a Harvard ventilator
(tidal volume=0.75 mL, respiratory rate=120/min) with room air,
followed by bilateral thoracotomy. The left hilum was cross-clamped
for a period of 1 hour after which the cross-clamp was released.
Reperfusion proceeded for 2 hours.
[0107] For all experiments, the surgical operator was blinded by a
colleague in the laboratory to the specific substance being
injected (vehicle or CD39, 8 mg/kg). Experimental procedures were
as follows. After one-hour ischemia followed by 2-hour reperfusion,
the contralateral (right hilum was permanently ligated, so that the
animal's survival and gas exchange depended solely upon the
reperfused lung, and observation continued for 1 hour. As the mouse
continued to be ventilated, death of the mouse was defined as a
combination of (1) cessation of regular cardiac activity; (2) the
apparent collapse of the left atrium; and (3) brief clonic activity
indicating cessation of cerebral blood flow.
[0108] In four experiments with CD39, mouse survival was 100%
following functional removal of the contralateral (nonischemic)
lung from the circulation. In contrast, control (vehicle-treated)
(n=7) demonstrated no survival under identical conditions. These
data indicate that CD39 has a marked protective effect (reduces
tissue injury and protects function) of the postischemic lung.
Discussion
[0109] Platelet and fibrin deposition downstream of an occlusive
lesion contribute significantly to the postischemic hypoperfusion
and tissue injury complicating stroke. It has been demonstrated for
the first time in vivo protection conferred by CD39 in this
platelet-dependent thrombotic disorder. CD39 improves cerebral
blood flow and reduces cerebral infarct volume when given
preoperatively. In addition, CD39 confers significant
cerebroprotection when administered three hours after onset of
stroke. Rendering cerebroprotection at this delayed time point is
significant because these effects occurred without an increase in
mortality or intracerebral hemorrhage. The CD39-/- mice had a
defect in thromboregulation in that they exhibited larger infarct
volume than their genotype-matched controls. The CD39-/- mice were
"reconstituted" by administration of CD39, thus fulfilling Koch's
postulates.sup.17.
[0110] CD39-/- mice did not have an obvious phenotype, with
completely normal baseline hematological and coagulation profiles,
including platelet counts. This contrasts with mice null for the
protein P-selectin, where leukocytosis is apparent in unperturbed
mice.sup.18. Moreover, spontaneous thrombotic events have not been
observed, as reported in PAI-1 overexpressing mice.sup.19. Rather,
CD39-/- mice appear to exhibit a latent prothrombotic phenotype,
elicited by inducing a platelet-dependent thrombotic disorder
(stroke). It is postulated that under basal conditions, vascular
homeostasis may be maintained by the endothelial thromboregulators
prostacyclin and nitric oxide.sup.8. However, a severe breach in
vascular integrity leads to platelet accumulation and consequent
fibrin deposition in the absence of CD39, as in the CD39-/- mice.
Reconstitution of the animal with CD39 appears to ameliorate this
defect (FIGS. 6A, 6B).
[0111] Although aspirin may be of benefit in primary prevention of
stroke, it does not appear to be efficacious in evolving
stroke.sup.12. Moreover, some patients obtain little benefit from
aspirin ("nonresponders"), even though it is efficacious in
others.sup.10,20. GPIIb/IIIa antagonists are potent inhibitors of
platelet aggregation, since they block a final step in platelet
accumulation, i.e. fibrinogen bridging of surface glycoprotein GP
IIb/IIIa receptors, thus abrogating platelet-platelet adherence.
Although useful in prevention of intravascular thrombosis following
percutaneous coronary intervention, these agents have not been
widely studied specifically in the setting of acute stroke. One
highly specific GP IIb/IIIa antagonist, GPI-562, had potent
antithrombotic effects in experimental stroke, and did reduce
cerebral infarction volume, but it was associated with
intracerebral hemorrhage.sup.2. Other platelet inhibitors useful in
management of acute ischemic syndromes, such as ticlopidine or
clopidogrel.sup.21, inhibit platelet aggregation mediated by the
low affinity P2Y1 ADP receptor on the platelet surface.sup.22. The
data herein show that endogenous CD39 is protective in stroke, and
that administration of pharmacological doses of CD39 is effective
in inhibiting thrombosis and tissue injury in stroke. Since CD39
inhibits all ADP-mediated platelet aggregation via metabolic
deletion of ADP from the activated platelet releasate, it may be
more potent than the ADP-receptor blockers currently in use.
[0112] The basis for the apparent superiority of CD39 to aspirin
may be that it induces more potent inhibition of ADP-induced
platelet aggregation. This latter mechanism is more efficient in
platelet-induced platelet recruitment than the
arachidonate/thromboxane pathway. Moreover, while platelet
reactivity to low-dose collagen is inhibited by CD39, platelets do
respond to higher doses of collagen. In contrast, aspirin had
little effect on platelet reactivity at any collagen dose. The
hemostatic effects of agonist-induced pathways are likely to
overlap with considerable redundancy in vivo. However, the
experimental data indicate that aspirin resulted in more bleeding
in response to vein injury, or stroke, than did CD39. Perhaps the
initial layer of platelets that adheres to an injured vessel wall
is essential for hemostasis, but in stroke, ADP-mediated
recruitment of platelets into an evolving thrombus results in
intravascular occlusion. CD39 disaggregates platelets that have
already responded to an agonist, but it does not have a deleterious
effect on primary hemostasis.
[0113] It has been previously demonstrated that microvascular
thrombosis is a continuing phenomenon after the onset of
stroke.sup.2. Therefore, this ongoing process can be modulated by
therapeusis with CD39, even three hours after stroke induction. The
data presented herein are especially pertinent in the setting of
clinical observations of increased intracerebral hemorrhage when
thrombolytic agents are administered beyond three hours following
stroke onset.sup.6. Thus, the results may constitute a possible new
approach to antithrombotic therapy, based upon metabolism of a
major agonist for vascular occlusion platelet-released ADP.
