U.S. patent application number 12/254940 was filed with the patent office on 2009-06-11 for platelet glycoprotein ib alpha fusion polypeptides and methods of use thereof.
This patent application is currently assigned to Genetics Institute, LLC. Invention is credited to Ravindra Kumar, Tom McDonagh, Dianne S. Sako, Gray D. Shaw, Francis Sullivan.
Application Number | 20090148427 12/254940 |
Document ID | / |
Family ID | 23016192 |
Filed Date | 2009-06-11 |
United States Patent
Application |
20090148427 |
Kind Code |
A1 |
Shaw; Gray D. ; et
al. |
June 11, 2009 |
PLATELET GLYCOPROTEIN IB ALPHA FUSION POLYPEPTIDES AND METHODS OF
USE THEREOF
Abstract
The present invention provides compositions and methods for
treating or preventing vascular-associated disorders.
Inventors: |
Shaw; Gray D.; (Milton,
MA) ; Sako; Dianne S.; (Medford, MA) ; Kumar;
Ravindra; (Acton, MA) ; Sullivan; Francis;
(Belmont, MA) ; McDonagh; Tom; (Acton,
MA) |
Correspondence
Address: |
Ivor R. Elrifi;MINTZ, LEVIN, COHN, FERRIS,
GLOVSKY AND POPEO, P.C., One Financial Center
Boston
MA
02111
US
|
Assignee: |
Genetics Institute, LLC
Cambridge
MA
|
Family ID: |
23016192 |
Appl. No.: |
12/254940 |
Filed: |
October 21, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11257581 |
Oct 24, 2005 |
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12254940 |
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10068426 |
Feb 6, 2002 |
6991796 |
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11257581 |
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60266838 |
Feb 6, 2001 |
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Current U.S.
Class: |
424/94.1 ;
514/1.1 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61P 11/04 20180101; A61P 9/00 20180101; C07K 14/705 20130101; C07K
2319/00 20130101; A61P 7/02 20180101; A61P 9/10 20180101 |
Class at
Publication: |
424/94.1 ;
514/12 |
International
Class: |
A61K 38/43 20060101
A61K038/43; A61K 38/16 20060101 A61K038/16 |
Claims
1-53. (canceled)
54. A method for treating a disorder associated with platelet
activation in a subject, the method comprising administering to a
subject in need thereof a polypeptide comprising the amino acid
sequence of SEQ ID NO:5.
55. A method for treating venous or coronary artery thrombosis in a
subject in need thereof comprising administering to the subject a
polypeptide comprising the amino acid sequence of SEQ ID NO:5.
56. The method of claim 54, wherein the disorder is selected from
the group consisting of restenosis, ischemic heart disease, angina,
unstable angina, atherosclerosis, acute myocardial infarction, or
vascular inflammation.
57. The method of claim 56, wherein the disorder is restenosis.
58. The method of claim 56, wherein the disorder is angina.
59. The method of claim 58, wherein the disorder is unstable
angina.
60. The method of claim 56, wherein the disorder is ischemic heart
disease.
61. The method of claim 56, wherein the disorder is
atherosclerosis.
62. The method of claim 56, wherein the disorder is acute
myocardial infarction.
63. The method of claim 56, wherein the disorder is vascular
inflammation.
64. The method of claim 54, wherein the method further comprises
administering a compound selected from the group consisting of
acetylsalicylic acid, heparin, and clopidogrel.
65. The method of claim 55, wherein the method further comprises
administering a compound selected from the group consisting of
acetylsalicylic acid, heparin, and clopidogrel.
66. The method of claim 54, wherein the method further comprises
administering a glycoprotein IIb/IIIa antagonist, a P-selectin
antagonist, a thrombin inhibitor, or a thrombolytic enzyme.
67. The method of claim 55, wherein the method further comprises
administering a glycoprotein IIb/IIIa antagonist, a P-selectin
antagonist, a thrombin inhibitor, or a thrombolytic enzyme.
68. The method of claim 54, wherein the subject is a mammal.
69. The method of claim 55, wherein the subject is a mammal.
70. The method of claim 54, wherein the subject is human.
71. The method of claim 55, wherein the subject is human.
72. The method of claim 54, wherein the polypeptide is a dimer.
73. The method of claim 55, wherein the polypeptide is a dimer.
Description
RELATED U.S. APPLICATION
[0001] The present application claims the benefit of priority from
U.S. application Ser. No. 11/257,581, filed Oct. 24, 2005, which is
a divisional of U.S. Ser. No. 10/068,426, filed Feb. 6, 2002, now
U.S. Pat. No. 6,991,796, which claims the benefit of U.S.
Provisional Application No. 60/266,838, filed Feb. 6, 2001, each of
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates generally to compositions and methods
for treating or preventing vascular-associated disorders and more
particularly to compositions including platelet glycoprotein
IB.alpha.-derived polypeptides and methods of using same.
BACKGROUND OF THE INVENTION
[0003] The deleterious effects of vascular-associated disorders
such as stroke, heart attack, and artheroseclerosis are thought to
be caused, at least in part, by the inappropriate triggering of a
vascular inflammation and repair response. The vascular
inflammation and repair response involves adhesive interactions
between various cell types normally found freely circulating in
blood. Examples of such interactions the interaction that can occur
between platelets, leukocytes and the inner wall of blood vessels
(i.e., the vascular endothelium). Under conditions of high fluid
shear forces, platelets adhere to the endothelium via an
interaction between the glycoprotein (GP) Ib-IX-V complex on their
surface and von Willebrand factor (vWF) present on exposed vessel
subendothelium. In contrast, leukocytes can adhere either directly
to activated endothelium or indirectly by first adhering to
vWF-immobilized platelets. In both instances, leukocyte cell
surface molecules that bind to either the selectins or integrins
classes of adhesion receptors mediate these adhesion events.
Leukocyte-platelet adhesion is thought to occur, in part, via
interaction of the leukocyte surface integrin molecule, MacI and
the GP1b component of the platelet surface GPIb-IX-V complex.
[0004] In response to vascular disturbances such as
artherosclerotic plaque rupture or mechanical injury, e.g., such as
that caused by angioplasty, stent placement, ischemic damage or
stenosis, leukocytes and platelets can accumulate at a vascular
lesion site and provide multiple adhesive substrates for one
another. This accumulation of leukocytes and platelets lead to the
local production of factors including, e.g., mitogens, cytokines
and chemokines, causing the further undesirable progression of a
vascular disease.
SUMMARY OF THE INVENTION
[0005] The invention is based in part on the discovery of
glycoprotein-Ib.alpha.-derived fusion proteins that inhibit the
adherence of platelets to leukocytes. Accordingly, the
glycoprotein-Ib.alpha.-derived fusion proteins can be used to treat
vascular conditions associated with vascular inflammation,
thrombosis, atherosclerosis, and angioplasty-related restenosis.
The polypeptides, referred to herein as glycoprotein Ib.alpha.
fusion polypeptides.
[0006] In one aspect, the invention provides a glycoprotein
Ib.alpha. fusion polypeptide that includes a first polypeptide,
comprising at least a region of a glycoprotein Ib.alpha.
polypeptide, operably linked to a second polypeptide. The second
polypeptide is preferably to form a multimer, e.g., a dimer. In
preferred embodiments, the second polypeptide comprising at least a
region of an immunoglobulin polypeptide. In some embodiments, the
fusion protein includes the sequences of GP1b302-Ig (SEQ ID NO: 1),
Gp1b302/2A-Ig (SEQ ID NO:2), GP1b302/4X-Ig (SEQ ID NO:3), GP1b290
Ig (SEQ ID NO:4), GP1b290/2V-Ig (SEQ ID NO:5), or GP1b290/1A-Ig
(SEQ ID NO:6), or a fragment, homolog, analog or derivative
thereof. The sequences of these polypeptides are provided
below:
TABLE-US-00001 GP1b302/Ig (SEQ ID NO:1)
MPLLLLLLLLPSPLHPHPICEVSKVASHLEVNCDKRNLTALPPDLPKDTT
ILHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDL
SHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGN
ELKTLPPGLLTPTPKLEKLSLANNNLTELPAGLLNGLENLDTLLLQENSL
YTIPKGFFGSHLLPFAFLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGV
DVKAMTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDT
EGDKVRATRTVVKFPTKARPHTCPPCPAPEALGAPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GP1b302/2A-Ig (SEQ ID
NO:2) MPLLLLLLLLPSPLHPHPICEVSKVASHLEVNCDKRNLTALPPDLPKDTT
TLHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDL
SHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGN
ELKTLPPGLLTPTPKLEKLSLANNNLTELPAGLLNGLENLDTLLLQENSL
YTIPKGFFGSHLLPFAFLHGNPWLCNCETLYFRRWLQDNAENVYVWKQGV
DVKAMTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDT
EGDKVAATATVVKFPTKARPHTCPPCPAPEALGAPSVFLFPPKPKDTLMT
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GP1b302/4X-Ig (SEQ ID
NO:3) MPLLLLLLLLPSPLHPHPICEVSKVASHLEVNCDKRNLTALPPDLPKDTT
ILHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDL
SHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGN
ELKTLPPGLLTPTPKLEKLSLANNNLTELPAGLLNGLENLDTLLLQENSL
YTIPKGFFGSHLLPFAFLHGNPWLCNCEILYFRRWLQDNAENVYVWKQVV
DVKAVTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDT
EGDKVAATATVVKFPTKARPHTCPPCPAPEALGAPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTTSKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDTAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GP1b290-Ig (SEQ ID
NO:4) MPLLLLLLLLPSPLHPHPICEVSKVASHLEVNCDKRNLTALPPDLPKDTT
ILHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDL
SHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGN
ELKTLPPGLLTPTPKLEKLSLANNNLTELPAGLLNGLENLDTLLLQENSL
YTIPKGFFGSHLLPFAFLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGV
DVKAMTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDT
EGDKVRPHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTIPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GP1b290/2V-Ig (SEQ ID NO:5)
MPLLLLLLLLPSPLHPHPICEVSKVASHLEVNCDKRNLTALPPDLPKDTT
ILHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDL
SHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGN
ELKTLPPGLLTPTPKLEKLSLANNNLTELPAGLLNGLENLDTLLLQENSL
YTIPKGFFGSHLLPFAFLHGNPWLCNCEILYFRRWLQDNAENVYVWKQVV
DVKAVTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDT
EGDKVRPHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GP1b290/1A-Ig (SEQ ID NO:6)
MPLLLLLLLLPSPLHPHPICEVSKVASHLEVNCDKRNLTALPPDLPKDTT
TLHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDL
SHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGN
ELKTLPPGLLTPTPKLEKLSLANNNLTELPAGLLNGLENLDTLLLQENSL
YTIPKGFFGSHLLPFAFLHGNPWLCNCETLYFRRWLQDNAENVYVWKQGV
DVAAMTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDT
EGDKVRPHTCPPCPAPEALGAPSVFLFPPKPKDTLMTSRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKKQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0007] Also provided by the invention is a method of inhibiting
leukocyte adhesion to a biological tissue contacting a leukocyte
with a glycoprotein Ib.alpha. fusion polypeptide according to the
invention. The leukocyte is contacted in an amount sufficient to
inhibit adherence of the leukocyte and the biological tissue
[0008] In another aspect, the invention provides a method of
treating a disorder associated with platelet activation. The method
includes administering to a subject an effective amount of a
glycoprotein Ib.alpha. fusion polypeptide.
[0009] Also included in the invention is a nucleic acid encoding a
glycoprotein Ib.alpha. fusion polypeptide, as well as a vector
containing glycoprotein Ib.alpha. fusion polypeptide-encoding
nucleic acids described herein, and a cell containing the vectors
or nucleic acids described herein.
[0010] Also included in the invention are pharmaceutical
compositions that include the glycoprotein Ib.alpha. fusion
polypeptides, as well as antibodies that specifically recognize the
glycoprotein Ib.alpha. fusion polypeptides.
[0011] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0012] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is an illustration of a coomasie blue stained gel
showing the purification of a GP1b302-Ig fusion protein secreted
from CHO cells stably transfected with a mammalian expression
vector containing a GP1b302-Ig coding region. Lanes 7, 9 show
protein A eluates containing tryptic fragments (lower band of
approximately 38 kD). Lane 10 is protein A eluate after gel
filtration column (GFC) as described in FIG. 2.
[0014] FIG. 2 is an illustration of a gel showing the purification
of a protein A eluted GP1b302-Ig fusion protein by gel filtration
column (GFC). GFC enables separation of upper band (intac fusion
protein, lane 4) from lower band (tryptic cleavage fragment, lane
7).
[0015] FIG. 3 is an illustration of a western blot of conditioned
cell culture medium demonstrating the extent of proteolysis for
various GP1b-Ig fusion proteins secreted from stability transfected
CHO cells.
[0016] FIG. 4 is a chart depicting an UV spectrum measuring
platelet aggregation.
[0017] FIG. 5 is a chart showing the effect of a single bolus
injection of a GPIb290/2V-Ig fusion protein at various
concentrations on mean LCX flow patterns during in vivo Folts model
experiments. Arrow shows time of drug injection.
[0018] FIG. 6 is a schematic illustration depicting an injured
coronary artery with high fluid shear blood flow.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The invention provides fusion proteins containing
glycoprotein Ib.alpha. protein-immunoglobulin fusion proteins that
are useful for inhibiting adherence of platelets and leukocytes to
biological tissues, such as for example the vascular endothelium.
