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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4152-4157
Antibodies to Protease-Activated Receptor 3 Inhibit Activation of
Mouse Platelets by Thrombin
By
Hiroaki Ishihara,
Dewan Zeng,
Andrew J. Connolly,
Carmen Tam, and
Shaun R. Coughlin
From the Cardiovascular Research Institute, Daiichi Research Center,
and the Departments of Medicine and Pharmacology, University of
California, San Francisco, San Francisco, CA.
 |
ABSTRACT |
Recent studies of mice deficient in the thrombin receptor,
protease-activated receptor 1 (PAR1), provided definitive evidence for
the existence of a second thrombin receptor in mouse platelets. We
recently identified a new thrombin receptor designated
protease-activated receptor 3 (PAR3). The mRNA encoding a mouse
homologue of PAR3 was highly expressed in mouse splenic megakaryocytes,
making it a good candidate for the missing mouse platelet thrombin
receptor. We now report that PAR3 protein is expressed on the surface
of mouse platelets and that PAR3 antibodies partially inhibit
activation of mouse platelets by thrombin but not U46619, a thromboxane
receptor agonist. These observations suggest that PAR3 contributes to
mouse platelet activation by thrombin.
| |
INTRODUCTION |
ACTIVATION OF PLATELETS by thrombin is
thought to be critical for both normal hemostasis and for
platelet-dependent arterial thrombosis in unstable angina and
myocardial infarction. Thrombin's actions on human platelets are
mediated at least in part by protease-activated receptor 1 (PAR1).1-4 Thrombin binds to and cleaves the amino terminal
exodomain of this unusual G protein-coupled receptor.1,5-8 Receptor cleavage serves to unmask a tethered peptide ligand that binds
intramolecularly to the body of the receptor, effecting transmembrane
signaling.1,5,9 Synthetic peptides mimicking this tethered
ligand sequence act as PAR1 agonists.1,3 Such peptides
mimic many of thrombin's actions on human platelets,1-4 and PAR1 antibodies partially inhibited activation of human platelets by thrombin.10,11 By contrast, synthetic peptides mimicking the tethered ligand domain of mouse PAR1 did not activate mouse platelets,12-14 and the thrombin responses of platelets
from PAR1 knockout and wild-type mice were
indistinguishable.14 These observations suggested that PAR1
was not important for thrombin signaling in mouse platelets and
prompted a search for a second thrombin receptor responsible for
activation of these cells.
We recently cloned a new protease-activated receptor designated
PAR3.15 Human PAR3 is clearly a second thrombin receptor; it conferred thrombin-stimulable calcium mobilization when expressed in
Xenopus oocytes and phosphoinositide hydrolysis when expressed in COS7
cells. By Northern analysis it was expressed in the heart, liver,
pancreas, thymus, small intestine, stomach, lymph node, trachea,
adrenal, and bone marrow. By contrast, a putative mouse homologue of
PAR3 was highly expressed in megakaryocytes with little expression
detected elsewhere. This tissue distribution made mouse PAR3 a strong
candidate for the missing mouse platelet thrombin
receptor.15 However, thrombin is known to cause rapid increases in intracellular calcium in mouse platelets, but attempts to
trigger calcium mobilization and phosphoinositide hydrolysis via mouse
PAR3 expressed in Xenopus oocytes or COS7 cells have been unsuccessful
thus far (unpublished data). We therefore sought to
determine whether PAR3 protein was expressed in mouse platelets and, if
so, to assess its functional importance. Polyclonal antisera were
raised to a peptide representing the thrombin cleavage site and
tethered ligand domains of mouse PAR3. Polyclonal10 and monoclonal11 antibodies to the cognate region of PAR1 were
previously shown to partially inhibit activation of PAR1-transfected
cells and human platelets by thrombin. We report that PAR3 protein was indeed expressed in mouse platelets and that PAR3 antibodies partially inhibited mouse platelet activation by thrombin, strongly suggesting an
important role for PAR3 in this process.
| |
MATERIALS AND METHODS |
In situ hybridization.