[0114] It was hypothesized that a latent prothrombotic phenotype
could be identified in a clinically relevant platelet-dependent
stroke model.sup.2. Indeed, CD39-/- mice, subjected to focal
cerebral ischemia, did exhibit diminished blood flow following
reperfusion as compared to genetically matched controls (FIG. 6A).
When CD39 (8 mg/kg) was administered to the CD39-/- mice, these
mice were "reconstituted" as shown by a postischemic blood flow
similar to untreated controls. Furthermore, CD39-/- mice
demonstrated increased cerebral infarction volume as compared to
genotype-matched controls following induced stroke (FIG. 6B).
CD39-/- mice "reconstituted" with CD39 had markedly diminished
infarct volume, indicating a protective effect of CD39. Other
parameters (neurological deficit scores, overall mortality, and
intracerebral hemorrhage) did not differ between groups (FIGS. 6C,
6D, & 6E). Moreover, CD39 should inhibit only platelet/platelet
recruitment mediated ADP, and attenuate recruitment by other
agonists such as collagen or arachidonate. As CD39 does not
interfere with the primary GPIb-mediated platelet adhesive event at
the site of vessel damage, CD39 should not prevent a subsequent
layer of platelets form forming at sites of vascular damage and
therefore not interfere with hemostatic regulatory mechanisms
required for prevention of intracerebral hemorrhage. I also
hypothesized that alternative methods of inhibiting
platelet-mediated amplification of intravascular thrombosis should
provide a strategy in which primary hemostasis at sites of vascular
injury may be maintained, in the setting of inhibition of
platelet-mediated autoamplification (recruitment).
[0115] The experimental data indicate that platelet and fibrin
deposition in the ipsilateral cerebral hemisphere contribute
significantly to the postischemic hypoperfusion and tissue injury
which occur in stroke. These studies identify for the first time an
in vivo protective role conferred by CD39 in a platelet-dependent
thrombotic disorder (stroke). CD39, which we show to be a potent
inhibitor of ADP-induced platelet aggregation, also has an extended
in vivo half life (elimination half-time in mice is 2 days.sup.11).
Not only does it improve cerebral blood flow and reduce cerebral
infarction volumes when given preoperatively, but it also confers
significant cerebroprotection when given 3 hours after the onset of
stroke. The effect of this agent in conferring cerebroprotection at
this delayed time point is both novel and important because the
cerebroprotective effects occurred without increasing intracerebral
hemorrhage or mortality. The CD39 null mice, which exhibit larger
infarct volumes than their genotype controls, were rescued by the
administration of CD39, fulfilling Koch's postulates.sup.32 for
demonstration that endogenous CD39 is a major thromboregulator.
[0116] CD39 is a 95 kD integral endothelial cell membrane
glycoprotein, comprised of two membrane spanning domains, a
centrally located hydrophobic domain, and four putative ADPase
(apyrase) domains which are highly conserved.sup.11. CD39 is
constitutively expressed on vascular endothelial cells, and appears
to exert an important antithrombotic activity on the endothelial
cell surface. This enzyme degrades nucleotide tri- and diphosphates
(but not monophosphates), and hence its expression at the
endothelial surface significantly blunts the ADP-mediated
recruitment phase of platelet reactivity.sup.11. In vitro studies
have shown that COS cells transfected with the cDNA encoding human
CD39 acquired the ability to inhibit ADP-induced platelet
aggregation.sup.9. As the initial step toward developing a
potentially useful antiplatelet therapeutic agent, a recombinant
soluble from of CD39 was prepared by transfecting CHO cells with a
cDNA construct containing the four apyrase domains but lacking the
two transmembrane regions of native CD39 and introducing a leader
sequence. The resulting peptide, isolated from conditioned medium
was affinity purified and shown to retain potent apyrase activity,
and to have an elimination half-time in mice of 2 days.sup.11. It
was this peptide, CD39, which was used in the experiments described
here. These studies confirm the ex vivo platelet inhibitory
activity of CD39. However, the current results extend the initial
observations in two ways: 1) I demonstrated platelet inhibitory
activity after administration in vivo; and 2) I documented the
effect of CD39 on primary hemostasis (dose-dependent increase in
bleeding time, and reduction of thrombosis in stroke).
[0117] In addition to demonstrating the effects of pharmalogical
doses of CD39, to our knowledge, the current studies are the first
to characterize the properties of CD39 null mice.
[0118] These mice do not have an obvious phenotype. Unlike mice
null for other unrelated alleles, such as P-selectin, in which
leukocytosis is apparent in unperturbed mice.sup.1, baseline
hematologic and coagulation profiles in these mice are completely
normal (including platelet counts). In addition, I have not
observed spontaneous thrombotic events, such as those seen in PAI-1
overexpressing mice.sup.33 which may exhibit spontaneous ischemic
events resulting in lost digits or the tip of the tail. Rather,
CD39 null mice to exhibit a latent prothrombotic phenotype. By
inducing a platelet-dependent thrombotic disorder (stroke), I was
able to elicit differences between appropriate genetic control mice
and CD39 null mice.
[0119] There is an important point to be considered in the setting
of the data presented herein. We and others have previously shown
that in studies of mice, uniformity of background strain is
critical for stroke research. Importantly, baseline cerebral
infarct volumes were smaller in the CD39 mice on the 129/C57Bl
background than in control mice which were pure C47Bl/6J. Because
of limitations in time for backbreeding, the CD39 null mice which
were used for the current experiments are on a mixed background
(C57Bl/129J). Therefore, genotype-matched control mice were
essential to establish the latent prothrombotic phenotype of these
mice and their relative susceptibility to cerebral infarction. Data
were also obtained or derived from experiments using pure C57Bl/6J
mice. As one would expect from published literature, C57Bl/6J mice
have larger absolute cerebral infarction volumes than "F1 control"
mice comprised of a mixed C57Bl/6J/129J background; therefore, it
is important to compare data only with genotype-matched controls.