The fusion proteins of the invention, or nucleic acids encoding
these fusion proteins, can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between an glycoprotein Ib.alpha. ligand (such as Von
Willebrand Factor, Mac-1, P-selectin or thrombin) and an
glycoprotein Ib.alpha. protein on the surface of a cell, such as a
platelet. Inhibition of binding suppresses glycoprotein Ib.alpha.
protein-mediated platelet aggregation and associated signal
transduction in vivo.
[0020] The glycoprotein Ib.alpha. protein-immunoglobulin fusion
proteins can be used to modulate the bioavailability of a
glycoprotein Ib.alpha. protein cognate ligand. Inhibition of the
glycoprotein Ib.alpha. protein ligand/glycoprotein Ib.alpha.
protein interaction are useful therapeutically for, inter alia, the
treatment of vascular inflammation and other vascular disorders
associated with platelet activation.
Glycoprotein Ib.alpha. Fusion Polypeptides
[0021] In various aspects the invention provides fusion proteins
that include a first polypeptide containing at least a portion of a
glycoprotein Ib.alpha. polypeptide operatively linked to a second
polypeptide. As used herein, an glycoprotein Ib.alpha. "fusion
protein" or "chimeric protein" includes at least a portion of a
glycoprotein Ib.alpha. polypeptide operatively linked to a
non-glycoprotein Ib.alpha. polypeptide. An "glycoprotein Ib.alpha.
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to at least a portion of a glycoprotein Ib.alpha.
polypeptide, whereas a "non-glycoprotein Ib.alpha. polypeptide"
refers to a polypeptide having an amino acid sequence corresponding
to a protein that is not substantially homologous to the
glycoprotein Ib.alpha. protein, e.g., a protein that is different
from the glycoprotein Ib.alpha. polypeptide or fragment and that is
derived from the same or a different organism. Within a
glycoprotein Ib.alpha. fusion protein the glycoprotein Ib.alpha.
polypeptide can correspond to all or a portion of an Ib.alpha.
protein.
[0022] In one embodiment, a glycoprotein Ib.alpha. fusion protein
comprises at least one biologically active portion of a
glycoprotein Ib.alpha. protein. In another embodiment, a
glycoprotein Ib.alpha. fusion protein comprises at least two
biologically active portions of a glycoprotein Ib.alpha. protein.
In yet another embodiment, a glycoprotein Ib.alpha. fusion protein
comprises at least three biologically active portions of a
glycoprotein Ib.alpha. protein. Within the fusion protein, the term
"operatively linked" is intended to indicate that the first and
second polypeptides are chemically linked (most typically via a
covalent bond such as a peptide bond) in a manner that allows for
at least one function associated with a glycoprotein Ib.alpha.
polypeptide. When used to refer to nucleic acids encoding a
glycoprotein Ib.alpha. fusion polypeptide, the term operatively
linked means that a nucleic acid encoding the glycoprotein
Ib.alpha. polypeptide and the non-glycoprotein Ib.alpha.
polypeptide are fused in-frame to each other. The non-glycoprotein
Ib.alpha. polypeptide can be fused to the N-terminus or C-terminus
of the glycoprotein Ib.alpha. polypeptide.
[0023] In a further embodiment, the glycoprotein Ib.alpha. fusion
protein may be linked to one or more additional moieties. For
example, the glycoprotein Ib.alpha. fusion protein may additionally
be linked to a GST fusion protein in which the glycoprotein
Ib.alpha. fusion protein sequences are fused to the C-terminus of
the GST (i.e., glutathione S-transferase) sequences. Such fusion
proteins can facilitate the purification of glycoprotein Ib.alpha.
fusion protein.
[0024] In another embodiment, the fusion protein is includes a
heterologous signal sequence (i.e., a polypeptide sequence that is
not present in a polypeptide encoded by a glycoprotein Ib.alpha.
nucleic acid) at its N-terminus. For example, the native
glycoprotein Ib.alpha. signal sequence can be removed and replaced
with a signal sequence from another protein. In certain host cells
(e.g., mammalian host cells), expression and/or secretion of
glycoprotein Ib.alpha. can be increased through use of a
heterologous signal sequence.
[0025] An chimeric or fusion protein of the invention can be
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are
ligated together in-frame in accordance with conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini
for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers that give rise to
complementary overhangs between two consecutive gene fragments that
can subsequently be annealed and reamplified to generate a chimeric
gene sequence (see, for example, Ausubel et al. (eds.) CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
Moreover, many expression vectors are commercially available that
encode a fusion moiety (e.g., an Fc region of an immunoglobulin
heavy chain). A glycoprotein Ib.alpha. encoding nucleic acid can be
cloned into such an expression vector such that the fusion moiety
is linked in-frame to the immunoglobulin protein.
[0026] In various embodiments, the glycoprotein Ib.alpha. fusion
polypeptide includes the amino acid sequence of one or more of SEQ
ID NOs: 1-6.
[0027] Glycoprotein Ib.alpha. fusion polypeptides may exist as
oligomers, such as dimers or trimers. Preferably the glycoprotein
Ib.alpha. fusion polypeptide is a dimer.
[0028] The first polypeptide, and/or nucleic acids encoding the
first polypeptide, can be constructed using GP Ib.alpha. encoding
sequences are known in the art and are described in, e.g. European
Patent Application Publication No. 0 317 278 A2, and Lopez et al.
84:5615-19, 1987. Other sources for GP Ib.alpha. polypeptides and
nucleic acids encoding GP Ib.alpha. polypeptides include GenBank
Accession Nos. BAB12038 and AB038516, D85894 and BAA12911,
respectively (human sequences), and GenBank Accession No. AAC53320
and U91967, respectively, and are incorporated herein by reference
in their entirety.
[0029] In some embodiments, the GP Ib .alpha. polypeptide moiety is
provided as a variant GP Ib .alpha. polypeptide having a mutation
in the naturally-occurring GP Ib .alpha. sequence (wild type) that
results in higher affinity (relative to the non-mutated sequence)
binding of the GP I.beta..alpha. polypeptide to a leukocyte cell
surface molecule. For example, the mutant polypeptide may bind with
higher affinity to Von Willebrand factor (vWF). This increased
reactivity, or hyperresponsiveness, can be assessed using low
concentrations of ristocetin. Alternately, any other suitable means
for determining the reactivity of the polypeptide with vWF can also
be utilized to identify polypeptides which are "more" reactive with
vWF, i.e. more reactive than naturally-occurring wild-type GP
Ib.alpha.. Examples of GP Ib .alpha. polypeptide variants that bind
with higher affinity to vWF include GP Ib.alpha. variants that
include sequence alterations in the hinge region of a GP Ib.alpha.
polypeptide. The hinge region is defined as the region including
residues 220 to 310 and is reported to be a major binding site for
vWF within the GP Ib .alpha. polypeptide. Mutations in the hinge
region include those at residue 233, which in the wild-type GP Ib
.alpha. encodes glycine. A substitution of valine for glycine 233
is preferred, but other amino acids could also be substituted. A
second site for mutation at the hinge region is at residue 239,
which in the wild-type GP Ib .alpha. encodes methionine. A
substitution of valine for glycine 239 is preferred, but other
amino acids can also be substituted. In addition, hinge region
variants of GP Ib .alpha. polypeptides suitable for use in a fusion
polypeptide of the invention have mutations oat residue both
positions 233 and 239. (see e.g., Dong et al., JBC 275:36
27663-27670 (2000)) Thus, the invention includes fusion proteins
that have a substitution at position 239, e.g., an M239V
substitution of a variant GP Ib .alpha. polypeptide. Also within
the invention is a fusion protein having a substitution at position
233, e.g., G233V, and a fusion protein that includes a a variant GP
Ib .alpha. polypeptide with positions at both 233 and 239, e.g, a
G233V and M239V substitution.
[0030] In some embodiments, the GP Ib .alpha. polypeptide moiety is
provided as a variant GP Ib .alpha. polypeptide having mutations in
the naturally-occurring GP Ib .alpha. sequence (wild type) that
results in a GP Ib .alpha. sequence more resistant to proteolysis
(relative to the non-mutated sequence). Tryptic cleavage sites in
the naturally-occurring GP Ib .alpha. sequence have been described.
(see e.g. Titani et al., PNAS 84: 5610-5614, (1987))
[0031] In some embodiments, the first polypeptide includes
full-length GP Ib .alpha. polypeptide. Alternatively, the first
polypeptide comprise less than full-length GP Ib .alpha.
polypeptide. For example the first polypeptide less than 600 amino
acids in length, e.g., less than or equal to 500, 250, 150, 100,
50, or 25 amino acids in length.
[0032] Examples of a first polypeptide include a polypeptide which
includes the amino acid sequence of any of the GP Ib .alpha.
polypeptide sequences of GP1b302 (SEQ ID NO:7), GP1b302/2A (SEQ ID
NO:8) GP1b/4.times. (SEQ ID NO:9), GP1b290 (SEQ ID NO:10),
GB1b290/2V (SEQ ID NO:11) and GB1b290/1A (SEQ ID NO:12).
TABLE-US-00002 (SEQ ID NO:7)
HPICEVSKVASHLEVNCDKRNLTALPPDLPKDTTILHLSENLLYTFSLAT
LMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDLSHNQLQSLPLLGQTLP
ALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTPTPKL
EKLSLANNNLTELPAGLLNGLENLDTLLLQENSLYTTPKGFFGSHLLPFA
FLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGVDVKANTSNVASVQCDN
SDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDTEGDKVRATRTVVKFPT KA (SEQ ID NO:8)
HPICEVSKVASHLEVNCDKRNLTALPPDLPKDTTILHLSENLLYTFSLAT
LMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDLSHNQLQSLPLLGQTLP
ALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTPTPKL
EKLSLANNNLTELPAGLLNGLENLDTLLLQENSLYTIPKGFFGSHLLPFA
FLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGVDVKAMTSNVASVQCDN
SDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDTEGDKVAATATVVKFPT KA (SEQ ID NO:9)
HPICEVSKVASHLEVNCDKRNLTALPPDLPKDTTILHLSENLLYTFSLAT
LMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDLSHNQLQSLPLLGQTLP
ALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTPTPKL
EKLSLANNNLTELPAGLLNGLENLDTLLLQENSLYTIPKGFFGSHLLPFA
FLHGNPWLCNCEILYFRRWLQDNAENVYVWKQVVDVKAVTSNVASVQCDN
SDKFPVKYPGKGCPTLGDEGDTDLYDYYPEEDTEGDKVAATATVVKFPTK A (SEQ ID NO:10)
HPICEVSKVASHLEVNCDKRNLTALPPDLPKDTTILHLSENLLYTFSLAT
LMPYTRLTQLNLDRCELTKLQVDOTLPVLGTLDLSHNQLQSLPLLGQTLP
ALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTPTPKL
EKLSLANNNLTELPAGLLNGLENLDTLLLQENSLYTIPKGFFGSHLLPFA
FLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGVDVKAMTSNVASVQCDN
SDKPPVYKYPGKGCPTLGDEGDTDLYDYYPEEDTEGDKVR (SEQ ID NO:11)
HPICEVSKVASHLEVNCDKRNLTALPPDLPKDTTILHLSENLLYTFSLAT
LMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDLSHNQLQSLPLLGQTLP
ALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTPTPKL
EKLSLANNNLTELPAGLLNGLENLDTLLLQENSLYTTPKGFFGSHLLPFA
FLHGNPWLCNCEILYFRRWLQDNAENVYVWKQVVDVKAVTSNVASVQCDN
SDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDTEGDKVR (SEQ ID NO:12)
HPICEVSKVASHLEVNCDKRNLTALPPDLPKDTTILHLSENLLYTFSLAT
LMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDLSHNQLQSLPLLGQTLP
ALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTPTPKL
EKLSLANNNLTELPAGLLNGLENLDTLLLQENSLYTIPKGFFGSHLLPFA
FLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGVDVAAMTSNVASVQCDN
SDKFPVYKYPGKGCPTLGDEGDTDLYDYYPEEDTEGDKVR
[0033] A signal peptide that can be included in the fusion protein
is MPLLLLLLLLPSPLHP (SEQ ID NO: 13). If desired, one or more amino
acids can additionally be inserted between the first polypeptide
moiety comprising the GP Ib .alpha. moiety and the second
polypeptide moiety.
[0034] The second polypeptide is preferably soluble. In some
embodiments, the second polypeptide enhances the half-life, (e.g.,
the serum half-life) of the linked polypeptide. In some
embodiments, the second polypeptide includes a sequence that
facilitates association of the fusion polypeptide with a second GP
Ib .alpha. polypeptide. In preferred embodiments, the second
polypeptide includes at least a region of an immunoglobulin
polypeptide. Immunoglobulin fusion polypeptide are known in the art
and are described in e.g., U.S. Pat. Nos. 5,516,964; 5,225,538;
5,428,130; 5,514,582; 5,714,147; and 5,455,165.
[0035] In some embodiments, the second polypeptide comprises a
full-length immunoglobulin polypeptide. Alternatively, the second
polypeptide comprise less than full-length immunoglobulin
polypeptide, e.g., a heavy chain, light chain, Fab, Fab.sub.2, Fv,
or Fc. Preferably, the second polypeptide includes the heavy chain
of an immunoglobulin polypeptide. More preferably the second
polypeptide includes the Fc region of an immunoglobulin
polypeptide.
[0036] In another aspect of the invention the second polypeptide
has less effector function that the effector function of a Fc
region of a wild-type immunoglobulin heavy chain. Fc effector
function includes for example, Fc receptor binding, complement
fixation and T cell depleting activity. (see for example, U.S. Pat.
