U.S. patent application number 11/492159 was filed with the patent office on 2006-11-16 for decorin proteoglycan inhibitor of fibrinogen blood clotting.
Invention is credited to Tracey A. Dugan, Magnus Hook.
Application Number | 20060258570 11/492159 |
Document ID | / |
Family ID | 34115354 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060258570 |
Kind Code |
A1 |
Hook; Magnus ; et
al. |
November 16, 2006 |
Decorin proteoglycan inhibitor of fibrinogen blood clotting
Abstract
The present invention provides compositions and methods of
inhibiting fibrin(ogen) clot formation by utilizing decorin
proteoglycan as an anticoagulating and antithrombotic agent. The
decorin proteoglycan comprises a decorin core protein or a fragment
thereof covalently linked to a galactosaminoglycan polysaccharide.
The decorin core protein acts as an anticoagulant and as a carrier
for the delivery of an antithrombotic galactosaminoglycan to
fibrinogen. Fibrin clotting is inhibited by the decorin
proteoglycan in a concentration-dependent fashion.
Inventors: |
Hook; Magnus; (Houston,
TX) ; Dugan; Tracey A.; (Houston, TX) |
Correspondence
Address: |
Benjamin Aaron Adler;ADLER & ASSOCIATES
8011 Candle Lane
Houston
TX
77071
US
|
Family ID: |
34115354 |
Appl. No.: |
11/492159 |
Filed: |
July 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10898841 |
Jul 26, 2004 |
7094880 |
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11492159 |
Jul 24, 2006 |
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60490081 |
Jul 25, 2003 |
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Current U.S.
Class: |
514/9.4 ;
514/13.6; 514/14.7 |
Current CPC
Class: |
A61K 47/61 20170801;
A61K 38/1709 20130101; A61K 2300/00 20130101; C07K 14/4725
20130101; A61K 38/1709 20130101 |
Class at
Publication: |
514/008 |
International
Class: |
A61K 38/36 20060101
A61K038/36 |
Goverment Interests
FEDERAL FUNDING LEGEND
[0002] This invention was produced in part using funds obtained
through a National Institutes of Health grant AR42919.
Consequently, the federal government has certain rights in this
invention.
Claims
1. A method of inhibiting fibrin clot formation, comprising:
contacting fibrinogen in a biological sample with a recombinant
anticoagulating and antithrombotic decorin proteoglycan comprising
decorin core protein linked to a galactosaminoglycan polysaccharide
within an N-terminal sequence thereof.
2. The method of claim 1, wherein said galactosaminoglycan
polysaccharide is covalently linked thereto.
3. The method of claim 1, wherein said decorin proteoglycan
comprises a fibrinogen-binding domain.
4. The method of claim 4, wherein said N-terminus has the sequence
of SEQ ID NO. 1.
5. The method of claim 1, wherein said decorin core protein has the
sequence of SEQ ID NO. 2.
6. The method of claim 1, wherein said decorin proteoglycan is
chemically derivatived.
7. The method of claim 1, wherein said decorin core protein or
N-terminus thereof contains a conservative amino acid
substitution.
8. The method of claim 1, wherein said galactosaminoglycan
polysaccharide is heparin, heparan sulfate, chondroitin sulfate,
dermatan sulfate, or combinations thereof.
9. The method of claim 1, wherein said biological sample is a blood
sample.
10. The method of claim 9, wherein said decorin proteoglycan is
attached to the surface of a medical device or a polymeric
biomaterial.
11. A method of inhibiting fibrin clot formation in a subject,
comprising: administering to said subject a pharmacologically
effective amount of a recombinant anticoagulating and
antithrombotic decorin proteoglycan comprising decorin core protein
or a fragment thereof linked to a galactosaminoglycan
polysaccharide.
12. The method of claim 11, wherein said galactosaminoglycan
polysaccharide is covalently linked thereto.
13. The method of claim 11, wherein said decorin proteoglycan
comprises a fibrinogen-binding domain.
14. The method of claim 11, wherein said N-terminus has the
sequence of SEQ ID NO. 1.
15. The method of claim 11, wherein said decorin core protein has
the sequence of SEQ ID NO. 2.