It has been shown that there are reproducible strain-related
differences in susceptibility to stroke. Thus, 129J is a
particularly resistant strain, and C57Bl is a particularly
susceptible strain.sup.25,34,35. For this reason, genetically
matched controls were performed.
[0120] Why should CD39 constitute a better therapeutic strategy in
stroke than other anti-platelet agents, with greater or lesser
efficacy different mechanisms of actions? Aspirin, which has clear
benefits in terms of primary prevention of stroke, has not been
shown to be efficacious for evolving stroke.sup.12. Furthermore,
there are a group of patients who are aspirin nonresponders.sup.36,
who obtain little benefit from aspirin even where it is efficacious
in others. GPIIb/IIIa antagonist which was tested in stroke,
GPI-562, had potent antithrombotic action and could diminished
cerebral infarction volumes.sup.2. However, the therapeutic window
for this agent was narrow. Thus only modest increases in dosage
were associated with unacceptably high rates of intracerebral
hemorrhage.sup.2. Although not specifically tested in current
experiments, other antiplatelet agents may be useful in the
treatment of evolving stroke. This includes agents such as
ticlodipine or clopidogrel, both of which inhibit platelet
aggregation mediated by the low-affinity type II ADP receptor on
the platelet surface. Experiments with the CD39 null mice and
recombinant CD39 show that endogenous CD39 is protective, and the
administration of pharmacologic doses of CD39 are effective in
terms of inhibiting thrombosis and tissue injury during stroke. It
remains to be seen whether sol39 is more potent than the other
ADP-receptor blockers, but CD39 should inhibit all ADP-mediated
platelet aggregation. Unlike ticlodipine or clopidogrel, CD39
metabolically deletes ADP from an activated platelet releasate.
[0121] Data obtained in the experiments give insights into reasons
for the superiority of CD39 to aspirin. First, it is clearly a more
potent antiplatelet agent with respect to ADP-induced platelet
aggregation, which may be of more importance in platelet-induced
platelet recruitment than the arachidonate/thromboxane axis.
Secondly, CD39 did not inhibit reactivity of platelets to low-dose
collagen, although at high dose collagen, there was minor
inhibition of collagen-induced platelet reactivity. Aspirin had no
effect on collagen-reactivity of platelet at any collagen dose. The
hemostatic effects agonist-induced pathways are likely to overlap
with considerable complexity in vivo; however, experimental data on
bleeding time and intracerebral hemorrhage indicated that aspirin
was more potent as an anticoagulant in response to specific stimuli
(such as cutting a vein, or stroke) than was CD39. Perhaps the
initial layer of platelets which is laid down is essential for
hemostasis, but the augmented accumulation of platelets via
recruitment results in the intravascular obstruction which is
deleterious in stroke. In this regard, CD39 is capable of
disaggregating platelets which have already aggregated in response
to all agonists.
[0122] How might CD39 prove to be therapeutically useful? It has
been shown that the therapeutic index of CD39 is high, i.e., even
twice the effective dose does not increase the occurrence of
intracerebral hemorrhage. Furthermore, when given even 3 hours
following stroke, therapeutic efficacy is apparent. These data
confirm previous research in which microvascular thrombosis was
demonstrated to be an ongoing process after the onset of stroke.
Inhibition of ongoing microvascular thrombosis is the therapeutic
target of the current CD39 strategy. These results are especially
important in light of current clinical observation, which show
increased intracerebral hemorrhage and mortality if a thrombolytic
agent is administered beyond three hours following the onset of
stroke. Even in the best of circumstances, few patients arrive at
an emergency room in sufficient time to qualify for thrombolytic
therapy.sup.6. An extended time window for administration of a
therapeutically useful agent may be an important first step towards
improving the current limited treatment paradigms for evolving
stroke. CD 39 may also have an inhibiting effect on white blood
cell accumulation or lucolyte accumulation
[0123] Table 1: Hematological profiles of CD39-/- and
genotype-matched control mice. N=f for each group. TABLE-US-00002
TABLE 1 Comparison of Blood Cell Components in Wildtype versus
CD39-deficient Mice. Segs Bands Lymphs Monos Eos Hgb WBC Plts
(cells/ (cells/ (cells/ (cells/ (cells/ Strain (mg/dl) (cells/.mu.l
.times. 10.sup.3) (cells/.mu.l .times. 10.sup.3) 100 WBCs) 100
WBCs) 100 WBCs) 100 WBCs) 100 WBCs) CD39 +/+ 13.6 .+-. 1.25 5.4
.+-. 0.74 799 .+-. 98.0 42 .+-. 2.0 1 .+-. 0.20 52 .+-. 2.4 5.2
.+-. 0.66 0.8 .+-. 0.37 CD39 -/- 13.2 .+-. 0.57 3.5 .+-. 0.40 776
.+-. 52.2 32.6 .+-. 3.4 0.4 .+-. 0.24 61.8 .+-. 3.2 5 .+-. 0.45 0.2
.+-. 0.2 p-value 0.47 0.11 0.82 0.39 0.37 0.28 0.85 0.21
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EXAMPLE 9
Cerebroprotective Role of CD39 (Endothelial EctoADPase) in Murine
Stroke
[0160] Endothelial cells express ectoADPase (CD39) which reduces
ADP-mediated platelet (plt) recruitment. The functional role of
CD39 was studied in a murine stroke model, in which platelet
recruitment is deleterious. In CD39 +/+ mice, recombinant soluble
human CD39 (sol CD39100 .mu.g/mouse) caused 100% inhibition of
ADP-mediated plt aggregation. When given prior to transient (45
min) intraluminal right middle cerebral artery occlusion, solCD39
(n=23) .dwnarw. ipsilateral fibrin deposition (by immunoblot).