No. 6,136,310) Methods of assaying T cell depleting activity, Fc
effector function, and antibody stability are known in the art. In
one embodiment the second polypeptide has low or no affinity for
the Fc receptor. In an alternative embodiment, the second
polypeptide has low or no affinity for complement protein C1q.
[0037] A preferred second polypeptide sequence includes the amino
acid sequence of SEQ ID NO: 12. This sequence includes a Fc region.
Underlined amino acids are those that differ from the amino acid
found in the corresponding position of the wild-type immunoglobulin
sequence:
TABLE-US-00003 (SEQ ID NO:14)
HTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK
[0038] DNA sequences encoding fusion polypeptides of SEQ ID NOs:
1-6 are disclosed below as sequences SEQ ID NOs;15-20,
respectively:
TABLE-US-00004 GP1b302-Ig nucleotide sequence (SEQ ID NO:15)
atgcctctcctcctcttgctgctcctgctgccaagccccttacaccccca
ccccatctgtgaggtctccaaagtggccagccacctagaagtgaactgtg
acaagaggaatctgacagcgctgcctccagacctgccgaaagacacaacc
atcctccacctgagtgagaacctcctgtacaccttctccctggcaaccct
gatgccttacactcgcctcactcagctgaacctagataggtgcgagctca
ccaagctccaggtcgatgggacgctgccagtgctggggaccctggatcta
tcccacaatcagctgcaaagcctgcccttgctagggcagacactgcctgc
tctcaccgtcctggacgtctccttcaaccggctgacctcgctgcctcttg
gtgccctgcgtggtcttggcgaactccaagagctctacctgaaaggcaat
gagctgaagaccctgcccccagggctcctgacgcccacacccaagctgga
gaagctcagtctggctaacaacaacttgactgagctccccgctgggctcc
tgaatgggctggagaatctcgacacccttctcctccaagagaactcgctg
tatacaataccaaagggcttttttgggtcccacctcctgccttttgcttt
tctccacgggaacccctggttatgcaactgtgagatcctctattttcgtc
gctggctgcaggacaatgctgaaaatgtctacgtatggaagcaaggtgtg
gacgtcaaggccatgacctctaacgtggccagtgtgcagtgtgacaattc
agacaagtttcccgtctacaaatacccaggaaaggggtgccccacccttg
gtgatgaaggtgacacagacctatatgattactacccagaagaggacact
gagggcgataaggtgcgtgccacaaggactgtggtcaagttccccaccaa
agcgcggccgcacacatgcccaccgtgcccagcacctgaagccctggggg
caccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatc
tcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaaga
ccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg
ccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc
agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaa
gtgcaaggtctccaacaaagccctcccagtccccatcgagaaaaccatct
ccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccca
tcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaa
aggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagc
cggagaacaactacaagaccacgcctcccgtgctggactccgacggcccc
ttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg
gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca
cgcagaagagcctctccctgtctccgggtaaa GP1b302/2A-Ig nucleotide sequence
(SEQ ID NO:16) atgcctctcctcctcttgctgctcctgctgccaagccccttacaccccca
ccccatctgtgaggtctccaaagtggccagccacctagaagtgaactgtg
acaagaggaatctgacagcgctgcctccagacctgccgaaagacacaacc
atcctccacctgagtgagaacctcctgtacaccttctccctggcaaccct
gatgccttacactcgcctcactcagctgaacctagataggtgcgagctca
ccaagctccaggtcgatgggacgctgccagtgctggggaccctggatcta
tcccacaatcagctgcaaagcctgcccttgctagggcagacactgcctgc
tctcaccgtcctggacgtctccttcaaccggctgacctcgctgcctcttg
gtgccctgcgtggtcttggcgaactccaagagctctacctgaaaggcaat
gagctgaagaccctgcccccagggctcctgacgcccacacccaagctgga
gaagctcagtctggctaacaacaacttgactgagctccccgctgggctcc
tgaatgggctggagaatctcgacacccttctcctccaagagaactcgctg
tatacaataccaaagggcttttttgggtcccacctcctgccttttgcttt
tctccacgggaacccctggttatgcaactgtgagatcctctattttcgtc
gctggctgcaggacaatgctgaaaatgtctacgtatggaagcaaggtgtg
gacgtcaaggccatgacctctaacgtggccagtgtgcagtgtgacaattc
agacaagtttcccgtctacaaatacccaggaaaggggtgccccacccttg
gtgatgaaggtgacacagacctatatgattactacccagaagaggacact
gagggcgataaggtggctgccacagcgactgtggtcaagttccccaccaa
agcgcggccgcacacatgcccaccgtgcccagcacctgaagccctggggg
caccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatc
tcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaaga
ccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg
ccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc
agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaa
gtgcaaggtctccaacaaagccctcccagtccccatcgagaaaaccatct
ccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccca
tcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaa
aggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagc
cggagaacaactacaagaccacgcctcccgtgctggactccgacggcccc
ttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg
gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca
cgcagaagagcctctccctgtctccgggtaaa GP1b302/4X-Ignucleotide sequence
(SEQ ID NO:17) atgcctctcctcctcttgctgctcctgctgccaagccccttacaccccca
ccccatctgtgaggtctccaaagtggccagccacctagaagtgaactgtg
acaagaggaatctgacagcgctgcctccagacctgccgaaagacacaacc
atcctccacctgagtgagaacctcctgtacaccttctccctggcaaccct
gatgccttacactcgcctcactcagctgaacctagataggtgcgagctca
ccaagctccaggtcgatgggacgctgccagtgctggggaccctggatcta
tcccacaatcagctgcaaagcctgcccttgctagggcagacactgcctgc
tctcaccgtcctggacgtctccttcaaccggctgacctcgctgcctcttg
gtgccctgcgtggtcttggcgaactccaagagctctacctgaaaggcaat
gagctgaagaccctgcccccagggctcctgacgcccacacccaagctgga
gaagctcagtctggctaacaacaacttgactgagctccccgctgggctcc
tgaatgggctggagaatctcgacacccttctcctccaagagaactcgctg
tatacaataccaaagggcttttttgggtcccacctcctgccttttgcttt
tctccacgggaacccctggttatgcaactgtgagatcctctattttcgtc
gctggctgcaggacaatgctgaaaatgtctacgtatggaagcaagtggtg
gacgtcaaggccgtgacctctaacgtggccagtgtgcagtgtgacaattc
agacaagtttcccgtctacaaatacccaggaaaggggtgccccacccttg
gtgatgaaggtgacacagacctatatgattactacccagaagaggacact
gagggcgataaggtggctgccacagcgactgtggtcaagttccccaccaa
agcgcggccgcacacatgcccaccgtgcccagcacctgaagccctggggg
caccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatc
tcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaaga
ccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg
ccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc
agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaa
gtgcaaggtctccaacaaagccctcccagtccccatcgagaaaaccatct
ccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccca
tcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaa
aggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagc
cggagaacaactacaagaccacgcctcccgtgctggactccgacggcccc
ttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg
gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca
cgcagaagagcctctccctgtctccgggtaaa GP1b290-Ig nucleotide sequence
(SEQ ID NO:18) atgcctctcctcctcttgctgctcctgctgccaagccccttacaccccca
ccccatctgtgaggtctccaaagtggccagccacctagaagtgaactgtg
acaagaggaatctgacagcgctgcctccagacctgccgaaagacacaacc
atcctccacctgagtgagaacctcctgtacaccttctccctggcaaccct
gatgccttacactcgcctcactcagctgaacctagataggtgcgagctca
ccaagctccaggtcgatgggacgctgccagtgctggggaccctggatcta
tcccacaatcagctgcaaagcctgcccttgctagggcagacactgcctgc
tctcaccgtcctggacgtctccttcaaccggctgacctcgctgcctcttg
gtgccctgcgtggtcttggcgaactccaagagctctacctgaaaggcaat
gagctgaagaccctgcccccagggctcctgacgcccacacccaagctgga
gaagctcagtctggctaacaacaacttgactgagctccccgctgggctcc
tgaatgggctggagaatctcgacacccttctcctccaagagaactcgctg
tatacaataccaaagggcttttttgggtcccacctcctgccttttgcttt
tctccacgggaacccctggttatgcaactgtgagatcctctattttcgtc
gctggctgcaggacaatgctgaaaatgtctacgtatggaagcaaggtgtg
gacgtcaaggccatgacctctaacgtggccagtgtgcagtgtgacaattc
agacaagtttcccgtctacaaatacccaggaaaggggtgccccacccttg
gtgatgaaggtgacacagacctatatgattactacccagaagaggacact
gagggcgataaggtgcggccgcacacatgcccaccgtgcccagcacctga
agccctgggggcaccgtcagtcttcctcttccccccaaaacccaaggaca
ccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtg
agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga
ggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgt
accgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggc
aaggagtacaagtgcaaggtctccaacaaagccctcccagtccccatcga
gaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtaca
ccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacc
tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagag
caatgggcagccggagaacaactacaagaccacgcctcccgtgctggact
ccgacggccccttcttcctctacagcaagctcaccgtggacaagagcagg
tggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgca
caaccactacacgcagaagagcctctccctgtctccgggtaaa GP1b290/2V-Ig
nucleotide sequence (SEQ ID NO:19)
atgcctctcctcctcttgctgctcctgctgccaagccccttacaccccca
ccccatctgtgaggtctccaaagtggccagccacctagaagtgaactgtg
acaagaggaatctgacagcgctgcctccagacctgccgaaagacacaacc
atcctccacctgagtgagaacctcctgtacaccttctccctggcaaccct
gatgccttacactcgcctcactcagctgaacctagataggtgcgagctca
ccaagctccaggtcgatgggacgctgccagtgctggggaccctggatcta
tcccacaatcagctgcaaagcctgcccttgctagggcagacactgcctgc
tctcaccgtcctggacgtctccttcaaccggctgacctcgctgcctcttg
gtgccctgcgtggtcttggcgaactccaagagctctacctgaaaggcaat
gagctgaagaccctgcccccagggctcctgacgcccacacccaagctgga
gaagctcagtctggctaacaacaacttgactgagctccccgctgggctcc
tgaatgggctggagaatctcgacacccttctcctccaagagaactcgctg
tatacaataccaaagggcttttttgggtcccacctcctgccttttgcttt
tctccacgggaacccctggttatgcaactgtgagatcctctattttcgtc
gctggctgcaggacaatgctgaaaatgtctacgtatggaagcaagtggtg
gacgtcaaggccgtgacctctaacgtggccagtgtgcagtgtgacaattc
agacaagtttcccgtctacaaatacccaggaaaggggtgccccacccttg
gtgatgaaggtgacacagacctatatgattactacccagaagaggacact
gagggcgataaggtgcggccgcacacatgcccaccgtgcccagcacctga
agccctgggggcaccgtcagtcttcctcttccccccaaaacccaaggaca
ccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtg
agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga
ggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgt
accgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggc
aaggagtacaagtgcaaggtctccaacaaagccctcccagtccccatcga
gaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtaca
ccctgcccccatcccgggaggagatgaccaagaaccagqtcagcctgacc
tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagag
caatgggcagccggagaacaactacaagaccacgcctcccgtgctggact
ccgacggccccttcttcctctacagcaagctcaccgtggacaagagcagg
tggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgca
caaccactacacgcagaagagcctctccctgtctccgggtaaa GP1b290/1A-Ig
nucleotide sequence (SEQ ID NO:20)
atgcctctcctcctcttgctgctcctgctgccaagccccttacaccccca
ccccatctgtgaggtctccaaagtggccagccacctagaagtgaactgtg
acaagaggaatctgacagcgctgcctccagacctgccgaaagacacaacc
atcctccacctgagtgagaacctcctgtacaccttctccctggcaaccct
gatgccttacactcgcctcactcagctgaacctagataggtgcgagctca
ccaagctccaggtcgatgggacgctgccagtgctggggaccctggatcta
tcccacaatcagctgcaaagcctgcccttgctagggcagacactgcctgc
tctcaccgtcctggacgtctccttcaaccggctgacctcgctgcctcttg
gtgccctgcgtggtcttggcgaactccaagagctctacctgaaaggcaat
gagctgaagaccctgcccccagggctcctgacgcccacacccaagctgga
gaagctcagtctggctaacaacaacttgactgagctccccgctgggctcc
tgaatgggctggagaatctcgacacccttctcctccaagagaactcgctg
tatacaataccaaagggcttttttgggtcccacctcctgccttttgcttt
tctccacgggaacccctggttatgcaactgtgagatcctctattttcgtc
gctggctgcaggacaatgctgaaaatgtctacgtatggaagcaaggtgtg
gacgtcgcggccatgacctctaacgtggccagtgtgcagtgtgacaattc
agacaagtttcccgtctacaaatacccaggaaaggggtgccccacccttg
gtgatgaaggtgacacagacctatatgattactacccagaagaggacact
gagggcgataaggtgcggccgcacacatgcccaccgtgcccagcacctga
agccctgggggcaccgtcagtcttcctcttccccccaaaacccaaggaca
ccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtg
agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga
ggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgt
accgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggc
aaggagtacaagtgcaaggtctccaacaaagccctcccagtccccatcga
gaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtaca
ccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacc
tgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagag
caatgggcagccggagaacaactacaagaccacgcctcccgtgctggact
ccgacggccccttcttcctctacagcaagctcaccgtggacaagagcagg
tggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgca
caaccactacacgcagaagagcctctccctgtctccgggtaaa
[0039] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
glycoprotein Ib.alpha. fusion polypeptides, or derivatives,
fragments, analogs or homologs thereof. As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One type of
vector is a "plasmid", which refers to a circular double stranded
DNA loop into which additional DNA segments can be ligated. Another
type of vector is a viral vector, wherein additional DNA segments
can be ligated into the viral genome. Certain vectors are capable
of autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively-linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0040] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operatively-linked to the nucleic acid sequence
to be expressed. Within a recombinant expression vector,
"operably-linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
that allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell).