C57BL/6 mice were anesthetized with pentobarbital and then
perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline.
Tissues were dissected free, immersion-fixed in 4% paraformaldehyde overnight, and then embedded in paraffin. Five-micrometer sections were
hybridized with a sense or antisense 33P-riboprobe
transcribed from full-length mPAR3 cDNA subcloned into pBluescript II
SK .14 The photographic emulsion was
developed after 1 week of exposure.
PAR polyclonal antibodies.
The synthetic peptides DATVNPR/SFFLRNPSENTFELVPLGDGC (mPAR1 amino acids
35-60 plus carboxyl glycine-cysteine) and
AKPTLTIK/SFNGGPQNTFEEFPLSDIEGC (mPAR3 amino acids 30-57 plus
carboxyl cysteine) were purified by reverse-phase high-pressure liquid
chromatography and conjugated via their carboxyl terminal cysteine to
keyhole limpet hemocyanin using m-maleimidobenzoyl-N-hydroxysuccinimide
ester (Pierce Chemical Co, Rockford, IL). Antigen conjugation, rabbit
immunization, and collection of sera were performed by AnimalPharm
Services (Healdsburg, CA) using standard techniques.16 The
immune sera used in these studies was collected on day 70 after the
primary and 6 booster immunizations. IgG was purified from preimmune
and immune sera using protein-A agarose and gel filtration (Pierce
Chemical Co). Using enzyme-linked immunosorbent assay (ELISA), PAR3 IgG
recognized the PAR3 peptide antigen but not the PAR1 antigen, and PAR1
IgG recognized the PAR1 antigen and not the PAR3 antigen (not shown, but see Fig 2). Binding of PAR1 and PAR3 IgG to the surface of transfected COS7 cells was examined as previously
described.17
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| Fig 2.
Binding of PAR3 IgG to the surface of mouse platelets by
flow cytometry. Washed platelets from C57BL/6J mice were incubated with
preimmune or PAR3 IgG as indicated and then washed and incubated with
FITC-goat antirabbit IgG. After three washes, platelets were analyzed
by flow cytometry. Each figure represents an analysis of 10,000 events
with SSC (side scatter) on the abscissa and FITC fluorescence intensity
on the ordinate. The median FITC fluorescence intensities were 3.5 for
preimmune IgG and 27.4 for PAR3 IgG. The same population of cells that
bound PAR3 IgG bound antibody to the platelet marker CD41; PAR1 IgG
showed no significant binding (not shown).
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Western blotting of platelets and transfected COS7 cells.
Washed mouse platelets were prepared as described below and then
resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer without reducing
agent. These samples were passed through a 27-g needle and then stored frozen as aliquots until analyzed by SDS-PAGE and immunoblot.
COS7 cells (105 per 3.8 cm2 well) were
transfected with 0.5 µg of mouse PAR1 or PAR3 cDNA in the mammalian
expression vector pBJ1 vector using LipofectAmine Reagent (GIBCO BRL,
Grand Island, NY). After 5 hours, the transfection medium
was replaced with Dulbecco's modified Eagle's medium (DME H-16)
containing 5% fetal bovine serum. After an additional 48 hours, cells
were lysed and lysates analyzed by SDS-PAGE (10% acrylamide) and
immunoblot. Western blotting was performed using protein-A
agarose-purified primary IgGs at the concentration of 1 µg/mL; bound
primary antibody was detected using the ECL system (Amersham, Arlington
Heights, IL).
Flow cytometry.
Washed mouse platelets were resuspended in platelet buffer (see below)
plus 1 µmol/L prostaglandin E1 and 5 mmol/L
EDTA and then incubated with 10 µg/mL primary antibodies at 4°C
for 1 hour followed by the appropriate fluorescein isothiocyanate
(FITC)-conjugated second antibody at 4 µg/mL for 0.5 hour. After
three washes, platelets were analyzed using flow cytometry. Rat
antimouse CD41 antibody (PharMingen 09911D; PharMingen, San Diego,
CA) that recognizes the abundant platelet surface protein
 IIb was used as a positive control in these studies.