.dwnarw. 111In-plt deposition (50% .dwnarw., p<0.04)
dose-dependently .uparw. post-ischemic laser doppler blood flow
(for 100 .mu.g/mouse, 2-fold .uparw. at 24 hours vs. Controls,
n=23, p<0.001) and I cerebral infarct volumes (44% smaller than
controls p=0.01, measured by TTC-stained/planimetered serial
cerebral sections). Bleeding times were not increased at this dose
(109 9 sec vs 100 11 sec for controls, p=NS) nor was there an
increase in intracerebral hemorrhage (ICH, measured
spectrophotometrically.) In contrast, aspirin (ASA, 5 mg/kg)
inhibited only arachidonate-mediated plt aggregation and markedly
.uparw. bleeding times (to 287.+-.31 sec for ASA, p<0.0001 vs
control): ASA did not significantly .uparw. postischemic reflow nor
.dwnarw. cerebral infarction volumes; however, ASA increased ICH
(74% .uparw., p<0.ol vs controls). Mice lacking CD39 (CD39-/-)
were made by deletion of exons 3-5 (ie apyrase conserved regions
1-3). They had normal phenotypes, hematologic profiles, and
bleeding time but exhibited .dwnarw. postischemic perfusion (43%
.dwnarw.) and .uparw. 3.5 fold cerebral infarct volumes compared
with their genotypic CD39 +/+ controls; CD39 -/- mice reonstituted
with solCD39 exhibited increased postischemic flows and rescue from
cerebral injury (16-fold smaller infarcts than untreated CD39 -/-
mice). SolCD39 was a potent agent to restore postischemic blood
flow and reduce cerebral infarction volumes even when given up to 3
hours after stroke. The efficacy of postischemic administration
confirms dynamic/ongoing microvascular thrombosis after stroke
onset. Not only is endogenous CD39 thromboprotective, but exogenous
solCD39 inhibits microvascular thrombosis in stroke without
increasing ICH.
EXAMPLE 10
Abstract
[0161] Endothelial CD39 metabolizes ADP released from activated
platelets. Recombinant soluble human CD39 (solCD39) potently
inhibited ex vivo platelet aggregation in response to ADP and
reduced cerebral infarct volumes in mice following transient middle
cerebral artery occlusion, even when given 3 hours after stroke.
Postischemic platelet and fibrin deposition were decreased and
perfusion increased without increasing intracerebral hemorrhage. In
contrast, aspirin did not increase postischemic blood flow nor
reduce infarction volume, but did increase intracerebral
hemorrhage. Mice lacking the enzymatically-active
(apyrase-conserved) extracellular portion of the CD39 molecule were
generated; they demonstrated normal phenotypes, hematological
profiles, and bleeding times, but exhibited increased cerebral
infarct volumes and reduced postischemic perfusion. solCD39
reconstituted these mice, restoring postischemic cerebral perfusion
and rescuing them from cerebral injury. Thus CD39 exerts a
protective thromboregulatory function in stroke.
Introduction
[0162] Stroke is the third leading cause of death and the main
cause of permanent morbidity in the United States, affecting over
450,000 patients annually(1). Our recent studies in a murine model
of ischemic stroke demonstrated 3 pivotal role for platelets in
progressive microvascular thrombosis distal to the primary
obstruction of a major cerebrovascular tributary(2,3). This
progressive microvascular thrombosis is characterized by distal
platelet and fibrin accumulation, resulting in postischemic
hypoperfusion ("no re-flow") and neuronal injury(2). While
leukocyte adhesion receptors and recruited neutrophils contribute
to postischemic hypoperfusion, postischemic hypoperfusion cannot be
completely abrogated because even in the absence of neutrophils,
progressive microvascular thrombosis persists(4,5). Two
thrombolytic agents have been approved for treatment of stroke
(recombinant tissue-type plasminogen activator [rtPA] and
pro-urokinase). However, their therapeutic utility is limited due
to risk of symptomatic and fatal intracranial hemorrhage(6). In the
United States, less than I % of patients presenting to community
hospitals with acute ischemic stroke receive rtPA(7). Inhibition of
the final common pathway of platelet accumulation via blockade of.
glycoprotein IIb/IIIa receptor-mediated platelet-platelet
interactions, does reduce microvascular thrombosis in experimental
stroke (2). However, as with thrombolytic agents, small excesses of
a GPIIb/IIIa receptor blocker culminated in serious intracerebral
hemorrhage. It is therefore important to identify novel strategies
for inhibition of platelet function in acute stroke that will
reduce intravascular thrombosis without increasing risk of
intracerebral hemorrhage.
[0163] When the integrity of a blood vessel wall is compromised,
platelets adhere to collagen in the subendothelium, leading to
platelet activation and release of additional agonists: ADP,
thromboxane A.sub.2, and serotonin. Of these, ADP is the most
important platelet agonist and recruiting agent present in the
microenviromnent of the thrombus(8). There are three primary
mechanisms by which endothelial cefls maintain blood fluidity.
These include local generation of NO, release of eicosanoids, and
CD39/ectoapyrase activity. The latter is a highly conserved
constitutively expressed enzyme, which strongly inhibits platelet
aggregation(9). Following transfection of CD39 into COS cells, they
acquire the ability to inhibit ADP-induced platelet aggregation,
establishing CD39 as a prime thromboregulator(10, 1 1). Recently, a
recombinant, soluble form of human CD39 (including a secretion
leader, but lacking transmembrane domains) was isolated from
stably. transfected CHO cells(12). This soluble CD39 (solCD39)
preparation blocked aggregation induced by ADP, as well as several
other agonists in vitro, and circulated in mice with a half life of
-2 days (12).