[0041] The term "regulatory sequence" is intended to includes
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described,
for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory sequences include those that direct constitutive
expression of a nucleotide sequence in many types of host cell and
those that direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein (e.g., glycoprotein Ib.alpha. fusion polypeptides,
mutant forms of glycoprotein Ib.alpha. fusion polypeptides,
etc.).
[0042] The recombinant expression vectors of the invention can be
designed for expression of glycoprotein Ib.alpha. fusion
polypeptides in prokaryotic or eukaryotic cells. For example,
glycoprotein Ib.alpha. fusion polypeptides can be expressed in
bacterial cells such as Escherichia coli, insect cells (using
baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0043] Expression of proteins in prokaryotes is most often carried
out in Escherichia coli with vectors containing constitutive or
inducible promoters directing the expression of either fusion or
non-fusion proteins. Fusion vectors add a number of amino acids to
a protein encoded therein, usually to the amino terminus of the
recombinant protein. Such fusion vectors typically serve three
purposes: (i) to increase expression of recombinant protein; (ii)
to increase the solubility of the recombinant protein; and (iii) to
aid in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin
and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0044] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and
pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
60-89).
[0045] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant
protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
119-128. Another strategy is to alter the nucleic acid sequence of
the nucleic acid to be inserted into an expression vector so that
the individual codons for each amino acid are those preferentially
utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids
Res. 20: 2111-2118). Such alteration of nucleic acid sequences of
the invention can be carried out by standard DNA synthesis
techniques.
[0046] In another embodiment, the glycoprotein Ib.alpha. fusion
polypeptide expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae
include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa
(Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et
al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San
Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
[0047] Alternatively, glycoprotein Ib.alpha. c fusion polypeptide
can be expressed in insect cells using baculovirus expression
vectors. Baculovirus vectors available for expression of proteins
in cultured insect cells (e.g., SF9 cells) include the pAc series
(Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL
series (Lucklow and Summers, 1989. Virology 170: 31-39).
[0048] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987.
EMBO J. 6: 187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0049] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes
Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton,
1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell
receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and
immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc.
Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters
(Edlund, et al., 1985. Science 230: 912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the
murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379)
and the .alpha.-fetoprotein promoter (Campes and Tilghman, 1989.
Genes Dev. 3: 537-546).
[0050] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively-linked to a regulatory sequence in a manner
that allows for expression (by transcription of the DNA molecule)
of an RNA molecule that is antisense to NOV glycoprotein Ib.alpha.
fusion polypeptide mRNA. Regulatory sequences operatively linked to
a nucleic acid cloned in the antisense orientation can be chosen
that direct the continuous expression of the antisense RNA molecule
in a variety of cell types, for instance viral promoters and/or
enhancers, or regulatory sequences can be chosen that direct
constitutive, tissue specific or cell type specific expression of
antisense RNA. The antisense expression vector can be in the form
of a recombinant plasmid, phagemid or attenuated virus in which
antisense nucleic acids are produced under the control of a high
efficiency regulatory region, the activity of which can be
determined by the cell type into which the vector is introduced.
For a discussion of the regulation of gene expression using
antisense genes see, e.g., Weintraub, et al., "Antisense RNA as a
molecular tool for genetic analysis," Reviews-Trends in Genetics,
Vol. 1(1) 1986.
[0051] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but also to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0052] A host cell can be any prokaryotic or eukaryotic cell. For
example, glycoprotein Ib.alpha. fusion polypeptides can be
expressed in bacterial cells such as E. coli, insect cells, yeast
or mammalian cells (such as human, Chinese hamster ovary cells
(CHO) or COS cells). Other suitable host cells are known to those
skilled in the art.
[0053] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A
LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0054] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Various selectable markers
include those that confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding glycoprotein Ib.alpha. fusion polypeptides or can be
introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g.,
cells that have incorporated the selectable marker gene will
survive, while the other cells die).
[0055] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) glycoprotein Ib.alpha. fusion polypeptides. Accordingly,
the invention further provides methods for producing glycoprotein
Ib.alpha. fusion polypeptides using the host cells of the
invention. In one embodiment, the method comprises culturing the
host cell of invention (into which a recombinant expression vector
encoding glycoprotein Ib.alpha. fusion polypeptides has been
introduced) in a suitable medium such that glycoprotein Ib.alpha.
fusion polypeptides is produced. In another embodiment, the method
further comprises isolating glycoprotein Ib.alpha. fusion
polypeptide from the medium or the host cell.
[0056] The fusion polypeptides may be isolated and purified in
accordance with conventional conditions, such as extraction,
precipitation, chromatography, affinity chromatography,
electrophoresis or the like. For example, the immunoglobulin fusion
proteins may be purified by passing a solution through a column
which contains immobilized protein A or protein G which selectively
binds the Fc portion of the fusion protein. See, for example, Reis,
K. J., et al., J. Immunol. 132:3098-3102 (1984); PCT Application,
Publication No. WO87/00329. The fusion polypeptide may the be
eluted by treatment with a chaotropic salt or by elution with
aqueous acetic acid (1 M).
[0057] Alternatively, fusion polypeptides according to the
invention can be chemically synthesized using methods known in the
art. Chemical synthesis of polypeptides is described in, e.g., A
variety of protein synthesis methods are common in the art,
including synthesis using a peptide synthesizer. See, e.g., Peptide
Chemistry, A Practical Textbook, Bodasnsky, Ed. Springer-Verlag,
1988; Merrifield, Science 232: 241-247 (1986); Barany, et al, Intl.
J. Peptide Protein Res. 30: 705-739 (1987); Kent, Ann. Rev.
Biochem. 57:957-989 (1988), and Kaiser, et al, Science 243: 187-198
(1989). The polypeptides are purified so that they are
substantially free of chemical precursors or other chemicals using
standard peptide purification techniques. The language
"substantially free of chemical precursors or other chemicals"
includes preparations of peptide in which the peptide is separated
from chemical precursors or other chemicals that are involved in
the synthesis of the peptide. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of peptide having less than about 30% (by dry
weight) of chemical precursors or non-peptide chemicals, more
preferably less than about 20% chemical precursors or non-peptide
chemicals, still more preferably less than about 10% chemical
precursors or non-peptide chemicals, and most preferably less than
about 5% chemical precursors or non-peptide chemicals.
[0058] Chemical synthesis of polypeptides facilitates the
incorporation of modified or unnatural amino acids, including
D-amino acids and other small organic molecules. Replacement of one
or more L-amino acids in a peptide with the corresponding D-amino
acid isoforms can be used to increase the resistance of peptides to
enzymatic hydrolysis, and to enhance one or more properties of
biologically active peptides, i.e., receptor binding, functional
potency or duration of action. See, e.g., Doherty, et al., 1993. J.
Med. Chem. 36: 2585-2594; Kirby, et al., 1993. J. Med. Chem.
36:3802-3808; Morita, et al., 1994. FEBS Lett. 353: 84-88; Wang, et
al., 1993. Int. J. Pept. Protein Res. 42: 392-399; Fauchere and
Thiunieau, 1992. Adv. Drug Res. 23: 127-159.
[0059] Introduction of covalent cross-links into a peptide sequence
can conformationally and topographically constrain the polypeptide
backbone. This strategy can be used to develop peptide analogs of
the fusion polypeptides with increased potency, selectivity and
stability. Because the conformational entropy of a cyclic peptide
is lower than its linear counterpart, adoption of a specific
conformation may occur with a smaller decrease in entropy for a
cyclic analog than for an acyclic analog, thereby making the free
energy for binding more favorable. Macrocyclization is often
accomplished by forming an amide bond between the peptide N- and
C-termini, between a side chain and the N- or C-terminus [e.g.,
with K.sub.3Fe(CN).sub.6 at pH 8.5] (Samson et al., Endocrinology,
137: 5182-5185 (1996)), or between two amino acid side chains. See,
e.g., DeGrado, Adv Protein Chem, 39: 51-124 (1988). Disulfide
bridges are also introduced into linear sequences to reduce their
flexibility. See, e.g., Rose, et al., Adv Protein Chem, 37: 1-109
(1985); Mosberg et al., Biochem Biophys Res Commun, 106: 505-512
(1982). Furthermore, the replacement of cysteine residues with
penicillamine (Pen, 3-mercapto-(D) valine) has been used to
increase the selectivity of some opioid-receptor interactions.
Lipkowski and Carr, Peptides. Synthesis, Structures, and
Applications, Gutte, ed., Academic Press pp. 287-320 (1995).
Pharmaceutical Compositions Including Glycoprotein Ib.alpha. Fusion
Polypeptides or Nucleic Acids Encoding Same
[0060] The glycoprotein Ib.alpha. fusion proteins, or nucleic acid
molecules encoding these fusion proteins, (also referred to herein
as "Therapeutics" or "active compounds") of the invention, and
derivatives, fragments, analogs and homologs thereof, can be
incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the nucleic
acid molecule, protein, or antibody and a pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Suitable carriers are described in
the most recent edition of Remington's Pharmaceutical Sciences, a
standard reference text in the field, which is incorporated herein
by reference. Preferred examples of such carriers or diluents
include, but are not limited to, water, saline, finger's solutions,
dextrose solution, and 5% human serum albumin. Liposomes and
non-aqueous vehicles such as fixed oils may also be used. The use
of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0061] The active agents disclosed herein can also be formulated as
liposomes. Liposomes are prepared by methods known in the art, such
as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:
3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030
(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0062] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0063] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0064] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0065] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a glycoprotein Ib.alpha.
fusion protein) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, methods of preparation are vacuum drying and
freeze-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0066] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0067] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0068] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0069] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0070] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0071] In some embodiments, oral or parenteral compositions are
formulated in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of
the active compound and the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an active compound for the treatment of individuals.
[0072] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see, e.g., U.S. Pat. No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al.,
1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells that
produce the gene delivery system.
[0073] Sustained-release preparations can be prepared, if desired.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
antibody, which matrices are in the form of shaped articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0074] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
Methods of Inhibiting Adherence of in a Biological System
[0075] Also included in the invention are methods of inhibiting
adherence of a blood cell to a biological tissue in a biological
system. The method includes adding to a biological system a fusion
polypeptide of the invention in an amount sufficient to inhibit
adherence of a blood cell to the biological tissue.
[0076] The blood cell can be for example, a leukocyte, platelet or
red blood cell. The leukocyte can be any leukocyte that is capable
of adhering to a biological tissue. In various aspects the
leukocyte is a granulocyte, (i.e., neutrophil, basophil or
eosinohil), monocyte (i.e., macrophage) or lymphocyte (e.g.,
T-lymphocyte, B-lymphocyte, tumor infiltrating lymphocytes or
natural killer cell). In some embodiments, the leukocytes express a
.beta.2 intergin, e.g. Mac-1. Alternately, the leuckocyte expresses
a selectin ligand.
[0077] Also included in the inventions are methods of inhibiting
adherence of a protein to a biological tissue in a biological
system. The method includes adding to a biological system a fusion
polypeptide of the invention in an amount sufficient to inhibit
adherence of the protein to the biological tissue.
[0078] The protein can be membrane associated (e.g., covalently,
non-covalently, ionicly). Alternatively, the protein can be in a
soluble form (i.e., in solution). The protein is von Willibrand
Factor, thrombin, P-selectin of glycoprotein Ib.alpha..
[0079] As used herein a "biological tissue" is meant to include one
or more cells with or without intracellular substances (e.g.,
extracellular matrix proteins, polysaccharides and proteoglycans. A
biological tissue also includes solely extracellular matrix
substances, such as the subendothelium connective tissue matrix. In
some aspects the biological tissue is the vascular endothelium. The
biological tissue can be one or more platelets or leukocytes. In
various aspects the biological tissue is complexed with a component
of the GP Ib-IX-V complex such as glycoprotein Ib .alpha., Mac-1,
P-selectin, thrombin or a von Willibrand Factor. By "complexed" is
meant that the biological tissue contains a soluble form of a
component of the GP Ib-IX-V complex. Alternatively, "complexed is
meant that the biological tissue contains a cell that expresses a
component of the GP Ib-IX-V complex.
[0080] As used herein a biological system is meant to include any
system that comprises biological components, e.g., cells, proteins,
carbohydrates, lipids or nucleic acids. The biological system can
be an in vivo, ex vivo or in vitro system.
[0081] By "adherence" is meant to include any leukocyte-biological
interaction, e.g., rolling, firm attachments or specific
interaction.
[0082] Inhibition of adherence of a blood cell or protein to a
biological tissue can be measured using methods known in the art.
For example, assays for detecting binding of glycoprotein Ib.alpha.
to a biological tissue are described in Simon et al., J. Exp. Med.
192:193-204, 2000, and references cited therein. In various
embodiments, binding of a GP Ib .alpha. fusion protein inhibits
binding of a blood cell or protein to a biological tissue by at
least 30%, 50%, 75%, 90%, 95%, 99% or 99.9%.
[0083] Adherence can also be assessed in condition of greater or
less than physiological flow conditions, including static
conditions and serial application of static and shear conditions.