16. The method of claim 11, wherein said decorin proteoglycan is
chemically derivatived.
17. The method of claim 11, wherein said decorin core protein or
N-terminus thereof contains a conservative amino acid
substitution.
18. The method of claim 11, wherein said galactosaminoglycan
polysaccharide is heparin, heparan sulfate, chondroitin sulfate,
dermatan sulfate, or combinations thereof.
19. The method of claim 11, wherein said subject is an animal or a
human.
20. The method of claim 11, wherein said decorin proteoglycan is
attached to the surface of a medical device or a polymeric
biomaterial.
21. The method of claim 11, wherein said decorin proteoglycan is
administered in a dosage of about 0.01 mg/kg to about 100 mg/kg of
the subject's body weight.
22. The method of claim 11, wherein said decorin proteoglycan is
administered topically, orally, intravenously, or
intraperitoneally.
23. The method of claim 11, wherein said decorin proteoglycan is
formulated as an ointment, a cream, a gel, a lotion, or wound
dressings.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a divisional of non-provisional application U.S.
Ser. No. 10/898,841, filed Jul. 26, 2004, which claims benefit of
provisional U.S. Ser. No. 60/490,081, filed Jul. 25, 2003, now
abandoned.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the field of
blood clotting research. More specifically, the present invention
provides methods of using decorin proteoglycan as an
anticoagulating and/or antithrombotic agent to inhibit fibrin(ogen)
clot formation.
[0005] 2. Description of the Related Art
[0006] Decorin is composed of a 40 kDa core protein and a
glycosaminoglycan chain. Many glycosaminoglycans affect blood
coagulation activity. For example, heparin and heparan sulfate have
been used as efficient anticoagulants clinically for decades. These
polysaccharides indirectly inhibit thrombin by activating serpins.
Heparin activates antithrombin III, the major physiological
regulator of thrombin, as well as heparin cofactor II. Both
heparin-induced thrombocytopenia and hemorrhage are side effects to
the use of heparin as an antithrombotic agent.
[0007] Although heparin/antithrombin III complexes efficiently
inhibit soluble thrombin, they only weakly inhibit thrombin bound
to surfaces such as membranes or a fibrin(ogen) clot. Fibrin-bound
thrombin represents a reservoir of active thrombin that can
exacerbate both venous and arterial thrombosis by generating more
fibrin locally or when released by fibrinolysis. Fibrin-bound
thrombin may also activate factor XIII, the transglutaminase that
crosslinks fibrin and thrombin-activatable fibrinolysis
inhibitor.
[0008] Fibrin-bound thrombin is inaccessible to heparin/heparin
cofactor II inactivation, but it is susceptible to dermatan
sulfate/heparin cofactor II inactivation. As opposed to heparin,
dermatan sulfate specifically activates heparin cofactor II
inhibition of thrombin. However, hemorrhage is also a side effect
of utilizing dermatan sulfate as an antithrombotic agent.
[0009] Accordingly, there is a need in the art for anticoagulating
and antithrombotic agents with high activity and low incidents of
side effects. The present invention fulfills this longstanding
need.
SUMMARY OF THE INVENTION
[0010] The present invention provides compositions and methods of
utilizing decorin proteoglycan to inhibit thrombin-induced
fibrin(ogen) clotting. Antithrombotic dermatan sulfate or dermatan
sulfate/heparin cofactor II complex is specifically targeted to
fibrin(ogen), an important site of thrombin action and a reservoir
of active thrombin.
[0011] The anticoagulating and antithrombotic decorin proteoglycan
of the present invention comprise (i) a decorin core protein or a
fragment thereof and (ii) a galactosaminoglycan polysaccharide. In
one embodiment, the decorin core protein or a fragment thereof is
covalently linked to the galactosaminoglycan polysaccharide. In one
specific embodiment, the decorin core protein fragment comprises
the N-terminus of the decorin core protein. According to one
particular embodiment, the decorin core protein fragment is a 45
amino acid residue recombinant Zn.sup.2+-binding peptide (SEQ ID
NO: 2) mimicking the N-terminal segment of decorin (SEQ ID NO: 1).