[0164] The present studies test the hypothesis that augmentation of
endogenous CD39 would inhibit ADP-mediated autoampfification of
platelet recruitment in distal microvessels and thereby reduce
thrombosis following stroke. Since solCD39 does not interfere with
primary GPIb-mediated platelet adhesion at the site of vessel
damage, theoretically, solCD39 administration should not prevent a
layer of platelets from forming at the site of injury or interfere
with hemostatic mechanisms that prevent intracerebral hemorrhage.
Our studies examine the thromboregulatory role of endogenous CD39
in stroke, and the ability of solCD39 to inhibit microvascular
thrombosis and confer cerebroprotection in stroke without inducing
intracerebral hemorrhage.
Methods
[0165] Murine platelet aggregation. C57/6J BL mice (6-8 wk) were
obtained from Jackson Laboratories, Bar Harbor, Me.). Untreated
mice, and mice treated with 4 mg/kg solCD39, with 5 mg/kg aspirin
or phosphate buffered saline, were anesthetized and heparinized (10
U/g), prior to blood collection via cardiac puncture; 80 AL 3.8%
trisodium citrate was added to each mL of blood. Samples from 6-8
mice were pooled and platelet-rich plasma (PRP) prepared by
centrifugation (90.0 g, 3 min, 20.degree. C. followed by 100 g, 2
min to eliminate residual erythrocytes and leukocytes). PRP
contained 400-700.times.10.sup.3 platelets per .mu.l. PRP (200 AL)
was preincubated (3 min, 37.degree. C.) with 100 AL Tris-buffered
saline (TSG) buffer (15 mM Tris, 134 mM NaCl, 5 mM glucose, pH 7.4)
(12-14) in an aggregometer cuvette (Lumiaggregometer; Chrono-Log,
Havertown, Pa.). Platelet agonists (ADP, collagen (Hormon Chemie,
Munchen, Germany), or sodium arachidonate (Nu-Check Prep, Elysian,
Minn.)) were added at the final concentrations indicated.
Aggregation responses were recorded for 2-4 min, and expressed as
area under the curve (height times width at 1/2 height). All
experiments were completed within 2 h of blood collection.
Murine Stroke Model.
[0166] A previously validated murine model of stroke injury was
employed(2-4,15). Anesthetized mice were maintained at
37.+-.2.degree. C. during and 90 min following surgery. A midline
neck incision was made and the right carotid artery exposed. Middle
cerebral artery occlusion was accomplished by advancing a 13-mm
heat-blunt tipped 6-0 nylon suture via an arteriotomy in the
external carotid stump. The external carotid artery was cauterized
to secure hemostasis, and arterial flow re-established. Carotid
artery occlusion never exceeded 3 min. 'Me occluding suture was
removed after 45 min and cautery was again locally applied to
prevent bleeding at the arteriotomy site. Surgical staples were
used for wound closure. Procedures for Doppler measurement of
cerebral cortical blood flow, neurological score(16), calculation
of infarct volumes, measurement of cerebral thrombosis using
.sup.111In-labeled platelets(2,17), detection of intracerebral
fibrin(2), and measurement of intracerebral hemorrhage(2,18) have
been previously described. Platelet accumulation was determined
using .sup.111Indium labeled platelets, collected and prepared as
previously described (2,17,19,20). Immediately prior to surgery,
mice were given 5.times.10.sup.6 .sup.111In-labeled-platelets
intravenously; deposition was quantified after 24 hours bv as
ipsilateral cpm/contralateral cpm. The accumulation of fibrin was
measured following sacrifice (of fully heparinized animals) using
an inununoblotting procedure which has been recently described and
validated (21,22). Because fibrin is extremely insoluble,
hemispheric brain tissue extracts were prepared by plasmin
digestion, then applied to a standard SDS-polyacrylamide gel for
electrophoresis, followed by immunoblotting using a polyclonal
rabbit anti-human antibody prepared to gamma-gamma chain dimers
present in cross-linked fibrin which can detect murine fibrin, with
relatively little cross-reactivity with fibrinogen. (2,21-23).
lntracerebral hemorrhage was quantified by a spectophotometric
assay recently developed and validated for use in murine stroked
8). In brief, mouse brains were homogenized, sonicated,
centrifuged, and methemoglobin in the supernatants converted (using
Drabkin's reagent) to cyanomethemoglobin, the concentration of
which was assessed by measuring O.D. at 550 nm. For each
experiment, the optical density relative to that obtained from a
group of control brains is reported.
[0167] Generation of CD39-/- mice by homologous recombination. A
gene targeting vector, in which a 4.1 KB Spel-BglIH fragment
containing exons 4-6 (encoding apyrase conserved regions 2-4)(24)
was replaced with a PGKneo cassette, was introduced into
129-derived ES cells, and cells were selected in G418 and
gancyclovir. ES clones with a disrupted CD39 abele were identified
by genomic Southern blot analyses of BglII digested DNA and were
injected into blastocysts. The resulting chimeras were crossed to
C57BL/6 to produce heterozygotes (CD39.+-.), which were used to
generate CD39-/- mice from CD39.+-. intercrosses.
[0168] Data Analysis: Values are expressed as means.+-.SEM, with
the numbers of experiments performed provided in the Figure
legends. For experiments in which 2 variables were compared,
unpaired Student's T test was used. For experiments in which more
than 2 variables were compared, one way ANOVA was used, with
Tukey's procedure used to test for significant differences.
Contigency analysis using Fisher's exact test % %, as performed to
test for differences in mortality between various treatments. Data
were considered significantly different when p<0.05.