Platelet aggregation, ATP secretion, and calcium mobilization.
Washed platelets were prepared from C57BL/6 mouse and resuspended at
the concentration of 6 × 108/mL in platelet buffer
(20 mmol/L Tris-HCl, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L KCl, 1 mmol/L
MgCl2, 1 mg/mL glucose, and 0.2% bovine serum
albumin). Platelet aggregation and ATP secretion were
evaluated in the platelet buffer with 2 mmol/L CaCl2 and Chromolume (Chrono-log Corp, Havertown, PA) using a dual channel lumiaggregometer (Chrono-log Corp) as changes of light transmittance or
chemiluminescence, respectively.14
For calcium mobilization studies, platelets were washed once with
platelet buffer and then incubated in the same buffer plus 1 mmol/L
EDTA and 4 µg/mL Fura-2 AM for 30 minutes at 37°C. Platelets were
then pelleted at 500g for 10 minutes and resuspended in
platelet buffer.14 EGTA (0.1 mmol/L) was added at zero time
in each scan to block aggregation. In all of the platelet experiments
shown, IgGs were added 5 minutes before the zero time at a final
protein concentration of 60 µg/mL.
| |
RESULTS AND DISCUSSION |
The specificity of the PAR3 and PAR1 peptide antisera used in these
studies was first characterized by immunoblot. Mouse PAR3 IgG
recognized several bands in PAR3-transfected COS7 cells but not in
PAR1-transfected COS7 cells or in untransfected cells
(Fig 1 and data not shown); PAR3 preimmune
IgG did not recognize these bands. The major bands recognized by PAR3
IgG migrated at approximately 40 and 90 kD. The transfection-dependence
of these bands and their recognition by immune but not preimmune IgG
suggests that they almost certainly represent PAR3 protein; their
aberrant migration is at least in part due to different levels of
glycosylation (D.Z. and S.R.C., unpublished results).
Mouse PAR1 IgG recognized a protein in PAR1-transfected COS7 cells, but
not in PAR3-transfected or untransfected cells (Fig 1).
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| Fig 1.
Recognition of PARs by immunoblot and cell surface
binding. (A) Immunoblotting of platelet and PAR-transfected COS7 cell
lysates with PAR antisera. COS7 cells were transfected with plasmids
directing expression of mouse PAR1 or PAR3 or with empty vector.
Transfected COS7 lysates and lysates of washed mouse platelets were
analyzed by SDS-PAGE and immunoblot with PAR1, PAR3, or preimmune IgG
(see the Materials and Methods). Preincubation of the PAR1 or PAR3 IgG
with the peptide antigen to which each was raised (final peptide concentration, 3.3 nmol/L; overnight at 4°C) ablated their ability to recognize PAR1 or PAR3, respectively (not shown). This experiment was replicated four times. (B) Selective recognition of PARs on the
COS7 cell surface. COS7 cells transfected with mPAR1 or mPAR3 expression vectors or with empty vector were fixed briefly with 2%
paraformaldehyde, incubated with PAR1 or PAR3 IgG (60 µg/mL) as
indicated, and then washed extensively. Antibody bound to the surface
of unpermeabilized cells was detected using horseradish peroxidase-conjugated second antibody by cell surface
ELISA.17 Data shown are the mean ± SEM (n = 4); binding
to empty expression vector-transfected cells (0.14 OD units for PAR3
IgG; 0.08 for PAR1 IgG) was subtracted. This experiment was replicated
twice.
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The anti-PAR3 peptide IgG also bound to the surface of unpermeabilized
COS7 cells expressing PAR3 but not to COS7 cells expressing PAR1; the
converse was found for the PAR1 peptide IgG (Fig 1). Thus, by both
immunoblot and surface binding studies, the PAR3 and PAR1 peptide
antisera recognized PAR3 and PAR1, respectively, with no evidence for
cross-reactivity.