Examples of galactosaminoglycan polysaccharides include heparin,
heparan sulfate, dermatan sulfate, chondroitin sulfate, or a
mixture of these galactosaminoglycan polysaccharides.
[0012] In another embodiment, there is provided an anticoagulating
and antithrombotic composition comprising the decorin proteoglycan
disclosed herein and a pharmaceutically acceptable carrier. This
anticoagulating and antithrombotic composition can be incorporated
into a kit for inhibiting fibrin clot formation.
[0013] In yet another embodiment, there is provided a method of
inhibiting fibrin clot formation, comprising the step of contacting
fibrinogen with the decorin proteoglycan disclosed herein. In one
embodiment, the decorin proteoglycan is attached to the surface of
a medical device or a polymeric biomaterial.
[0014] In yet another embodiment, there is provided a method of
inhibiting fibrin clot formation in a subject such as an animal or
a human, comprising the step of administering the decorin
proteoglycan disclosed herein to said subject.
[0015] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention. These
embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The appended drawings have been included herein so that the
above-recited features, advantages and objects of the invention
will become clear and can be understood in detail. These drawings
form a part of the specification. It is to be noted, however, that
the appended drawings illustrate preferred embodiments of the
invention and should not be considered to limit the scope of the
invention.
[0017] FIG. 1 shows the concentration-dependent effects of decorin
on the progress of fibrin clotting. Two .mu.M fibrinogen (Fg) was
pre-incubated alone for 4 hours at ambient temperature in HBS, 0.1%
CHAPS, 20 .mu.M ZnSO.sub.4 or with increasing concentrations of
decorin proteoglycan (DcnPg). Clotting was initiated by the
addition of thrombin to a final concentration of 0.25 U/ml and
monitored by absorbance at 405 nm. This example shows that
contacting fibrinogen with increasing concentrations of decorin
proteoglycan prior to introducing thrombin slows the progress of
fibrin clotting in a concentration-dependent fashion.
[0018] FIGS. 2A-2B show Zn.sup.2+-dependent effect of decorin on
fibrin clotting. Two .mu.M fibrinogen was pre-incubated in HBS,
0.1% CHAPS containing either 0.2 .mu.M, 20 .mu.M, or 100 .mu.M
ZnSO.sub.4. Decorin proteoglycan (DcnPg) (0.4 .mu.M) derived from
cultured mammalian cells (FIG. 2A), or 2 .mu.M decorin core protein
(Dcn) expressed by vaccinia virus-infected mammalian cell line
CHO745 that lacks xylosyl transferase activity (FIG. 2B) were also
added to the medium. Time course of fibrin clotting was monitored
spectroscopically after adding thrombin to a final concentration of
0.25 U/ml.
[0019] FIGS. 3A-3B show the comparison of inhibitory action of
decorin proteoglycan and heparin cofactor II towards thrombin
cleavage of fibrinogen or a chromogenic substrate. Two .mu.M
fibrinogen was pre-incubated in HBS, 0.1% CHAPS, 20 .mu.M
ZnSO.sub.4 with or without 2.5 U/ml heparin cofactor II (HCII); 0.4
.mu.M decorin proteoglycan (DcnPg) expressed by mammalian cells
capable of producing dermatan sulfate; 0.4 .mu.M decorin
proteoglycan plus 2.5 U/ml heparin cofactor II; 0.4 .mu.M
chondroitin sulfate B (CSB) plus 2.5 U/ml HCII; 0.4 .mu.M
chondroitin sulfate A (CSA) plus 2.5 U/ml HCII (FIG. 3A); or 2.5
U/ml hirudin or 0.6 mM chromogenic thrombin substrate S-2238 (FIG.
3B). After adding thrombin to 0.25 U/ml, the progress of each
reaction was traced by the absorbance at 405 nm which detects
fibrin clotting (FIG. 3A) or p-nitroaniline release (FIG. 3B).
[0020] FIG. 4 shows the concentration-dependent effects of decorin
core protein (decorin) on fibrin clotting. Two .mu.M fibrinogen was
pre-incubated for 1.5 hours at ambient temperature in HBS, 0.1%
CHAPS, 20 uM ZnCl.sub.2, 5 mM CaCl.sub.2, 5 mM EACA, pH 7.4 with or
without increasing concentrations of decorin core protein (decorin)
expressed by CHO745 cells. All subsequent steps were performed as
described above.