Results
Platelet Aggregation: solCD39 vs Aspirin
[0169] Ex vivo platelet aggregation was studied in platelet-rich
plasma (PRP) obtained from mice 1 hour following injection of
saline (vehicle), solCD39 (prepared as described(12)), or aspirin
(FIG. 7). This was done to ascertain the relative potency of
solCD39 as compared to aspirin, an agent which improves outcomes
following transient ischemic attacks (TIAS) in humans.(25).
Platelets from control as well as aspirin-treated mice strongly
aggregated to either ADP (FIG. 7a) or collagen (FIG. 7b). SolCD39
administration abrogated platelet aggregation to ADP, and
attenuated aggregation to collagen (FIG. 7b,d) and arachidonate
(FIG. 7c,d). Aspirin treatment, in contrast, only blocked platelet
reactivity to arachidonate (FIG. 7c,d). Platelets from mice
pretreated with solCD39 showed a brisk initial phase of aggregation
to arachidonate, but before a full response occurred, the platelets
rapidly disaggregated and returned to the resting state (FIG. 7c).
Thus arachidonate evoked an initial reaction, but the released ADP
(required for sustaining aggregation) was metabolized by solCD39 in
the PRP. This was responsible for the disaggregation observed.
Reduction in Sequelae of Stroke by solCD39
[0170] Experiments were performed to demonstrate the therapeutic
utility of intravenously injected solCD39 in stroke SolCD39
inhibited platelet as well as fibrin accumulation in the
ipsilateral cerebral hemisphere following induction of stroke (FIG.
8a,b). As postulated, the ability of solCD39 to reduce thrombosis
was accompanied by improved postischemic cerebral perfusion (FIG.
9a). In contrast, aspirin, when administered at a clinically
relevant dose that inhibited the ex vivo response of platelets to
arachidonate, did not improve postischemic cerebral blood flow
(FIG. 9b). Preoperatively administered solCD39 conferred a
dose-dependent diminution of cerebral infarct volumes (FIG. 9c).
This reduction was sustained even when solCD39 was administered 3
hours after stroke (FIG. 9c). In contrast, although aspirin showed
a tendency to decrease cerebral infarct volumes, the effect was not
statistically significant. SolCD39 treatment (either prior to; or
up to 3 hr following stroke) reduced both neurological deficit
(FIG. 9d) and mortality (FIG. 9e)
[0171] SolCD39 and aspirin were examined with regard to development
of intracerebral hemorrhage following stroke (FIG. 9f). Whereas
aspirin increased, intracerebral hemorrhage significantly, there
was no statistically. significant increase in intracerebral
hemorrhage at any dose of solCD39 tested (FIG. 9f). At these doses
solCD39 inhibited both platelet and fibrin accumulation and
promoted an increase in postischemic blood flow (FIGS. 8a,b &
9a). A covariate plot of cerebral infarct volume vs. intracerebral
hemorrhage for each treatment indicates that aspirin is less
capable of reducing infarct volume and preventing intracerebral
hemorrhage than solCD39 treatment (FIG. 10).
Reconstitution of CD39-/- Mice with CD39
[0172] To further characterize the contributions of endogenous CD39
to hemostasis and thrombosis, CD39-/- mice were generated by a gene
targeting vector in which exons 4-6, encoding apyrase conserved
regions 2-4(24,26-28), were replaced with a PGKneo cassette (FIG.
11). Homozygous CD39-/- mice did not display an obvious phenotype
in the unperturbed state. Hematological profiles were normal,
including erythrocyte parameters, platelet counts, leukocyte counts
and differentials, and coagulation screening tests. Bleeding times
of CD39-/- mice were normal, in contrast to the markedly increased
bleeding time following aspirin treatment, and a dose-dependent
increase in bleeding time induced by solCD39 (FIG. 12).
[0173] We hypothesized that a latent prothrombotic phenotype could
be identified in a clinically relevant platelet-dependent stroke
model(2). Indeed, CD39-/- mice, subjected to focal cerebral
ischemia, did exhibit diminished blood flow following reperfusion
as compared to genetically matched controls (FIG. 13a). When
solCD39 (4 mg/kg) was administered to the CD39-/- mice, these mice
were "reconstituted" as shown by increased postischemic blood flow.
Furthermore, CD39-/- mice demonstrated increased cerebral
infarction volumes as compared to genotype matched controls
following induced stroke (FIG. 13b). CD39-/- mice "reconstituted"
with solCD39 had markedly diminished infarct volumes, similar to
untreated CD39+/+controls, indicating a protective effect of
solCD39. Other parameters (neurological deficit scores, overall
mortality, and intracerebral hemorrhage) did not differ between
groups (FIG. 13c, d, e). A covariate plot of infarct volume and
intracerebral hemorrhage revealed that the large infarcts in CD39
null mice were reduced by "reconstitution" with solCD39 to become
similar to those in CD39 treated animals with respect to infarct
volume and intracerebral hemorrhage (FIG. 10).
Discussion
[0174] Platelet and fibrin deposition downstream of an occlusive
lesion contribute significantly to the postischemic hypoperfusion
and tissue injury complicating stroke. Our results demonstrate for
the first time in vivo protection conferred by CD39 in this
platelet-dependent thrombotic disorder. SolCD39 improves cerebral
blood flow and reduces cerebral infarct volume when given
preoperatively. In addition, solCD39 confers significant
cerebroprotection when administered 3 h after onset of stroke.
Rendering cerebroprotection at this delayed time point is
significant because these effects occurred without an increase in
mortality or intracerebral hemorrhage. The CD39-/- mice had a
defect in thromboregulation in that they exhibited larger infarct
volumes than their genotype-matched controls. The CD39-/- mice were
"reconstituted" by administration of solCD39, thus fulfilling
Koch's postulates (29).