PAR3 and PAR1 IgGs were next used to assess expression of PAR3 and PAR1
protein by mouse platelets. By immunoblot of mouse platelet lysate,
PAR3 recognized a broad band centered at a molecular weight of
approximately 60 kD and minor bands at approximately 50 kD. The upper
band probably represents fully processed glycosylated PAR3 and the
lower band precursor and partially glycosylated forms. These bands were
not detected by PAR1 IgG or by PAR3 preimmune IgG. PAR3 IgG also bound
to the surface of mouse platelets as assessed by flow cytometry
(Fig 2); PAR1 IgG showed little or no
binding above background. These observations strongly suggest that PAR3
protein is indeed expressed by mouse platelets and are consistent with
previous in situ hybridization experiments showing PAR3 mRNA in mouse
splenic megakaryocytes15 and with recent studies confirming
PAR3 mRNA expression in megakaryocytes from mouse bone marrow (data not
shown). In contrast to the case with PAR3, PAR1 IgG failed to recognize
a specific band in mouse platelets. The absence of detectable PAR1
protein in mouse platelets is consistent with its apparent lack of
functional importance in these cells.12-14
Given the ability of PAR3 peptide IgG to bind specifically to the
surface of PAR3-transfected COS7 cells and to mouse platelets, we next
determined whether this IgG could block thrombin signaling in mouse
platelets. Thrombin triggered rapid increases in cytoplasmic calcium in
washed mouse platelets. The EC50 for this response was
approximately 0.5 nmol/L (data not shown). Preincubation of mouse
platelets with PAR3 IgG, but not preimmune IgG, markedly attenuated
calcium mobilization by 0.5 nmol/L thrombin
(Fig 3). By contrast, PAR3 IgG did not
attenuate responses to an EC50 concentration the
thromboxane receptor agonist U46619 (Fig 3).
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| Fig 3.
Inhibitory effect of PAR3 IgG on -thrombin-induced
calcium mobilization in mouse platelets. Mouse platelets were loaded
with fura-2 and cytosolic free Ca2+ concentration
[Ca2+]i (nmol/L) was measured
fluorometrically.14 Extracellular calcium was chelated to
prevent platelet aggregation. Platelets were preincubated with the
indicated IgG preparations for 5 minutes at 37°C and then
transferred to a fluorometer at room temperature. -Thrombin (0.5 nmol/L) or U46619 (3 µmol/L) was added at
approximately 100 seconds. The data shown are representative of three
experiments with thrombin and two experiments with U46619.
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PAR3 IgG also inhibited thrombin-triggered ATP secretion and
aggregation in mouse platelets in response to 0.5 nmol/L thrombin (Fig 4); preimmune IgG was without
activity. Preincubation with PAR3 antigen ablated the inhibitory
activity of PAR3 IgG preparations; preincubation with the cognate PAR1
antigen had no effect. These data strongly suggest that the inhibitory
effects of PAR3 IgG on thrombin signaling were mediated by antibodies
that recognize PAR3.
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| Fig 4.
Inhibitory effect of PAR3 IgG on mouse platelet responses
to -thrombin and ablation of inhibition by the peptide antigen. -Thrombin-induced (0.5 nmol/L) platelet aggregation (A) and
secretion (B) are shown. IgGs were preincubated with peptide antigens
for 5 minutes at 37°C. Platelets were incubated with this mixture (final concentration of IgG, 60 µg/mL; antigen, 0.1 µmol/L) for 5 minutes at 37°C; responses were then analyzed in a
lumiaggregometer. -Thrombin was added at approximately 3 minutes
(arrow). Responses were expressed as the percentage of maximum, with
100% defined as the response induced by 10 nmol/L -thrombin in
platelets incubated with preimmune IgG only. The latter had no effect
on platelet responses. The data shown were replicated in two separate
experiments.