[0021] FIGS. 5A-5C show decorin core protein alters the structure
of a fibrin(ogen) clot. Fibrin(ogen) clotting was induced by the
addition of thrombin (final concentration 0.4 U/ml) to fibrinogen
pre-incubated in the absence or presence of decorin core protein
under the solution conditions described in FIG. 4. Scanning
electron microscopy was utilized to obtain images of clots formed
from 1.2 mM fibrin(ogen) alone (FIG. 5A) or in the presence of 0.24
mM (FIG. 5B) or 1.2 mM (FIG. 5C) decorin core protein.
(16,000.times. magnification).
[0022] FIG. 6 shows the concentration-dependent effects of a 45
amino acid residue peptide mimetic of the N-terminal region of
decorin core protein (DcnNTD) on fibrin clotting. Clotting assays
were performed as described above in FIG. 4.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides a method of inhibiting
fibrin(ogen) clot formation by utilizing decorin proteoglycan as an
anticoagulating and antithrombotic agent. In one embodiment,
decorin proteoglycan consists of decorin core protein and a
covalently attached antithrombotic galactosaminoglycan
polysaccharide. A method of inhibiting fibrin clotting using
decorin core protein, or related peptides, which act as an
anticoagulating agents has been described in U.S. Pat. No.
6,413,931, the entire content of which is incorporated herein by
reference. Likewise, dermatan sulfate polysaccharide has been
utilized as an effective antithrombotic agent both in vivo and in
vitro. Decorin proteoglycan of the present invention, which is a
combination of decorin core protein and an attached antithrombotic
galactosaminoglycan polysaccharide, is an improvement over either
decorin core protein or dermatan sulfate alone.
[0024] Features that contribute to the mode of action of the
present decorin proteoglycan derive from each structural component
and include: 1) the fibrinogen-binding structure of the decorin
core protein and 2) the galactosaminoglycan composition of the
polysaccharide. Decorin-fibrinogen binding is mediated by the
N-terminal segment of the core protein, a segment that forms
multimers in the presence of zinc (Dugan et al., 2003). The
N-terminal segment of decorin core protein binds zinc at
physiological concentrations (Yang et al., 1999). Furthermore, both
decorin proteoglycan and decorin core protein slow the progress of
fibrin clotting in a zinc-dependent fashion and at physiological
zinc concentrations.
[0025] The galactosaminoglycan component of decorin proteoglycan
may consist of chondroitin sulfate or dermatan sulfate, or a
mixture thereof, depending upon the cell-type in which it is
expressed. Acting alone, chondroitin sulfate polysaccharide
promotes fibrin clotting, while dermatan sulfate polysaccharide
indirectly inhibits thrombin through heparin cofactor II
activation. Dermatan sulfate-containing galactosaminoglycans
isolated from decorin as well as type V collagen-bound decorin
proteoglycan enhance the inhibitory activity of heparin cofactor II
toward thrombin cleavage of a chromogenic substrate. Decorin
proteoglycan according to the present invention enhances heparin
cofactor II inhibition of thrombin-induced fibrin(ogen) clotting.
Significantly, decorin proteoglycan is a more potent enhancer than
dermatan sulfate polysaccharide. Accordingly, decorin proteoglycan
produced in HEK 293 cells contains an antithrombotic component.
Taken together, the decorin core protein acts as an anticoagulant
and as a carrier for the delivery of an antithrombotic
galactosaminoglycan to fibrinogen.
[0026] The present invention provides several methods and related
biological compositions involved in the inhibition of fibrin(ogen)
clot formation. The invention provides a method of inhibiting
fibrin clot formation, comprising contacting fibrinogen with a
decorin proteoglycan composition. As used herein, the term
"inhibiting clot formation" refers to any prolonging in the time of
clot formation or any reduction in the extent of clot formation as
compared to control conditions in the absence of decorin
proteoglycan composition.