Adherence can be determined for example colormetrically,
fluorometrically, by flow cytometry or using a parrallel plate flow
chamber assay.
[0084] Also included in the invention are methods of treating
platelet activation associated disorders in a subject by
administering to a subject a biologically-active therapeutic
compound (hereinafter "Therapeutic"). Alternatively, the subject is
also administered one or more of the following acetylsalicylic
acid, e.g., aspirin heparin, e.g., unfractionated or low-molecular
weight heparins, glycoprotein IIb/IIIa antagonists, clopidogrel,
P-selectin antagonists, thrombin inhibitors or thrombolytic
enzymes.
[0085] The subject can be e.g., any mammal, e.g., a human, a
primate, mouse, rat, dog, cat, cow, horse, pig.
[0086] The Therapeutics include, e.g.: (i) any one or more of the
glycoprotein Ib.alpha. fusion polypeptides, and derivative,
fragments, analogs and homologs thereof, (ii) antibodies directed
against the glycoprotein Ib.alpha. fusion polypeptides; and (iii)
nucleic acids encoding a glycoprotein Ib.alpha. fusion polypeptide,
and derivatives, fragments, analogs and homologs thereof.
[0087] Essentially, any disorder, which is etiologically linked to
platelet activation, is considered amenable to prevention or to
treatment. The disorder can be, e.g., vascular inflammation,
atherosclerosis, restenosis (e.g., angioplasty-related restenosis)
and/or a condition associated with thrombotic disease, e.g.,
angina, (i.e., stable angina and unstable angia) acute myocardial
infarction, stoke, venous thrombosis or arterial thrombosis.
[0088] The invention will be further illustrated in the following
non-limiting examples.
EXAMPLE 1
Production and Purification of Recombinant GP1b-IG Fusion
Proteins
[0089] Three GP1b-Ig fusion proteins, GP1b320-Ig (SEQ ID NO: 1),
GP1b290Ig (SEQ ID NO:4), and GP1b290/2V-Ig (SEQ ID NO:5), were
produced by recombinant methods and purified. Chinese hamster ovary
(CHO) cells lacking dihydrofolate reductase (DHFR) activity were
stability transfected with linearized plasmid DNA consisting of a
mammalian expression vector directing the transcription of a
GP1b-Ig coding regions in polycistronic fashion with a DHFR
selectable maker gene. Candidate expressing cells were selected in
medium containing increasing concentrations of methotrexate (MTX)
essentially as described in Kaufman et al. Nucleic Acids Res.
(1991) 19:4485-90. For collection of GP1b-Ig conditioned medium,
CHO cells were grown to near confluent levels on 5-20 culture
dishes (150 mm diameter), the cell monolayer was washed twice with
PBS and cells were cultured for approximately 24 hrs in medium
lacking fetal bovine serum. The medium was then collected and cells
discarded.
[0090] CHO cell condition media (CM) was adjusted to 50 mM Tris
pH8.0, 200 mM NaCl, filtered through a 0.2 um filter, and applied
to a Poros Protein A column. The column was washed with 10 column
volumes of 50 mM Tris pH 8.0, 200 mM NaCl and eluted with Pierce
IgG elution buffer. The protein peak was followed by absorbance at
280 nM. The pH of the elute was adjusted with 0.1 volumes of 1 M
Tris, pH 8.0. The protein was then concentrated and the buffer
exchanged by finger dialysis (25 kD MWCO) against TBS (10 mM Tris,
pH 8.0, 150 mM NaCl). The concentrated protein was then further
purified by gel filtration chromatography on a TosoHaas G3000SW
column run in TBS.
[0091] The purified protein was analyzed by Western Blots. Briefly,
13 microliters of CHO cell conditioned medium was loaded per lane
on a 4-20% reducing SDS PAGE gel. Western transfer was performed
using Electroblot apparatus and nitrocellulose membrane (Novex, San
Diego, Calif.). The primary detection antibody was monoclonal AP1,
and secondary antibody was an HRP-conjugated goat anti-murine IgG
(GTI, Brookfield, Wis.). HRP detection was via ECL system
(Amersham-Pharmacia Biotech).
EXAMPLE 2
In Vitro Inhibition of Platelet Aggregation
[0092] The ability of the glycoprotein Ib.alpha.
polypeptide-immunoglobulin fusion polypeptide to inhibit platelet
aggregation in vitro, was determined. Platelet rich plasma (PRP)
from freshly drawn, citrate blood was prepared by differential
centrifugation for 10 minutes at 900 rpm. 0.4 mls of PRP
(3.times.10.sup.8/ml ) was preincubated for 5 minutes at 37.degree.
C. with various concentrations of GP1b290/2v-Ig. Ristocetin was
added to 1.5 mg/ml to induce platelet aggregation. Aggregation was
measured using a Sienco DP247E aggregometer. Aggregation was
quantified and recorded on a chart recorder by monitoring the
increase in light transmittance with stirring at 1000 rpm. As
illustrated in FIG. 4, GP1b290/2v-Ig inhibited ristocetin induced
platelet aggregation.
EXAMPLE 3
In Vivo Inhibition of Repetitive Coronary Artery Thrombosis
[0093] The ability of a glycoprotein Ib.alpha. GP1b290/2V-Ig
polypeptide-immunoglobulin fusion polypeptide to inhibit coronary
artery thrombosis in vivo was determined using the procedure
described by Folts at al., Circulation 54:365-70, 1976.
[0094] Mongrel dogs, weighing 20-25 kg, were anesthetized with
sodium pentobarbital (30 mg/kg i.v.), then intubated and ventilated
with room air using a respirator. Venous and arterial cathetors
were placed. The heart was approached by left thoracotomy through
the fifth intercostal space. The pericardium was opened and sutured
to the wound edges to provide a cradle without displacing the
heart. About 2 cm of the left circumflex coronary artery (LCX) was
isolated. Mean and dynamic LCX flow was continuously monitored
using a perivascular ultrasonic flow probe placed proximally on the
artery. After a stabilization period, the endothelium of the LCX
was injured by squeezing with a hemostat. A plastic constrictor was
placed distal and overlying the area of injured endothelium to
provide approximately 70-80% vessel stenosis. When blood flow
decreased to zero, the blood flow was restored by shaking the
constrictor to dislodge aggregated platelets. This decrease and
restoration of blood flow are termed CFRs. At least five
consecutive CFRs were recorded prior to administering the test
drug.
[0095] Representative results are shown in FIG. 5. The tracings
indicate that increasing amounts of glycoprotein Ib.alpha.
GPIb290/2V-Ig resulted in higher blood flow. These results
demonstrate that glycoprotein Ib.alpha. GPIb290/2V-Ig inhibits
thrombosis in the animal model.
[0096] A schematic illustration depicting an injured coronary
artery with high fluid shear blood flow is presented in FIG. 6. The
figure depicts an injured coronary arterty with high fluid shear
blood flow. The vessel has a segment of damaged endothelium that
exposes subendothelial matrix proteins, including immobilized vWF.
In the presence of GP1b alpha fusion polypeptide (GPIb-Ig), the vWF
binding site is blocked, thereby preventing platelet adherence via
the platelet-bound GPIb alpha within the GPIb-V-IX complex.
Lukocyte capture is also diminished.
OTHER EMBODIMENTS
[0097] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
Sequence CWU 1
1
201544PRTHomo sapiensDOMAIN(1)..(544)GP1b302-Ig 1Met Pro Leu Leu
Leu Leu Leu Leu Leu Leu Pro Ser Pro Leu His Pro 1 5 10 15His Pro
Ile Cys Glu Val Ser Lys Val Ala Ser His Leu Glu Val Asn 20 25 30Cys
Asp Lys Arg Asn Leu Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp 35 40
45Thr Thr Ile Leu His Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu
50 55 60Ala Thr Leu Met Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp
Arg 65 70 75 80Cys Glu Leu Thr Lys Leu Gln Val Asp Gly Thr Leu Pro
Val Leu Gly 85 90 95Thr Leu Asp Leu Ser His Asn Gln Leu Gln Ser Leu
Pro Leu Leu Gly 100 105 110Gln Thr Leu Pro Ala Leu Thr Val Leu Asp
Val Ser Phe Asn Arg Leu 115 120 125Thr Ser Leu Pro Leu Gly Ala Leu
Arg Gly Leu Gly Glu Leu Gln Glu 130 135 140Leu Tyr Leu Lys Gly Asn
Glu Leu Lys Thr Leu Pro Pro Gly Leu Leu145 150 155 160Thr Pro Thr
Pro Lys Leu Glu Lys Leu Ser Leu Ala Asn Asn Asn Leu 165 170 175Thr
Glu Leu Pro Ala Gly Leu Leu Asn Gly Leu Glu Asn Leu Asp Thr 180 185
190Leu Leu Leu Gln Glu Asn Ser Leu Tyr Thr Ile Pro Lys Gly Phe Phe
195 200 205Gly Ser His Leu Leu Pro Phe Ala Phe Leu His Gly Asn Pro
Trp Leu 210 215 220Cys Asn Cys Glu Ile Leu Tyr Phe Arg Arg Trp Leu
Gln Asp Asn Ala225 230 235 240Glu Asn Val Tyr Val Trp Lys Gln Gly
Val Asp Val Lys Ala Met Thr 245 250 255Ser Asn Val Ala Ser Val Gln
Cys Asp Asn Ser Asp Lys Phe Pro Val 260 265 270Tyr Lys Tyr Pro Gly
Lys Gly Cys Pro Thr Leu Gly Asp Glu Gly Asp 275 280 285Thr Asp Leu
Tyr Asp Tyr Tyr Pro Glu Glu Asp Thr Glu Gly Asp Lys 290 295 300Val
Arg Ala Thr Arg Thr Val Val Lys Phe Pro Thr Lys Ala Arg Pro305 310
315 320His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu Gly Ala Pro
Ser 325 330 335Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 340 345 350Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro 355 360 365Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 370 375 380Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val385 390 395 400Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 405 410 415Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys Thr 420 425
430Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
435 440 445Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys 450 455 460Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser465 470 475 480Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp 485 490 495Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 500 505 510Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 515 520 525Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535
5402544PRTHomo sapiensDOMAIN(1)..(544)GP1b302/2A-Ig 2Met Pro Leu
Leu Leu Leu Leu Leu Leu Leu Pro Ser Pro Leu His Pro 1 5 10 15His
Pro Ile Cys Glu Val Ser Lys Val Ala Ser His Leu Glu Val Asn 20 25
30Cys Asp Lys Arg Asn Leu Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp
35 40 45Thr Thr Ile Leu His Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser
Leu 50 55 60Ala Thr Leu Met Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu
Asp Arg 65 70 75 80Cys Glu Leu Thr Lys Leu Gln Val Asp Gly Thr Leu
Pro Val Leu Gly 85 90 95Thr Leu Asp Leu Ser His Asn Gln Leu Gln Ser
Leu Pro Leu Leu Gly 100 105 110Gln Thr Leu Pro Ala Leu Thr Val Leu
Asp Val Ser Phe Asn Arg Leu 115 120 125Thr Ser Leu Pro Leu Gly Ala
Leu Arg Gly Leu Gly Glu Leu Gln Glu 130 135 140Leu Tyr Leu Lys Gly
Asn Glu Leu Lys Thr Leu Pro Pro Gly Leu Leu145 150 155 160Thr Pro
Thr Pro Lys Leu Glu Lys Leu Ser Leu Ala Asn Asn Asn Leu 165 170
175Thr Glu Leu Pro Ala Gly Leu Leu Asn Gly Leu Glu Asn Leu Asp Thr
180 185 190Leu Leu Leu Gln Glu Asn Ser Leu Tyr Thr Ile Pro Lys Gly
Phe Phe 195 200 205Gly Ser His Leu Leu Pro Phe Ala Phe Leu His Gly
Asn Pro Trp Leu 210 215 220Cys Asn Cys Glu Ile Leu Tyr Phe Arg Arg