[0175] CD39 -/- mice generated by our group did not have an obvious
phenotype, with completely normal baseline hematological and
coagulation profiles. Very recently, a study was published which
reported development of a CD39 gene-deleted mouse(30). These mice
paradoxically demonstrated both thrombosis and hemorrhage, with
marked platelet dysfunction at baseline. This phenotype is in sharp
contrast with that observed in our CD39 -/- mice, described for the
first time here, in which the prothrombotic phenotype was
demonstrable after induction of stroke, but baseline hematologic
and hemostatic parameters (including platelet function) were
normal. Platelets from our CD39 -/- mice retain the ability to
aggregate in response to ADP (data not shown), and bleeding times
were normal. In contrast, platelets taken from Enjyoji et al. 's
CD39 gene-deleted mice exhibited hemorrhagic shock with tail vein
bleeding experiments, and ex vivo reactivity of platelets to ADP
was virtually absent. Our knockout strategy focused on elimination
of the enzymatically active extracellular domain (specifically,
apyrase conserved regions 2-4) of the CD39 gene. In contrast,
Enjyoji et al. Targeted the ATG start site and a portion of the 5'
UTR. Gene disruption may affect cell populations which may
secondarily affect phenotype. For instance, mild thrombocytopenia
was reported in the Enjyoji et al. 's CD39 gene-deleted mice(30).
Mice null for the protein P-selectin exhibit baseline
leukocytosis(31). In contrast, our mice with targeted deletion of
the enzymatically-active region of the CD39 gene exhibited both
normal platelet and leukocyte counts. Moreover, we did not observe
spontaneous thrombotic events, as reported in PAI-1 overexpressing
mice(32). Rather, our CD39-/- mice appear to exhibit a latent
prothrombotic phenotype, elicited by inducing a platelet-dependent
thrombotic disorder (stroke). We postulate that under basal
conditions, vascular homeostasis may be maintained by the
endothelial thromboregulators prostacyclin and nitric oxide(9).
However, a severe breach in vascular integrity leads to platelet
accumulation and consequent fibrin deposition in the absence of
CD39, as in the CD39-/- mice. Functional reconstitution of our CD39
-/- mice with solCD39 normalizes the phenotype, providing a
compelling case for deletion of CD39 activity as the basis for the
latent prothrombotic phenotype observed in our knockout mice.
[0176] Although aspirin may be of benefit in primary prevention of
stroke, it does not appear to be efficacious in evolving
stroke(25). Moreover, some patients obtain little benefit from
aspirin ("nonresponders"), even though it is efficacious in
others(11,33). GPIIb/IIIa antagonists are potent inhibitors of
platelet aggregation, since they block a final step in platelet
accumulation, i.e. fibrinogen bridging of surface glycoprotein GP
IIb/IIIa receptors, thus abrogating platelet-platelet adherence.
Although useful in prevention of intravascular thrombosis following
percutaneous coronary intervention, these agents have not been
widely studied specifically in the setting of acute stroke. One
highly specific GP IIb/IIIa antagonist, GPI-562, had potent
antithrombotic effects in experimental stroke, and did reduce
cerebral infarction volumes, but it was associated with
intracerebral hemorrhage(2). Other platelet inhibitors useful in
management of acute ischemic syndromes, such as ticlopidine or
clopidogrel(34), inhibit platelet aggregation mediated by the low
affinity P2Y1 ADP receptor-on the platelet surface(35). Our data
show that endogenous CD39 is protective in stroke, and that
administration of pharmacological doses of solCD39 is effective in
inhibiting thrombosis and tissue injury in stroke. Since solCD39
inhibits all ADP-mediated platelet aggregation via metabolic
deletion of ADP from the activated platelet releasate, it may be
more potent than the ADP-receptor blockers currently in use.
[0177] The basis for the apparent superiority of solCD39 to aspirin
may be that it induces more potent inhibition of ADP-induced
platelet aggregation. This latter mechanism is more efficient in
platelet-induced platelet recruitment than the
arachidonate/thromboxane pathway. Moreover, while platelet
reactivity to low-dose collagen is inhibited by solCD39, platelets
do respond to higher doses of collagen. In contrast, aspirin had
little effect on platelet reactivity at any collagen dose. The
hemostatic effects of agonist-induced pathways are likely to
overlap with considerable redundancy in vivo; however, our data
indicate that aspirin resulted in more bleeding in response to vein
injury, or stroke, than did solCD39. Perhaps the initial layer of
platelets that adheres to an injured vessel wall is essential for
hemostasis, but in stroke, ADP-mediated recruitment of platelets
into an evolving thrombus results in intravascular occlusion.
SolCD39 disaggregates platelets that have already responded to an
agonist, but it does not have a deleterious effect on primary
hemostasis.
[0178] We previously demonstrated that microvascular thrombosis is
a continuing phenomenon after the onset of stroke(2). Therefore,
this ongoing process can be modulated by therapeusis with solCD39,
even 3 h after stroke induction. Our data are especially pertinent
in the setting of clinical observations of increased intracerebral
hemorrhage when thrombolytic agents are administered beyond 3 h
following stroke onset(7). Thus, our results may constitute a
possible new approach to antithrombotic therapy, based upon
metabolism of a major agonist for vascular occlusion
platelet-released ADP.
[0179] Acknowledgements: We thank Steven D. Gimpel, R. Guy Caspary,
Louis J. Kim, and Alexander Coon for expert technical assistance.
This work was supported in part by a Merit Review grant from the
Department of Veterans Affairs, the National Institutes of Health
grants HL47073, HL-46403, and HL-07423 (AJM, MJB, JHFD), NS 02038
(ESC), HL-59488 and HL-55397 (DJP), a New York Academy of Medicine
Glomey-Raisbeck Medical Student Research Fellowship (RAM), and an
Established Investigator of the American Heart Association
(DJP).