[0027] In one aspect of the present invention, there is provided an
anticoagulating and antithrombotic decorin proteoglycan comprising
decorin core protein or a fragment thereof and a
galactosaminoglycan polysaccharide. In one embodiment, the decorin
core protein or a fragment thereof is covalently linked to the
galactosaminoglycan polysaccharide. Preferably, the decorin core
protein or a fragment thereof comprises a fibrinogen-binding
domain. In general, the decorin core protein fragment comprises the
N-terminus (SEQ ID NO: 1), or fragments or derivatives of SEQ ID
NO: 1, of the decorin core protein. In one embodiment, the decorin
core protein fragment has the sequence of SEQ ID NO: 2 or or
fragments or derivatives thereof. Representative examples of
galactosaminoglycan polysaccharides contained in the present
decorin proteoglycan include heparin, heparan sulfate, chondroitin
sulfate, dermatan sulfate, or a mixtures of these or other
galactosaminoglycan polysaccharides.
[0028] The present invention also provides a kit for inhibiting
fibrin clot formation, comprising the present decorin proteoglycan
which is dispersed in a pharmaceutically acceptable carrier.
[0029] In another embodiment, there is provided a method of
inhibiting fibrin clot formation, comprising the step of contacting
fibrinogen with a decorin proteoglycan composition effective to
inhibit fibrin clot formation in a sample. In general, the fibrin
clot formation takes place in a biological sample such as a blood
sample. In another embodiment, the decorin proteoglycan is attached
to the surface of a medical device or a polymeric biomaterial.
[0030] In yet another embodiment, there is provided a method of
inhibiting fibrin clot formation in a subject such as an animal or
a human, comprising the step of providing to the subject a decorin
proteoglycan composition effective to inhibit fibrin clot
formation. In one embodiment, the decorin proteoglycan is attached
to the surface of a medical device or a polymeric biomaterial.
[0031] It should be noted that all amino-acid residue sequences are
represented herein by formulae whose left and right orientation is
in the conventional direction of amino-terminus to
carboxy-terminus. Furthermore, it should be noted that a dash at
the beginning or end of an amino acid residue sequence indicates a
peptide bond to a further sequence of one or more amino-acid
residues.
[0032] The amino acids described herein are preferred to be in the
"L" isomeric form. However, residues in the "D" isomeric form can
be substituted for any L-amino acid residue, as long as the desired
functional property of immunoglobulin binding is retained by the
polypeptide. NH.sub.2 refers to the free amino group present at the
amino terminus of a polypeptide. COOH refers to the free carboxy
group present at the carboxy terminus of a polypeptide. In keeping
with standard polypeptide nomenclature, J Biol. Chem., 243:3552-59
(1969), abbreviations for amino acid residues are known in the
art.
[0033] Nonstandard amino acids may be incorporated into proteins by
chemical modification of existing amino acids or by de novo
synthesis of a protein/peptide. A nonstandard amino acid refers to
an amino acid that differs in chemical structure from the twenty
standard amino acids encoded by the genetic code.
Post-translational modification in vivo can also lead to the
presence of a nonstandard or amino acid derivative in a protein.
The N-terminal NH.sub.2 and C-terminal COOH groups of a protein can
also be modified, for example, by natural or artificial
post-translational modification of a protein.
[0034] Proteins/peptides, such as the decorin core protein or
fragments thereof, may be modified by amino acids substitutions.
Often, some changes result in significant changes in the activity
(agonists versus antagonists) and potency/affinity of
proteins/peptides while others have little or no effect.