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To further test the specificity of the PAR3 IgG preparation's
inhibitory actions, we compared its effects on the
concentration-response curves for thrombin and U46619 using platelet
secretion as an endpoint (Fig 5). PAR3 IgG
reliably right-shifted the concentration-response to thrombin. At
thrombin concentrations at or below EC50, thrombin-induced secretion was virtually ablated. By contrast, PAR3 IgG had no effect on
platelet secretion induced by U46619, even at sub-EC50 agonist concentrations. Mouse PAR1 IgG had no effect on thrombin signaling and did not enhance the inhibitory effect of PAR3 IgG (Fig 5
and data not shown). Taken together, these observations strongly
suggest that PAR3 signaling is necessary for normal activation of mouse
platelets by thrombin.
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| Fig 5.
Effect of PAR3 IgG on mouse platelet ATP secretion in
response to -thrombin or U46619. Washed platelets were incubated
with PAR3 IgG (60 µg/mL) for 5 minutes at 37°C and then
stimulated with the indicated concentration of -thrombin (A) or
U46619 (B). The maximum response to each concentration of agonist was
quantitated and expressed as a percentage of the response of platelets
incubated with preimmune IgG only to 10 nmol/L -thrombin. (In
separate experiments, preimmune IgG had no significant effect on
platelet responses.) Each point represents a single aggregometry
tracing. The data shown were replicated in two separate experiments.
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The failure of mouse PAR1 agonists to activate mouse
platelets,12-14 the lack of an effect of the PAR1 knockout
on mouse platelet activation,14 and our inability to detect
PAR1 protein or alter platelet thrombin responses with mouse PAR1
antibodies all suggest that PAR1 plays little or no role in mouse
platelet activation. Does the observation that inhibition of mouse
platelet activation by PAR3 IgG is overcome at high thrombin
concentrations imply the existence of yet another thrombin receptor? We
previously showed that a human PAR1 IgG inhibited human platelet
activation by thrombin and that this inhibition was overcome at high
thrombin concentrations.10 The same phenomenon was observed
for signaling via PAR1 heterologously expressed in Xenopus oocytes, a
system in which all thrombin signaling requires the exogenous
receptor.1,10 The ability of high concentrations of
thrombin to overcome the blocking antibody may simply be due to
competition; the irreversibility of the proteolytic activation
mechanism versus the reversibility of antibody binding might favor
thrombin in this regard. It is thus not necessary to invoke the
existence of yet another mouse platelet thrombin receptor to explain
available data. However, it is critical to point out that neither do
these data exclude the existence of such a receptor. The presence or
absence of persistent thrombin responses in platelets derived from
Par3g  / mice will be definitive in this regard.
Synthetic peptides that mimic the tethered ligand domains of PAR1 and
PAR2 function as agonists for these receptors. Unfortunately, this was
not the case for human and mouse PAR3.15 It is thus not
possible to use a PAR3 tethered ligand peptide to determine whether
PAR3 activation is sufficient for activation of mouse platelets.
Previous studies showed that antibodies to human PAR1's thrombin
cleavage site and agonist peptide domain inhibited PAR1
signaling.10,11 The present study provides similar data for
antibodies to the cognate domain of PAR3. Antibodies that bind to the
protease cleavage site and/or tethered ligand domain of
protease-activated receptors may thus be generally useful for
inhibiting signaling by PAR2 or perhaps as yet undiscovered members of
this budding receptor subfamily.