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Sequence CWU 1
1
2 1 510 PRT HOMO SAPIENS 1 Met Glu Asp Thr Lys Glu Ser Asn Val Lys
Thr Phe Cys Ser Lys Asn 1 5 10 15 Ile Leu Ala Ile Leu Gly Phe Ser
Ser Ile Ile Ala Val Ile Ala Leu 20 25 30 Leu Ala Val Gly Leu Thr
Gln Asn Lys Ala Leu Pro Glu Asn Val Lys 35 40 45 Tyr Gly Ile Val
Leu Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile 50 55 60 Tyr Lys
Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val His Gln 65 70 75 80
Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln 85
90 95 Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg
Ala 100 105 110 Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu Thr Pro
Val Tyr Leu 115 120 125 Gly Ala Thr Ala Gly Met Arg Leu Leu Arg Met
Glu Ser Glu Glu Leu 130 135 140 Ala Asp Arg Val Leu Asp Val Val Glu
Arg Ser Leu Ser Asn Tyr Pro 145 150 155 160 Phe Asp Phe Gln Gly Ala
Arg Ile Ile Thr Gly Gln Glu Glu Gly Ala 165 170 175 Tyr Gly Trp Ile
Thr Ile Asn Tyr Leu Leu Gly Lys Phe Ser Gln Lys 180 185 190 Thr Arg
Trp Phe Ser Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr 195 200 205
Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val 210
215 220 Pro Gln Asn Gln Thr Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe
Arg 225 230 235 240 Leu Tyr Gly Lys Asp Tyr Asn Val Tyr Thr His Ser
Phe Leu Cys Tyr 245 250 255 Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu
Ala Lys Asp Ile Gln Val 260 265 270 Ala Ser Asn Glu Ile Leu Arg Asp
Pro Cys Phe His Pro Gly Tyr Lys 275 280 285 Lys Val Val Asn Val Ser
Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg 290 295 300 Phe Glu Met Thr
Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly 305 310 315 320 Asn
Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser 325 330
335 Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro
340 345 350 Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe Val
Met Lys 355 360 365 Phe Leu Asn Leu Thr Ser Glu Lys Val Ser Gln Glu
Lys Val Thr Glu 370 375 380 Met Met Lys Lys Phe Cys Ala Gln Pro Trp
Glu Glu Ile Lys Thr Ser 385 390 395 400 Tyr Ala Gly Val Lys Glu Lys
Tyr Leu Ser Glu Tyr Cys Phe Ser Gly 405 410 415 Thr Tyr Ile Leu Ser
Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp 420 425 430 Ser Trp Glu
His Ile His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala 435 440 445 Gly
Trp Thr Leu Gly Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala 450 455
460 Glu Gln Pro Leu Ser Thr Pro Leu Ser His Ser Thr Tyr Val Phe Leu
465 470 475 480 Met Val Leu Phe Ser Leu Val Leu Phe Thr Val Ala Ile
Ile Gly Leu 485 490 495 Leu Ile Phe His Lys Pro Ser Tyr Phe Trp Lys
Asp Met Val 500 505 510 2 439 PRT HOMO SAPIENS 2 Thr Gln Asn Lys
Ala Leu Pro Glu Asn Val Lys Tyr Gly Ile Val Leu 1 5 10 15 Asp Ala
Gly Ser Ser His Thr Ser Leu Tyr Ile Tyr Lys Trp Pro Ala 20 25 30
Glu Lys Glu Asn Asp Thr Gly Val Val His Gln Val Glu Glu Cys Arg 35
40 45 Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln Lys Val Asn Glu
Ile 50 55 60 Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala Arg Glu
Val Ile Pro 65 70 75 80 Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu
Gly Ala Thr Ala Gly 85 90 95 Met Arg Leu Leu Arg Met Glu Ser Glu
Glu Leu Ala Asp Arg Val Leu 100 105 110 Asp Val Val Glu Arg Ser Leu
Ser Asn Tyr Pro Phe Asp Phe Gln Gly 115 120 125 Ala Arg Ile Ile Thr
Gly Gln Glu Glu Gly Ala Tyr Gly Trp Ile Thr 130 135 140 Ile Asn Tyr
Leu Leu Gly Lys Phe Ser Gln Lys Thr Arg Trp Phe Ser 145 150 155 160
Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr Phe Gly Ala Leu Asp 165
170 175 Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val Pro Gln Asn Gln
Thr 180 185 190 Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg Leu Tyr
Gly Lys Asp 195 200 205 Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr
Gly Lys Asp Gln Ala 210 215 220 Leu Trp Gln Lys Leu Ala Lys Asp Ile
Gln Val Ala Ser Asn Glu Ile 225 230 235 240 Leu Arg Asp Pro Cys Phe
His Pro Gly Tyr Lys Lys Val Val Asn Val 245 250 255 Ser Asp Leu Tyr
Lys Thr Pro Cys Thr Lys Arg Phe Glu Met Thr Leu 260 265 270 Pro Phe
Gln Gln Phe Glu Ile Gln Gly Ile Gly Asn Tyr Gln Gln Cys 275 280 285
His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser Tyr Cys Pro Tyr Ser 290
295 300 Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro Leu Gln Gly Asp
Phe 305 310 315 320 Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys Phe
Leu Asn Leu Thr 325 330 335 Ser Glu Lys Val Ser Gln Glu Lys Val Thr
Glu Met Met Lys Lys Phe 340 345 350 Cys Ala Gln Pro Trp Glu Glu Ile
Lys Thr Ser Tyr Ala Gly Val Lys 355 360 365 Glu Lys Tyr Leu Ser Glu
Tyr Cys Phe Ser Gly Thr Tyr Ile Leu Ser 370 375 380 Leu Leu Leu Gln
Gly Tyr His Phe Thr Ala Asp Ser Trp Glu His Ile 385 390 395 400 His
Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala Gly Trp Thr Leu Gly 405 410
415 Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala Glu Gln Pro Leu Ser
420 425 430 Thr Pro Leu Ser His Ser Thr 435
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