Conservative substitutions are least likely to drastically alter
the activity of a protein. A "conservative amino acid substitution"
refers to replacement of amino acid with a chemically similar amino
acid, i.e. replacing nonpolar amino acids with other nonpolar amino
acids; substitution of polar amino acids with other polar amino
acids, acidic residues with other acidic amino acids, etc. Examples
of preferred conservative substitutions are set forth in Table I:
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions
Original Preferred Conservative Most Preferred Residue
Substitutions Conservative Substitution Ala (A) Val; Leu; Ile Val
Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg, Ser Gln Asp
(D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G)
Pro, Ala, DAla Pro His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Nle Leu Leu (L) Ile; Val; Met; Ala; Phe; Nle Ile Lys
(K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile, Nle Leu Phe (F) Leu;
Val; Ile; Ala Leu Pro (P) Gly, Sar Gly Ser (S) Thr Thr Thr (T) Ser
Ser Trp (W) Tyr, Nal, Cpa Tyr Tyr (Y) Trp; Phe; Thr; Ser, His Phe
Val (V) Ile; Leu; Met; Phe; Ala; Nle Leu Sar = sarcasine, Nal =
naphthylalanine, Cpa = 4-chloro-phenylalanine
[0035] "Chemical derivative" refers to a subject polypeptide, such
as the decorin core protein or fragments thereof, having one or
more residues chemically derivatized by reaction of a functional
side group. Such derivatized polypeptides include, for example,
those in which free amino groups have been derivatized to form
specific salts or derivatized by alkylation and/or acylation,
p-toluene sulfonyl groups, carbobenzoxy groups, t-butylocycarbonyl
groups, chloroacetyl groups, formyl or acetyl groups among others.
Free carboxyl groups may be derivatized to form organic or
inorganic salts, methyl and ethyl esters or other types of esters
or hydrazides and preferably amides (primary or secondary).
Chemical derivatives may include those peptides which contain one
or more naturally occurring amino acids derivatives of the twenty
standard amino acids. For example, 4-hydroxyproline may be
substituted for serine; and ornithine may be substituted for
lysine. Peptides embraced by the present invention also include
peptides having one or more residue additions and/or deletions
relative to the specific peptide whose sequence is shown herein, so
long as the modified peptide maintains the requisite biological
activity.
[0036] The preferred dose for human administration can be
determined based on the needs of the individual patient and the
nature of the disorder being treated. The dose should be adjusted
to suit the individual to whom the composition is administered and
will vary with age, weight and metabolism of the individual. In
general, decorin proteoglycan is administered in a dosage range of
from about 0.01 mg/kg to about 100 mg/kg of the individual's body
weight.
[0037] Suitable methods of administration of any pharmaceutical
composition disclosed in this application include, but are not
limited to, topical, oral, intravenous, and intraperitoneal
administration.
[0038] For topical administration, the composition can be
formulated in the form of an ointment, cream, gel, or lotion. Wound
or surgical dressings, or sutures may be impregnated with the
composition. The composition may contain conventional additives,
such as preservatives or solvents to promote penetration and
emollients. Topical formulations may also contain conventional
carriers such as cream or ointment bases, ethanol, or oleyl
alcohol.
[0039] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion. The present
examples, along with the methods, procedures, treatments,
molecules, and specific compounds described herein are presently
representative of preferred embodiments. One skilled in the art
will appreciate readily that the present invention is well adapted
to carry out the objects and obtain the ends and advantages
mentioned, as well as those objects, ends and advantages inherent
herein. Changes therein and other uses which are encompassed within
the spirit of the invention as defined by the scope of the claims
will occur to those skilled in the art.
EXAMPLE 1
Concentration-Dependent Effect of Decorin Proteoglycan On The
Progress of Fibrin Clotting
[0040] Two .mu.M fibrinogen was pre-incubated alone for 4 hours at
ambient temperature in HBS, 0.1% CHAPS, 20 .mu.M ZnSO.sub.4 or with
increasing concentrations of decorin proteoglycan derived from
transfected mammalian cells. Subsequently, aliquots of each
pre-incubation mixture were dispensed into a microliter plate.
Clotting was initiated by the addition of thrombin to a final
concentration of 0.25 U/ml and monitored by absorbance at 405
nm.
[0041] FIG. 1 shows that contacting fibrinogen with increasing
concentrations of decorin proteoglycan prior to introducing
thrombin slows the progress of fibrin clotting in a
concentration-dependent fashion.
EXAMPLE 2
Zn.sup.2+-Dependent Effect of Decorin Proteoglycan On Fibrin
Clotting
[0042] Two .mu.M fibrinogen was pre-incubated in HBS, 0.1% CHAPS
containing either 0.2 .mu.M, 20 .mu.M, or 100 .mu.M ZnSO.sub.4.