In summary, PAR3 mRNA and protein are expressed in mouse megakaryocytes
and platelets, and PAR3 antibodies partially inhibit thrombin responses
in mouse platelets. These results strongly suggest that PAR3 plays an
important role in activation of mouse platelets by thrombin. This
result motivates investigation of mouse PAR3 signaling in platelets and
begs the question of why mouse PAR3 has thus far not shown signaling in
transfected COS7 cells and other systems. Our data supporting a role
for PAR3 in mouse platelet activation also motivate generation of a
Par3g / mouse. If viable, this mouse will either
provide evidence for the existence of yet another platelet thrombin
receptor or will constitute a valuable system for determining the
relative importance of thrombin signaling in various models of
thrombosis. Lastly, the finding that mouse PAR3 appears to play an
important signaling role prompts studies to define the role of human
PAR3. Using Northern blot analysis, the tissue distribution of human PAR3 appears to be distinct from that of mouse PAR3.14
Studies to identify which human cell types express PAR3 are needed. In particular, it is unknown whether PAR3 contributes to human platelet function. Human PAR3 IgG did not inhibit thrombin responses in human
platelets and did not enhance the inhibition seen with human PAR1 IgG
(H.I. and S.R.C., unpublished results), but such negative results must be interpreted with caution. Nonetheless, given the dichotomy in the roles of PAR1 in human versus mouse
platelets,14 it would not be surprising if PAR3 also served
distinct roles in mice and humans. How such distinct tissue-specific
roles for PAR1 and perhaps PAR3 might have evolved remains an
interesting question.
| |
FOOTNOTES |
Submitted August 4, 1997;
accepted January 16, 1998.
H.I. and D.Z. contributed equally to this work.
Supported by the Daiichi Research Center at the University of
California, San Francisco, and by National Institutes of Health Grants
No. HL44907, DK50267, and HL59202. D.Z. was supported by NRSA HL09256. A.J.C. was supported by KO8 HL03234.
Address reprint requests to Shaun R. Coughlin, MD, PhD, HSE-1300,
University of California, San Francisco, 505 Parnassus Ave, San
Francisco, CA 94143-0130.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr JoAnn Trejo for her help with computer art, Linda
Prentice for paraffin sections, Prof Henry Bourne (UCSF) for critical
reading of this manuscript, and Prof John Fenton II (Albany Medical
College, Albany, NY) for purified -thrombin.
| |
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C. D. Major, R. J. Santulli, C. K. Derian, and P. Andrade-Gordon
Extracellular Mediators in Atherosclerosis and Thrombosis: Lessons From Thrombin Receptor Knockout Mice
Arterioscler Thromb Vasc Biol,
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[Abstract]
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S. R. Macfarlane, M. J. Seatter, T. Kanke, G. D. Hunter, and R. Plevin
Proteinase-Activated Receptors
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M. L. Kahn, T. G. Diacovo, D. F. Bainton, F. Lanza, J. Trejo, and S. R. Coughlin
Glycoprotein V-Deficient Platelets Have Undiminished Thrombin Responsiveness and Do Not Exhibit a Bernard-Soulier Phenotype
Blood,
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[Abstract]
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W.-m. Cheung, M. R. D’Andrea, P. Andrade-Gordon, and B. P. Damiano
Altered Vascular Injury Responses in Mice Deficient in Protease-Activated Receptor-1
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S. R. Coughlin
How the protease thrombin talks to cells
PNAS,
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[Abstract]
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M. L. Kahn, S. R. Hammes, C. Botka, and S. R. Coughlin
Gene and Locus Structure and Chromosomal Localization of the Protease-activated Receptor Gene Family
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[Abstract]
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M. J. Shapiro, E. J. Weiss, T. R. Faruqi, and S. R. Coughlin
Protease-activated Receptors 1 and 4 Are Shut Off with Distinct Kinetics after Activation by Thrombin
J. Biol. Chem.,
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[Abstract]
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T. R. Faruqi, E. J. Weiss, M. J. Shapiro, W. Huang, and S. R. Coughlin
Structure-Function Analysis of Protease-activated Receptor 4 Tethered Ligand Peptides. DETERMINANTS OF SPECIFICITY AND UTILITY IN ASSAYS OF RECEPTOR FUNCTION
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[Abstract]
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Abstract
Introduction
Abstract