Trp Leu Gln Asp Asn Ala225 230 235 240Glu Asn Val Tyr Val Trp Lys
Gln Gly Val Asp Val Lys Ala Met Thr 245 250 255Ser Asn Val Ala Ser
Val Gln Cys Asp Asn Ser Asp Lys Phe Pro Val 260 265 270Tyr Lys Tyr
Pro Gly Lys Gly Cys Pro Thr Leu Gly Asp Glu Gly Asp 275 280 285Thr
Asp Leu Tyr Asp Tyr Tyr Pro Glu Glu Asp Thr Glu Gly Asp Lys 290 295
300Val Ala Ala Thr Ala Thr Val Val Lys Phe Pro Thr Lys Ala Arg
Pro305 310 315 320His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu
Gly Ala Pro Ser 325 330 335Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 340 345 350Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro 355 360 365Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala 370 375 380Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val385 390 395 400Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 405 410
415Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys Thr
420 425 430Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 435 440 445Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 450 455 460Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser465 470 475 480Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 485 490 495Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 500 505 510Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 515 520 525Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535
5403544PRTHomo sapiensDOMAIN(1)..(544)GP1b302/4X-Ig 3Met Pro Leu
Leu Leu Leu Leu Leu Leu Leu Pro Ser Pro Leu His Pro 1 5 10 15His
Pro Ile Cys Glu Val Ser Lys Val Ala Ser His Leu Glu Val Asn 20 25
30Cys Asp Lys Arg Asn Leu Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp
35 40 45Thr Thr Ile Leu His Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser
Leu 50 55 60Ala Thr Leu Met Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu
Asp Arg 65 70 75 80Cys Glu Leu Thr Lys Leu Gln Val Asp Gly Thr Leu
Pro Val Leu Gly 85 90 95Thr Leu Asp Leu Ser His Asn Gln Leu Gln Ser
Leu Pro Leu Leu Gly 100 105 110Gln Thr Leu Pro Ala Leu Thr Val Leu
Asp Val Ser Phe Asn Arg Leu 115 120 125Thr Ser Leu Pro Leu Gly Ala
Leu Arg Gly Leu Gly Glu Leu Gln Glu 130 135 140Leu Tyr Leu Lys Gly
Asn Glu Leu Lys Thr Leu Pro Pro Gly Leu Leu145 150 155 160Thr Pro
Thr Pro Lys Leu Glu Lys Leu Ser Leu Ala Asn Asn Asn Leu 165 170
175Thr Glu Leu Pro Ala Gly Leu Leu Asn Gly Leu Glu Asn Leu Asp Thr
180 185 190Leu Leu Leu Gln Glu Asn Ser Leu Tyr Thr Ile Pro Lys Gly
Phe Phe 195 200 205Gly Ser His Leu Leu Pro Phe Ala Phe Leu His Gly
Asn Pro Trp Leu 210 215 220Cys Asn Cys Glu Ile Leu Tyr Phe Arg Arg
Trp Leu Gln Asp Asn Ala225 230 235 240Glu Asn Val Tyr Val Trp Lys
Gln Val Val Asp Val Lys Ala Val Thr 245 250 255Ser Asn Val Ala Ser
Val Gln Cys Asp Asn Ser Asp Lys Phe Pro Val 260 265 270Tyr Lys Tyr
Pro Gly Lys Gly Cys Pro Thr Leu Gly Asp Glu Gly Asp 275 280 285Thr
Asp Leu Tyr Asp Tyr Tyr Pro Glu Glu Asp Thr Glu Gly Asp Lys 290 295
300Val Ala Ala Thr Ala Thr Val Val Lys Phe Pro Thr Lys Ala Arg
Pro305 310 315 320His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu
Gly Ala Pro Ser 325 330 335Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 340 345 350Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro 355 360 365Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala 370 375 380Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val385 390 395 400Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 405 410
415Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys Thr
420 425 430Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 435 440 445Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 450 455 460Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser465 470 475 480Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 485 490 495Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 500 505 510Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 515 520 525Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535
5404531PRTHomo sapiensDOMAIN(1)..(531)GP1b290-Ig 4Met Pro Leu Leu
Leu Leu Leu Leu Leu Leu Pro Ser Pro Leu His Pro 1 5 10 15His Pro
Ile Cys Glu Val Ser Lys Val Ala Ser His Leu Glu Val Asn 20 25 30Cys
Asp Lys Arg Asn Leu Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp 35 40
45Thr Thr Ile Leu His Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu
50 55 60Ala Thr Leu Met Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp
Arg 65 70 75 80Cys Glu Leu Thr Lys Leu Gln Val Asp Gly Thr Leu Pro
Val Leu Gly 85 90 95Thr Leu Asp Leu Ser His Asn Gln Leu Gln Ser Leu
Pro Leu Leu Gly 100 105 110Gln Thr Leu Pro Ala Leu Thr Val Leu Asp
Val Ser Phe Asn Arg Leu 115 120 125Thr Ser Leu Pro Leu Gly Ala Leu
Arg Gly Leu Gly Glu Leu Gln Glu 130 135 140Leu Tyr Leu Lys Gly Asn
Glu Leu Lys Thr Leu Pro Pro Gly Leu Leu145 150 155 160Thr Pro Thr
Pro Lys Leu Glu Lys Leu Ser Leu Ala Asn Asn Asn Leu 165 170 175Thr
Glu Leu Pro Ala Gly Leu Leu Asn Gly Leu Glu Asn Leu Asp Thr 180 185
190Leu Leu Leu Gln Glu Asn Ser Leu Tyr Thr Ile Pro Lys Gly Phe Phe
195 200 205Gly Ser His Leu Leu Pro Phe Ala Phe Leu His Gly Asn Pro
Trp Leu 210 215 220Cys Asn Cys Glu Ile Leu Tyr Phe Arg Arg Trp Leu
Gln Asp Asn Ala225 230 235 240Glu Asn Val Tyr Val Trp Lys Gln Gly
Val Asp Val Lys Ala Met Thr 245 250 255Ser Asn Val Ala Ser Val Gln
Cys Asp Asn Ser Asp Lys Phe Pro Val 260 265 270Tyr Lys Tyr Pro Gly
Lys Gly Cys Pro Thr Leu Gly Asp Glu Gly Asp 275 280 285Thr Asp Leu
Tyr Asp Tyr Tyr Pro Glu Glu Asp Thr Glu Gly Asp Lys 290 295 300Val
Arg Pro His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu Gly305 310
315 320Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 325 330 335Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 340 345 350Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 355 360 365His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 370 375 380Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly385 390 395 400Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile 405 410 415Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 420 425
430Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
435 440 445Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 450 455 460Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro465 470 475 480Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 485 490 495Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 500 505 510His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 515 520 525Pro Gly Lys
5305531PRTHomo sapiensDOMAIN(1)..(531)GP1b290/2V-Ig 5Met Pro Leu
Leu Leu Leu Leu Leu Leu Leu Pro Ser Pro Leu His Pro 1 5 10 15His
Pro Ile Cys Glu Val Ser Lys Val Ala Ser His Leu Glu Val Asn 20 25
30Cys Asp Lys Arg Asn Leu Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp
35 40 45Thr Thr Ile Leu His Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser
Leu 50 55 60Ala Thr Leu Met Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu
Asp Arg 65 70 75 80Cys Glu Leu Thr Lys Leu Gln Val Asp Gly Thr Leu
Pro Val Leu Gly 85 90 95Thr Leu Asp Leu Ser His Asn Gln Leu Gln Ser
Leu Pro Leu Leu Gly 100 105 110Gln Thr Leu Pro Ala Leu Thr Val Leu
Asp Val Ser Phe Asn Arg Leu 115 120 125Thr Ser Leu Pro Leu Gly Ala
Leu Arg Gly Leu Gly Glu Leu Gln Glu 130 135 140Leu Tyr Leu Lys Gly
Asn Glu Leu Lys Thr Leu Pro Pro Gly Leu Leu145 150 155 160Thr Pro
Thr Pro Lys Leu Glu Lys Leu Ser Leu Ala Asn Asn Asn Leu 165 170
175Thr Glu Leu Pro Ala Gly Leu Leu Asn Gly Leu Glu Asn Leu Asp Thr
180 185 190Leu Leu Leu Gln Glu Asn Ser Leu Tyr Thr Ile Pro Lys Gly
Phe Phe 195 200 205Gly Ser His Leu Leu Pro Phe Ala Phe Leu His Gly
Asn Pro Trp Leu 210 215 220Cys Asn Cys Glu Ile Leu Tyr Phe Arg Arg
Trp Leu Gln Asp Asn Ala225 230 235 240Glu Asn Val Tyr Val Trp Lys
Gln Val Val Asp Val Lys Ala Val Thr 245 250 255Ser Asn Val Ala Ser
Val Gln Cys Asp Asn Ser Asp Lys Phe Pro Val 260 265 270Tyr Lys Tyr
Pro Gly Lys Gly Cys Pro Thr Leu
Gly Asp Glu Gly Asp 275 280 285Thr Asp Leu Tyr Asp Tyr Tyr Pro Glu
Glu Asp Thr Glu Gly Asp Lys 290 295 300Val Arg Pro His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Leu Gly305 310 315 320Ala Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 325 330 335Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 340 345
350Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
355 360 365His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 370 375 380Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly385 390 395 400Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Val Pro Ile 405 410 415Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 420 425 430Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 435 440 445Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 450 455 460Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro465 470
475 480Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 485 490 495Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 500 505 510His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 515 520 525Pro Gly Lys 5306531PRTHomo
sapiensDOMAIN(1)..(531)GP1b290/1A-Ig 6Met Pro Leu Leu Leu Leu Leu
Leu Leu Leu Pro Ser Pro Leu His Pro 1 5 10 15His Pro Ile Cys Glu
Val Ser Lys Val Ala Ser His Leu Glu Val Asn 20 25 30Cys Asp Lys Arg
Asn Leu Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp 35 40 45Thr Thr Ile
Leu His Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu 50 55 60Ala Thr
Leu Met Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp Arg 65 70 75
80Cys Glu Leu Thr Lys Leu Gln Val Asp Gly Thr Leu Pro Val Leu Gly
85 90 95Thr Leu Asp Leu Ser His Asn Gln Leu Gln Ser Leu Pro Leu Leu
Gly 100 105 110Gln Thr Leu Pro Ala Leu Thr Val Leu Asp Val Ser Phe
Asn Arg Leu 115 120 125Thr Ser Leu Pro Leu Gly Ala Leu Arg Gly Leu
Gly Glu Leu Gln Glu 130 135 140Leu Tyr Leu Lys Gly Asn Glu Leu Lys
Thr Leu Pro Pro Gly Leu Leu145 150 155 160Thr Pro Thr Pro Lys Leu
Glu Lys Leu Ser Leu Ala Asn Asn Asn Leu 165 170 175Thr Glu Leu Pro
Ala Gly Leu Leu Asn Gly Leu Glu Asn Leu Asp Thr 180 185 190Leu Leu
Leu Gln Glu Asn Ser Leu Tyr Thr Ile Pro Lys Gly Phe Phe 195 200
205Gly Ser His Leu Leu Pro Phe Ala Phe Leu His Gly Asn Pro Trp Leu
210 215 220Cys Asn Cys Glu Ile Leu Tyr Phe Arg Arg Trp Leu Gln Asp
Asn Ala225 230 235 240Glu Asn Val Tyr Val Trp Lys Gln Gly Val Asp
Val Ala Ala Met Thr 245 250 255Ser Asn Val Ala Ser Val Gln Cys Asp
Asn Ser Asp Lys Phe Pro Val 260 265 270Tyr Lys Tyr Pro Gly Lys Gly
Cys Pro Thr Leu Gly Asp Glu Gly Asp 275 280 285Thr Asp Leu Tyr Asp
Tyr Tyr Pro Glu Glu Asp Thr Glu Gly Asp Lys 290 295 300Val Arg Pro
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu Gly305 310 315
320Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
325 330 335Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 340 345 350Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 355 360 365His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 370 375 380Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly385 390 395 400Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile 405 410 415Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 420 425 430Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 435 440
445Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
450 455 460Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro465 470 475 480Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 485 490 495Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 500 505 510His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 515 520 525Pro Gly Lys
5307302PRTHomo sapiensDOMAIN(1)..(302)GP1b302 7His Pro Ile Cys Glu
Val Ser Lys Val Ala Ser His Leu Glu Val Asn 1 5 10 15Cys Asp Lys
Arg Asn Leu Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp 20 25 30Thr Thr
Ile Leu His Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu 35 40 45Ala
Thr Leu Met Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp Arg 50 55
60Cys Glu Leu Thr Lys Leu Gln Val Asp Gly Thr Leu Pro Val Leu Gly
65 70 75 80Thr Leu Asp Leu Ser His Asn Gln Leu Gln Ser Leu Pro Leu
Leu Gly 85 90 95Gln Thr Leu Pro Ala Leu Thr Val Leu Asp Val Ser Phe
Asn Arg Leu 100 105 110Thr Ser Leu Pro Leu Gly Ala Leu Arg Gly Leu
Gly Glu Leu Gln Glu 115 120 125Leu Tyr Leu Lys Gly Asn Glu Leu Lys
Thr Leu Pro Pro Gly Leu Leu 130 135 140Thr Pro Thr Pro Lys Leu Glu
Lys Leu Ser Leu Ala Asn Asn Asn Leu145 150 155 160Thr Glu Leu Pro
Ala Gly Leu Leu Asn Gly Leu Glu Asn Leu Asp Thr 165 170 175Leu Leu
Leu Gln Glu Asn Ser Leu Tyr Thr Ile Pro Lys Gly Phe Phe 180 185
190Gly Ser His Leu Leu Pro Phe Ala Phe Leu His Gly Asn Pro Trp Leu
195 200 205Cys Asn Cys Glu Ile Leu Tyr Phe Arg Arg Trp Leu Gln Asp
Asn Ala 210 215 220Glu Asn Val Tyr Val Trp Lys Gln Gly Val Asp Val
Lys Ala Met Thr225 230 235 240Ser Asn Val Ala Ser Val Gln Cys Asp
Asn Ser Asp Lys Phe Pro Val 245 250 255Tyr Lys Tyr Pro Gly Lys Gly
Cys Pro Thr Leu Gly Asp Glu Gly Asp 260 265 270Thr Asp Leu Tyr Asp
Tyr Tyr Pro Glu Glu Asp Thr Glu Gly Asp Lys 275 280 285Val Arg Ala
Thr Arg Thr Val Val Lys Phe Pro Thr Lys Ala 290 295 3008302PRTHomo
sapiensDOMAIN(1)..(302)GP1b302/2A 8His Pro Ile Cys Glu Val Ser Lys
Val Ala Ser His Leu Glu Val Asn 1 5 10 15Cys Asp Lys Arg Asn Leu
Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp 20 25 30Thr Thr Ile Leu His
Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu 35 40 45Ala Thr Leu Met
Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp Arg 50 55 60Cys Glu Leu
Thr Lys Leu Gln Val Asp Gly Thr Leu Pro Val Leu Gly 65 70 75 80Thr
Leu Asp Leu Ser His Asn Gln Leu Gln Ser Leu Pro Leu Leu Gly 85 90
95Gln Thr Leu Pro Ala Leu Thr Val Leu Asp Val Ser Phe Asn Arg Leu
100 105 110Thr Ser Leu Pro Leu Gly Ala Leu Arg Gly Leu Gly Glu Leu
Gln Glu 115 120 125Leu Tyr Leu Lys Gly Asn Glu Leu Lys Thr Leu Pro
Pro Gly Leu Leu 130 135 140Thr Pro Thr Pro Lys Leu Glu Lys Leu Ser
Leu Ala Asn Asn Asn Leu145 150 155 160Thr Glu Leu Pro Ala Gly Leu
Leu Asn Gly Leu Glu Asn Leu Asp Thr 165 170 175Leu Leu Leu Gln Glu
Asn Ser Leu Tyr Thr Ile Pro Lys Gly Phe Phe 180 185 190Gly Ser His
Leu Leu Pro Phe Ala Phe Leu His Gly Asn Pro Trp Leu 195 200 205Cys
Asn Cys Glu Ile Leu Tyr Phe Arg Arg Trp Leu Gln Asp Asn Ala 210 215
220Glu Asn Val Tyr Val Trp Lys Gln Gly Val Asp Val Lys Ala Met
Thr225 230 235 240Ser Asn Val Ala Ser Val Gln Cys Asp Asn Ser Asp
Lys Phe Pro Val 245 250 255Tyr Lys Tyr Pro Gly Lys Gly Cys Pro Thr
Leu Gly Asp Glu Gly Asp 260 265 270Thr Asp Leu Tyr Asp Tyr Tyr Pro
Glu Glu Asp Thr Glu Gly Asp Lys 275 280 285Val Ala Ala Thr Ala Thr
Val Val Lys Phe Pro Thr Lys Ala 290 295 3009301PRTHomo
sapiensDOMAIN(1)..(301)GP1b/4X 9His Pro Ile Cys Glu Val Ser Lys Val
Ala Ser His Leu Glu Val Asn 1 5 10 15Cys Asp Lys Arg Asn Leu Thr
Ala Leu Pro Pro Asp Leu Pro Lys Asp 20 25 30Thr Thr Ile Leu His Leu
Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu 35 40 45Ala Thr Leu Met Pro
Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp Arg 50 55 60Cys Glu Leu Thr
Lys Leu Gln Val Asp Gly Thr Leu Pro Val Leu Gly 65 70 75 80Thr Leu
Asp Leu Ser His Asn Gln Leu Gln Ser Leu Pro Leu Leu Gly 85 90 95Gln
Thr Leu Pro Ala Leu Thr Val Leu Asp Val Ser Phe Asn Arg Leu 100 105
110Thr Ser Leu Pro Leu Gly Ala Leu Arg Gly Leu Gly Glu Leu Gln Glu
115 120 125Leu Tyr Leu Lys Gly Asn Glu Leu Lys Thr Leu Pro Pro Gly
Leu Leu 130 135 140Thr Pro Thr Pro Lys Leu Glu Lys Leu Ser Leu Ala
Asn Asn Asn Leu145 150 155 160Thr Glu Leu Pro Ala Gly Leu Leu Asn
Gly Leu Glu Asn Leu Asp Thr 165 170 175Leu Leu Leu Gln Glu Asn Ser
Leu Tyr Thr Ile Pro Lys Gly Phe Phe 180 185 190Gly Ser His Leu Leu
Pro Phe Ala Phe Leu His Gly Asn Pro Trp Leu 195 200 205Cys Asn Cys
Glu Ile Leu Tyr Phe Arg Arg Trp Leu Gln Asp Asn Ala 210 215 220Glu
Asn Val Tyr Val Trp Lys Gln Val Val Asp Val Lys Ala Val Thr225 230
235 240Ser Asn Val Ala Ser Val Gln Cys Asp Asn Ser Asp Lys Phe Pro
Val 245 250 255Lys Tyr Pro Gly Lys Gly Cys Pro Thr Leu Gly Asp Glu
Gly Asp Thr 260 265 270Asp Leu Tyr Asp Tyr Tyr Pro Glu Glu Asp Thr
Glu Gly Asp Lys Val 275 280 285Ala Ala Thr Ala Thr Val Val Lys Phe
Pro Thr Lys Ala 290 295 30010290PRTHomo
sapiensDOMAIN(1)..(290)GP1b290 10His Pro Ile Cys Glu Val Ser Lys
Val Ala Ser His Leu Glu Val Asn 1 5 10 15Cys Asp Lys Arg Asn Leu
Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp 20 25 30Thr Thr Ile Leu His
Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu 35 40 45Ala Thr Leu Met
Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp Arg 50 55 60Cys Glu Leu
Thr Lys Leu Gln Val Asp Gly Thr Leu Pro Val Leu Gly 65 70 75 80Thr
Leu Asp Leu Ser His Asn Gln Leu Gln Ser Leu Pro Leu Leu Gly 85 90
95Gln Thr Leu Pro Ala Leu Thr Val Leu Asp Val Ser Phe Asn Arg Leu
100 105 110Thr Ser Leu Pro Leu Gly Ala Leu Arg Gly Leu Gly Glu Leu
Gln Glu 115 120 125Leu Tyr Leu Lys Gly Asn Glu Leu Lys Thr Leu Pro
Pro Gly Leu Leu 130 135 140Thr Pro Thr Pro Lys Leu Glu Lys Leu Ser
Leu Ala Asn Asn Asn Leu145 150 155 160Thr Glu Leu Pro Ala Gly Leu
Leu Asn Gly Leu Glu Asn Leu Asp Thr 165 170 175Leu Leu Leu Gln Glu
Asn Ser Leu Tyr Thr Ile Pro Lys Gly Phe Phe 180 185 190Gly Ser His
Leu Leu Pro Phe Ala Phe Leu His Gly Asn Pro Trp Leu 195 200 205Cys
Asn Cys Glu Ile Leu Tyr Phe Arg Arg Trp Leu Gln Asp Asn Ala 210 215
220Glu Asn Val Tyr Val Trp Lys Gln Gly Val Asp Val Lys Ala Met
Thr225 230 235 240Ser Asn Val Ala Ser Val Gln Cys Asp Asn Ser Asp
Lys Phe Pro Val 245 250 255Tyr Lys Tyr Pro Gly Lys Gly Cys Pro Thr
Leu Gly Asp Glu Gly Asp 260 265 270Thr Asp Leu Tyr Asp Tyr Tyr Pro
Glu Glu Asp Thr Glu Gly Asp Lys 275 280 285Val Arg 29011290PRTHomo
sapiensDOMAIN(1)..(290)GB1b290/2V 11His Pro Ile Cys Glu Val Ser Lys
Val Ala Ser His Leu Glu Val Asn 1 5 10 15Cys Asp Lys Arg Asn Leu
Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp 20 25 30Thr Thr Ile Leu His
Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu 35 40 45Ala Thr Leu Met
Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp Arg 50 55 60Cys Glu Leu
Thr Lys Leu Gln Val Asp Gly Thr Leu Pro Val Leu Gly 65 70 75 80Thr
Leu Asp Leu Ser His Asn Gln Leu Gln Ser Leu Pro Leu Leu Gly 85 90
95Gln Thr Leu Pro Ala Leu Thr Val Leu Asp Val Ser Phe Asn Arg Leu
100 105 110Thr Ser Leu Pro Leu Gly Ala Leu Arg Gly Leu Gly Glu Leu
Gln Glu 115 120 125Leu Tyr Leu Lys Gly Asn Glu Leu Lys Thr Leu Pro
Pro Gly Leu Leu 130 135 140Thr Pro Thr Pro Lys Leu Glu Lys Leu Ser
Leu Ala Asn Asn Asn Leu145 150 155 160Thr Glu Leu Pro Ala Gly Leu
Leu Asn Gly Leu Glu Asn Leu Asp Thr 165 170 175Leu Leu Leu Gln Glu
Asn Ser Leu Tyr Thr Ile Pro Lys Gly Phe Phe 180 185 190Gly Ser His
Leu Leu Pro Phe Ala Phe Leu His Gly Asn Pro Trp Leu 195 200 205Cys
Asn Cys Glu Ile Leu Tyr Phe Arg Arg Trp Leu Gln Asp Asn Ala 210 215
220Glu Asn Val Tyr Val Trp Lys Gln Val Val Asp Val Lys Ala Val
Thr225 230 235 240Ser Asn Val Ala Ser Val Gln Cys Asp Asn Ser Asp
Lys Phe Pro Val 245 250 255Tyr Lys Tyr Pro Gly Lys Gly Cys Pro Thr
Leu Gly Asp Glu Gly Asp 260 265 270Thr Asp Leu Tyr Asp Tyr Tyr Pro
Glu Glu Asp Thr Glu Gly Asp Lys 275 280 285Val Arg 29012290PRTHomo
sapiensDOMAIN(1)..(290)GB1b290/1A 12His Pro Ile Cys Glu Val Ser Lys
Val Ala Ser His Leu Glu Val Asn 1 5 10 15Cys Asp Lys Arg Asn Leu
Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp 20 25 30Thr Thr Ile Leu His
Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu 35 40 45Ala Thr Leu Met
Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp Arg 50 55 60Cys Glu Leu
Thr Lys Leu Gln Val Asp Gly Thr Leu Pro Val Leu Gly 65 70 75 80Thr
Leu Asp Leu Ser His Asn Gln Leu Gln Ser Leu Pro Leu Leu Gly 85 90
95Gln Thr Leu Pro Ala Leu Thr Val Leu Asp Val Ser Phe Asn Arg Leu
100 105 110Thr Ser Leu Pro Leu Gly Ala Leu Arg Gly Leu Gly Glu Leu
Gln Glu 115 120 125Leu Tyr Leu Lys Gly Asn Glu Leu Lys Thr Leu Pro
Pro Gly Leu Leu 130 135 140Thr Pro Thr Pro Lys Leu Glu Lys Leu Ser
Leu Ala Asn Asn Asn Leu145 150 155 160Thr Glu Leu Pro Ala Gly Leu
Leu Asn Gly Leu Glu Asn Leu Asp Thr
165 170 175Leu Leu Leu Gln Glu Asn Ser Leu Tyr Thr Ile Pro Lys Gly
Phe Phe 180 185 190Gly Ser His Leu Leu Pro Phe Ala Phe Leu His Gly
Asn Pro Trp Leu 195 200 205Cys Asn Cys Glu Ile Leu Tyr Phe Arg Arg
Trp Leu Gln Asp Asn Ala 210 215 220Glu Asn Val Tyr Val Trp Lys Gln
Gly Val Asp Val Ala Ala Met Thr225 230 235 240Ser Asn Val Ala Ser
Val Gln Cys Asp Asn Ser Asp Lys Phe Pro Val 245 250 255Tyr Lys Tyr
Pro Gly Lys Gly Cys Pro Thr Leu Gly Asp Glu Gly Asp 260 265 270Thr
Asp Leu Tyr Asp Tyr Tyr Pro Glu Glu Asp Thr Glu Gly Asp Lys 275 280
285Val Arg 2901316PRTHomo sapiensDOMAIN(1)..(16)Signal Peptide
13Met Pro Leu Leu Leu Leu Leu Leu Leu Leu Pro Ser Pro Leu His Pro 1
5 10 1514224PRTHomo sapiensDOMAIN(1)..(224) 14His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Leu Gly Ala Pro Ser 1 5 10 15Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55
60Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
65 70 75 80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr 85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile
Glu Lys Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 115 120 125Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys 130 135 140Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185
190Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 210 215 220151632DNAHomo sapiens 15atgcctctcc tcctcttgct
gctcctgctg ccaagcccct tacaccccca ccccatctgt 60gaggtctcca aagtggccag
ccacctagaa gtgaactgtg acaagaggaa tctgacagcg 120ctgcctccag
acctgccgaa agacacaacc atcctccacc tgagtgagaa cctcctgtac
180accttctccc tggcaaccct gatgccttac actcgcctca ctcagctgaa
cctagatagg 240tgcgagctca ccaagctcca ggtcgatggg acgctgccag
tgctggggac cctggatcta 300tcccacaatc agctgcaaag cctgcccttg
ctagggcaga cactgcctgc tctcaccgtc 360ctggacgtct ccttcaaccg
gctgacctcg ctgcctcttg gtgccctgcg tggtcttggc 420gaactccaag
agctctacct gaaaggcaat gagctgaaga ccctgccccc agggctcctg
480acgcccacac ccaagctgga gaagctcagt ctggctaaca acaacttgac
tgagctcccc 540gctgggctcc tgaatgggct ggagaatctc gacacccttc
tcctccaaga gaactcgctg 600tatacaatac caaagggctt ttttgggtcc