Decorin proteoglycan (DcnPg) (0.4 .mu.M) derived from cultured
mammalian cells (FIG. 2A), or 2 .mu.M decorin core protein (Dcn)
expressed by vaccinia virus-infected mammalian cell line CHO745
that lacks xylosyl transferase activity (FIG. 2B) were also added
to the medium. Time course of fibrin clotting was monitored
spectroscopically after adding thrombin to a final concentration of
0.25 U/ml. This example demonstrates that Zn.sup.2+ enhances fibrin
clotting in the presence or absence of decorin proteoglycan or
decorin core protein. The results also show that contacting
fibrinogen with a sufficient concentration of decorin proteoglycan
in the presence of physiological levels of zinc will inhibit
fibrin(ogen) clotting in vitro.
EXAMPLE 3
Comparison of The Inhibitory Action of Decorin Proteoglycan And
Heparin Cofactor II Towards Thrombin Cleavage of Fibrinogen
[0043] Two .mu.M fibrinogen was pre-incubated in HBS, 0.1% CHAPS,
20 .mu.M ZnSO.sub.4 with or without 2.5 U/ml heparin cofactor II
(HCII); 0.4 .mu.M decorin proteoglycan (DcnPg) expressed by
mammalian cells capable of producing dermatan sulfate; 0.4 .mu.M
decorin proteoglycan plus 2.5 U/ml HCII; 0.4 .mu.M chondroitin
sulfate B (CSB) plus 2.5 U/ml HCII; 0.4 .mu.M chondroitin sulfate A
(CSA) plus 2.5 U/ml HCII (FIG. 3A); or 2.5 U/ml hirudin or 0.6 mM
chromogenic thrombin substrate S-2238 (FIG. 3B). After adding
thrombin to 0.25 U/ml, the progress of each reaction was traced by
the absorbance at 405 nm which detects fibrin clotting (FIG. 3A) or
p-nitroaniline release (FIG. 3B).
[0044] As shown in FIG. 3A, the combination of decorin proteoglycan
with heparin cofactor II inhibits clotting more potently than
either compound alone and also more potently than dermatan sulfate
with heparin cofactor II. According to FIG. 3B, the polysaccharide
component of decorin may not be rich in dermatan sulfate since the
effect of decorin on heparin cofactor II inhibition of thrombin was
minimal in a system where thrombin cleaves a chromogenic substrate.
These findings show that the potency of decorin proteoglycan
derives from the ability of the decorin core protein to bind
fibrinogen and target the antithrombotic polysaccharide and perhaps
heparin cofactor II to the site of thrombin action.
EXAMPLE 4
Concentration-Dependent Effects of Decorin Core Protein On Fibrin
Clotting
[0045] Two .mu.M fibrinogen was pre-incubated for 1.5 hours at
ambient temperature in HBS, 0.1% CHAPS, 20 uM ZnCl.sub.2, 5 mM
CaCl.sub.2, 5 mM EACA, pH 7.4 with or without increasing
concentrations of decorin core protein expressed by CHO745 cells, a
recombinant vaccinia virus-infected mutant CHO cell line lacking
xylosyl-transferase activity. All subsequent steps were performed
as described under Example 1.
[0046] The results show that contacting fibrinogen with increasing
concentrations of decorin core protein prior to introducing
thrombin slows the progress of fibrin(ogen) clotting and alters the
structure of the clot in a concentration-dependent fashion (FIG.
4). The progress of clotting and final clot structure are described
quantitatively as the rate and final absorbance values,
respectively (Table 2). TABLE-US-00002 TABLE 2 Thrombin-Induced
Clotting of Buffered Fibrin(ogen) in the Absence or Presence of
Decorin Core Protein [Decorin] mM Rate.sup.a (mAbs/min) Final
Abs.sup.a (405 nm) 0 90.0 .+-. 4.3 0.206 .+-. 0.005 0.2 61.8 .+-.