cacctcctgc cttttgcttt tctccacggg 660aacccctggt tatgcaactg
tgagatcctc tattttcgtc gctggctgca ggacaatgct 720gaaaatgtct
acgtatggaa gcaaggtgtg gacgtcaagg ccatgacctc taacgtggcc
780agtgtgcagt gtgacaattc agacaagttt cccgtctaca aatacccagg
aaaggggtgc 840cccacccttg gtgatgaagg tgacacagac ctatatgatt
actacccaga agaggacact 900gagggcgata aggtgcgtgc cacaaggact
gtggtcaagt tccccaccaa agcgcggccg 960cacacatgcc caccgtgccc
agcacctgaa gccctggggg caccgtcagt cttcctcttc 1020cccccaaaac
ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg
1080gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga
cggcgtggag 1140gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
acagcacgta ccgtgtggtc 1200agcgtcctca ccgtcctgca ccaggactgg
ctgaatggca aggagtacaa gtgcaaggtc 1260tccaacaaag ccctcccagt
ccccatcgag aaaaccatct ccaaagccaa agggcagccc 1320cgagaaccac
aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc
1380agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga
gtgggagagc 1440aatgggcagc cggagaacaa ctacaagacc acgcctcccg
tgctggactc cgacggcccc 1500ttcttcctct acagcaagct caccgtggac
aagagcaggt ggcagcaggg gaacgtcttc 1560tcatgctccg tgatgcatga
ggctctgcac aaccactaca cgcagaagag cctctccctg 1620tctccgggta aa
1632161632DNAHomo sapiens 16atgcctctcc tcctcttgct gctcctgctg
ccaagcccct tacaccccca ccccatctgt 60gaggtctcca aagtggccag ccacctagaa
gtgaactgtg acaagaggaa tctgacagcg 120ctgcctccag acctgccgaa
agacacaacc atcctccacc tgagtgagaa cctcctgtac 180accttctccc
tggcaaccct gatgccttac actcgcctca ctcagctgaa cctagatagg
240tgcgagctca ccaagctcca ggtcgatggg acgctgccag tgctggggac
cctggatcta 300tcccacaatc agctgcaaag cctgcccttg ctagggcaga
cactgcctgc tctcaccgtc 360ctggacgtct ccttcaaccg gctgacctcg
ctgcctcttg gtgccctgcg tggtcttggc 420gaactccaag agctctacct
gaaaggcaat gagctgaaga ccctgccccc agggctcctg 480acgcccacac
ccaagctgga gaagctcagt ctggctaaca acaacttgac tgagctcccc
540gctgggctcc tgaatgggct ggagaatctc gacacccttc tcctccaaga
gaactcgctg 600tatacaatac caaagggctt ttttgggtcc cacctcctgc
cttttgcttt tctccacggg 660aacccctggt tatgcaactg tgagatcctc
tattttcgtc gctggctgca ggacaatgct 720gaaaatgtct acgtatggaa
gcaaggtgtg gacgtcaagg ccatgacctc taacgtggcc 780agtgtgcagt
gtgacaattc agacaagttt cccgtctaca aatacccagg aaaggggtgc
840cccacccttg gtgatgaagg tgacacagac ctatatgatt actacccaga
agaggacact 900gagggcgata aggtggctgc cacagcgact gtggtcaagt
tccccaccaa agcgcggccg 960cacacatgcc caccgtgccc agcacctgaa
gccctggggg caccgtcagt cttcctcttc 1020cccccaaaac ccaaggacac
cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 1080gtggacgtga
gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag
1140gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta
ccgtgtggtc 1200agcgtcctca ccgtcctgca ccaggactgg ctgaatggca
aggagtacaa gtgcaaggtc 1260tccaacaaag ccctcccagt ccccatcgag
aaaaccatct ccaaagccaa agggcagccc 1320cgagaaccac aggtgtacac
cctgccccca tcccgggagg agatgaccaa gaaccaggtc 1380agcctgacct
gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc
1440aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc
cgacggcccc 1500ttcttcctct acagcaagct caccgtggac aagagcaggt
ggcagcaggg gaacgtcttc 1560tcatgctccg tgatgcatga ggctctgcac
aaccactaca cgcagaagag cctctccctg 1620tctccgggta aa
1632171632DNAHomo sapiens 17atgcctctcc tcctcttgct gctcctgctg
ccaagcccct tacaccccca ccccatctgt 60gaggtctcca aagtggccag ccacctagaa
gtgaactgtg acaagaggaa tctgacagcg 120ctgcctccag acctgccgaa
agacacaacc atcctccacc tgagtgagaa cctcctgtac 180accttctccc
tggcaaccct gatgccttac actcgcctca ctcagctgaa cctagatagg
240tgcgagctca ccaagctcca ggtcgatggg acgctgccag tgctggggac
cctggatcta 300tcccacaatc agctgcaaag cctgcccttg ctagggcaga
cactgcctgc tctcaccgtc 360ctggacgtct ccttcaaccg gctgacctcg
ctgcctcttg gtgccctgcg tggtcttggc 420gaactccaag agctctacct
gaaaggcaat gagctgaaga ccctgccccc agggctcctg 480acgcccacac
ccaagctgga gaagctcagt ctggctaaca acaacttgac tgagctcccc
540gctgggctcc tgaatgggct ggagaatctc gacacccttc tcctccaaga
gaactcgctg 600tatacaatac caaagggctt ttttgggtcc cacctcctgc
cttttgcttt tctccacggg 660aacccctggt tatgcaactg tgagatcctc
tattttcgtc gctggctgca ggacaatgct 720gaaaatgtct acgtatggaa
gcaagtggtg gacgtcaagg ccgtgacctc taacgtggcc 780agtgtgcagt
gtgacaattc agacaagttt cccgtctaca aatacccagg aaaggggtgc
840cccacccttg gtgatgaagg tgacacagac ctatatgatt actacccaga
agaggacact 900gagggcgata aggtggctgc cacagcgact gtggtcaagt
tccccaccaa agcgcggccg 960cacacatgcc caccgtgccc agcacctgaa
gccctggggg caccgtcagt cttcctcttc 1020cccccaaaac ccaaggacac
cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 1080gtggacgtga
gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag
1140gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta
ccgtgtggtc 1200agcgtcctca ccgtcctgca ccaggactgg ctgaatggca
aggagtacaa gtgcaaggtc 1260tccaacaaag ccctcccagt ccccatcgag
aaaaccatct ccaaagccaa agggcagccc 1320cgagaaccac aggtgtacac
cctgccccca tcccgggagg agatgaccaa gaaccaggtc 1380agcctgacct
gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc
1440aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc
cgacggcccc 1500ttcttcctct acagcaagct caccgtggac aagagcaggt
ggcagcaggg gaacgtcttc 1560tcatgctccg tgatgcatga ggctctgcac
aaccactaca cgcagaagag cctctccctg 1620tctccgggta aa
1632181593DNAHomo sapiens 18atgcctctcc tcctcttgct gctcctgctg
ccaagcccct tacaccccca ccccatctgt 60gaggtctcca aagtggccag ccacctagaa
gtgaactgtg acaagaggaa tctgacagcg 120ctgcctccag acctgccgaa
agacacaacc atcctccacc tgagtgagaa cctcctgtac 180accttctccc
tggcaaccct gatgccttac actcgcctca ctcagctgaa cctagatagg
240tgcgagctca ccaagctcca ggtcgatggg acgctgccag tgctggggac
cctggatcta 300tcccacaatc agctgcaaag cctgcccttg ctagggcaga
cactgcctgc tctcaccgtc 360ctggacgtct ccttcaaccg gctgacctcg
ctgcctcttg gtgccctgcg tggtcttggc 420gaactccaag agctctacct
gaaaggcaat gagctgaaga ccctgccccc agggctcctg 480acgcccacac
ccaagctgga gaagctcagt ctggctaaca acaacttgac tgagctcccc
540gctgggctcc tgaatgggct ggagaatctc gacacccttc tcctccaaga
gaactcgctg 600tatacaatac caaagggctt ttttgggtcc cacctcctgc
cttttgcttt tctccacggg 660aacccctggt tatgcaactg tgagatcctc
tattttcgtc gctggctgca ggacaatgct 720gaaaatgtct acgtatggaa
gcaaggtgtg gacgtcaagg ccatgacctc taacgtggcc 780agtgtgcagt
gtgacaattc agacaagttt cccgtctaca aatacccagg aaaggggtgc
840cccacccttg gtgatgaagg tgacacagac ctatatgatt actacccaga
agaggacact 900gagggcgata aggtgcggcc gcacacatgc ccaccgtgcc
cagcacctga agccctgggg 960gcaccgtcag tcttcctctt ccccccaaaa
cccaaggaca ccctcatgat ctcccggacc 1020cctgaggtca catgcgtggt
ggtggacgtg agccacgaag accctgaggt caagttcaac 1080tggtacgtgg
acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac
1140aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg
gctgaatggc 1200aaggagtaca agtgcaaggt ctccaacaaa gccctcccag
tccccatcga gaaaaccatc 1260tccaaagcca aagggcagcc ccgagaacca
caggtgtaca ccctgccccc atcccgggag 1320gagatgacca agaaccaggt
cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1380atcgccgtgg
agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
1440gtgctggact ccgacggccc cttcttcctc tacagcaagc tcaccgtgga
caagagcagg 1500tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg
aggctctgca caaccactac 1560acgcagaaga gcctctccct gtctccgggt aaa
1593191593DNAHomo sapiens 19atgcctctcc tcctcttgct gctcctgctg
ccaagcccct tacaccccca ccccatctgt 60gaggtctcca aagtggccag ccacctagaa
gtgaactgtg acaagaggaa tctgacagcg 120ctgcctccag acctgccgaa
agacacaacc atcctccacc tgagtgagaa cctcctgtac 180accttctccc
tggcaaccct gatgccttac actcgcctca ctcagctgaa cctagatagg
240tgcgagctca ccaagctcca ggtcgatggg acgctgccag tgctggggac
cctggatcta 300tcccacaatc agctgcaaag cctgcccttg ctagggcaga
cactgcctgc tctcaccgtc 360ctggacgtct ccttcaaccg gctgacctcg
ctgcctcttg gtgccctgcg tggtcttggc 420gaactccaag agctctacct
gaaaggcaat gagctgaaga ccctgccccc agggctcctg 480acgcccacac
ccaagctgga gaagctcagt ctggctaaca acaacttgac tgagctcccc
540gctgggctcc tgaatgggct ggagaatctc gacacccttc tcctccaaga
gaactcgctg 600tatacaatac caaagggctt ttttgggtcc cacctcctgc
cttttgcttt tctccacggg 660aacccctggt tatgcaactg tgagatcctc
tattttcgtc gctggctgca ggacaatgct 720gaaaatgtct acgtatggaa
gcaagtggtg gacgtcaagg ccgtgacctc taacgtggcc 780agtgtgcagt
gtgacaattc agacaagttt cccgtctaca aatacccagg aaaggggtgc
840cccacccttg gtgatgaagg tgacacagac ctatatgatt actacccaga
agaggacact 900gagggcgata aggtgcggcc gcacacatgc ccaccgtgcc
cagcacctga agccctgggg 960gcaccgtcag tcttcctctt ccccccaaaa
cccaaggaca ccctcatgat ctcccggacc 1020cctgaggtca catgcgtggt
ggtggacgtg agccacgaag accctgaggt caagttcaac 1080tggtacgtgg
acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac
1140aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg
gctgaatggc 1200aaggagtaca agtgcaaggt ctccaacaaa gccctcccag
tccccatcga gaaaaccatc 1260tccaaagcca aagggcagcc ccgagaacca
caggtgtaca ccctgccccc atcccgggag 1320gagatgacca agaaccaggt
cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1380atcgccgtgg
agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
1440gtgctggact ccgacggccc cttcttcctc tacagcaagc tcaccgtgga
caagagcagg 1500tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg
aggctctgca caaccactac 1560acgcagaaga gcctctccct gtctccgggt aaa
1593201593DNAHomo sapiens 20atgcctctcc tcctcttgct gctcctgctg
ccaagcccct tacaccccca ccccatctgt 60gaggtctcca aagtggccag ccacctagaa
gtgaactgtg acaagaggaa tctgacagcg 120ctgcctccag acctgccgaa
agacacaacc atcctccacc tgagtgagaa cctcctgtac 180accttctccc
tggcaaccct gatgccttac actcgcctca ctcagctgaa cctagatagg
240tgcgagctca ccaagctcca ggtcgatggg acgctgccag tgctggggac
cctggatcta 300tcccacaatc agctgcaaag cctgcccttg ctagggcaga
cactgcctgc tctcaccgtc 360ctggacgtct ccttcaaccg gctgacctcg
ctgcctcttg gtgccctgcg tggtcttggc 420gaactccaag agctctacct
gaaaggcaat gagctgaaga ccctgccccc agggctcctg 480acgcccacac
ccaagctgga gaagctcagt ctggctaaca acaacttgac tgagctcccc
540gctgggctcc tgaatgggct ggagaatctc gacacccttc tcctccaaga
gaactcgctg 600tatacaatac caaagggctt ttttgggtcc cacctcctgc
cttttgcttt tctccacggg 660aacccctggt tatgcaactg tgagatcctc
tattttcgtc gctggctgca ggacaatgct 720gaaaatgtct acgtatggaa
gcaaggtgtg gacgtcgcgg ccatgacctc taacgtggcc 780agtgtgcagt
gtgacaattc agacaagttt cccgtctaca aatacccagg aaaggggtgc
840cccacccttg gtgatgaagg tgacacagac ctatatgatt actacccaga
agaggacact 900gagggcgata aggtgcggcc gcacacatgc ccaccgtgcc
cagcacctga agccctgggg 960gcaccgtcag tcttcctctt ccccccaaaa
cccaaggaca ccctcatgat ctcccggacc 1020cctgaggtca catgcgtggt
ggtggacgtg agccacgaag accctgaggt caagttcaac 1080tggtacgtgg
acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac
1140aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg
gctgaatggc 1200aaggagtaca agtgcaaggt ctccaacaaa gccctcccag
tccccatcga gaaaaccatc 1260tccaaagcca aagggcagcc ccgagaacca
caggtgtaca ccctgccccc atcccgggag 1320gagatgacca agaaccaggt
cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1380atcgccgtgg
agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
1440gtgctggact ccgacggccc cttcttcctc tacagcaagc tcaccgtgga
caagagcagg 1500tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg
aggctctgca caaccactac 1560acgcagaaga gcctctccct gtctccgggt aaa
1593
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