4.3 (p < 0.01) 0.160 .+-. 0.004 (p < 0.01) 0.4 55.0 .+-. 3.0
(p < 0.002) 0.145 .+-. 0.003 (p < 0.002) 1.0 38.8 .+-. 4.3 (p
< 0.0003) 0.120 .+-. 0.002 (p < 0.0003) 2.0 34.5 .+-. 2.5 (p
< 0.0006) 0.113 .+-. 0.001 (p < 0.0006) .sup.aValues in each
column represent the mean (n = 4). p values indicate a
statistically significant difference when compared to fibrinogen
alone.
EXAMPLE 5
Decorin Core Protein Alters The Structure of Fibrin(ogen) Clot
[0047] Fibrin(ogen) clotting was induced by the addition of
thrombin (final concentration 0.4 U/ml) to fibrinogen pre-incubated
in the absence or presence of decorin core protein under the
solution conditions described in Example 4. Scanning electron
microscopy was utilized to obtain images of clots formed from 1.2
mM fibrin(ogen) alone (FIG. 5A) or in the presence of 0.24 mM (FIG.
5B) or 1.2 mM (FIG. 5C) decorin core protein. The results show that
contacting fibrinogen with decorin core protein prior to
introducing thrombin results in an altered fibrin(ogen) clot
structure. Fibrin(ogen) fibril growth is attenuated by decorin in a
concentration-dependent fashion.
EXAMPLE6
Concentration-Dependent Effects of A 45 Amino Acid Residue Peptide
Mimetic of The N-Terminal Region of Decorin Core Protein On Fibrin
Clotting
[0048] Clotting assays were performed in the absence or presence of
a 45 amino acid residue peptide mimetic of the N-terminal region of
decorin core protein (DcnNTD). Assay conditions were the same as
described above. The results show that contacting fibrinogen with
increasing concentrations of the 45 amino acid residue peptide
mimetic of the N-terminal region of decorin core protein prior to
introducing thrombin slows the progress of fibrin(ogen) clotting
and alters the structure of the clot in a concentration-dependent
fashion (FIG. 6). The progress of clotting and final clot structure
are described quantitatively as the rate and final absorbance
values, respectively (Table 3). TABLE-US-00003 TABLE 3
Thrombin-Induced Clotting of Buffered Fibrin(ogen) in the Absence
or Presence of Decorin Peptide (DcnNTD) [DcnNTD] mM Rate.sup.a
(mAbs/min) Final Abs.sup.a (405 nm) 0 110 .+-. 6 0.24 .+-. 0.01 2.0
102 .+-. 3 0.23 .+-. 0.01 6.0 91 .+-. 3 0.22 .+-. 0.01 (p <
0.03) 12 80 .+-. 2 0.213 .+-. 0.002 (p < 0.05) (p < 0.01) 30
70 .+-. 3 0.197 .+-. 0.005 (p < 0.002) (p < 0.01)
.sup.aValues in a column represent the mean (n = 4). p values
indicate a statistically significant difference when compared to
fibrinogen alone.
[0049] The following references were cited herein: [0050] Dugan et
al., J. Biol. Chem. 278:13655-13662 (2003). [0051] Yang et al., J.
Biol. Chem. 274:12454-12460 (1999).
[0052] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually
incorporated by reference.
[0053] One skilled in the art will appreciate readily that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those objects,
ends and advantages inherent herein. Changes therein and other uses
which are encompassed within the spirit of the invention as defined
by the scope of the claims will occur to those skilled in the art.
Sequence CWU 1
1
2 1 41 PRT Artificial Sequence DOMAIN N-terminus region of decorin
core protein 1 Asp Glu Ala Ser Gly Ile Ile Pro Tyr Asp Pro Asp Asn
Pro Leu 5 10 15 Ile Ser Met Cys Pro Tyr Arg Cys Gln Cys His Leu Arg
Val Val 20 25 30 Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro 35 40
2 45 PRT Artificial Sequence DOMAIN recombinant decorin core
protein N-terminus sequence 2 Gly Ser Asn Gly Asp Glu Ala Ser Gly
Ile Ile Pro Tyr Asp Pro 5 10 15 Asp Asn Pro Leu Ile Ser Met Cys Pro
Tyr Arg Cys Gln Cys His 20 25 30 Leu Arg Val Val Gln Cys Ser Asp
Leu Gly Leu Asp Lys Val Pro 35 40 45
* * * * *