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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From INSERM U.311, Etablissement Français du
Sang-Alsace, Strasbourg, France; University of Pennsylvania,
Philadelphia, PA; and Witten/Herdecke University, Klinikum Wuppertal,
Germany.
Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX
receptor for von Willebrand factor and thrombin. GPV is cleaved from
the platelet surface during activation by thrombin, but its role in
hemostasis is still unknown. It is reported that GPV knockout mice had
a decreased tendency to form arterial occluding thrombi in an
intravital thrombosis model and abnormal platelet interaction with the
subendothelium. In vitro, GPV-deficient platelets exhibited defective
adhesion to a collagen type I-coated surface under flow or static
conditions. Aggregation studies demonstrated a decreased response of
the GPV-deficient platelets to collagen, reflected by an increased lag
phase and reduced amplitude of aggregation. Responses to adenosine
diphosphate, arachidonic acid, and the thromboxane analog U46619 were
normal but were enhanced to low thrombin concentrations. The defect of
GPV null platelets made them more sensitive to inhibition by the
anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in
aspirin- or apyrase-treated platelets. Moreover, an mAb (V.3) against
the extracellular domain of human GPV selectively inhibited
collagen-induced aggregation in human or rat platelets. V.3
injected in rats as a bolus decreased the ex vivo collagen aggregation
response without affecting the platelet count. Finally, surface plasmon
resonance studies demonstrated binding of recombinant soluble GPV on a
collagen-coupled matrix. In conclusion, GPV binds to collagen and
appears to be required for normal platelet responses to this agonist.
(Blood. 2001;98:1038-1046) Platelets play a central role in hemostasis through
their ability to adhere to a damaged vessel wall and to aggregate in
response to agonists such as thrombin, collagen, or adenosine
diphosphate (ADP),1 and these properties are known to be
mediated by cell surface glycoproteins.2,3 However, the
exact functions of numerous platelet glycoproteins that have been
characterized biochemically remain unknown. Glycoprotein V (GPV, Mr 82 kd) is one of the most abundant glycoproteins at the surface of blood
platelets and has long been identified,4-6 but its
functional role is still subject to speculation. GPV is noncovalently
linked to the GPIb-IX von Willebrand factor (vWF) receptor on the
platelet surface.7,8 This type I transmembrane protein has
a large extracellular domain comprising 15 Leu-rich motifs followed by
a thrombin cleavage site.9,10 The specific release of a
soluble 69-kd extracellular domain fragment (GPVf1) by thrombin has led
to its proposal as a thrombin receptor,6 whereas cloning
of the gene in rat and mouse revealed a well-conserved thrombin
cleavage site.11 Studies in transfected cells have shown
that GPV is required for efficient thrombin binding, possibly through a
direct interaction with GPIb The role of GPV as a thrombin receptor has been questioned on the basis
of efficient platelet responses to high doses of thrombin when GPV had
been enzymatically removed or cleavage was prevented by a blocking
antibody.15,16 More recently, the functions of GPV have
been reassessed in mouse strains lacking the GPV
gene.17,18 These studies demonstrated that the
absence of GPV did not provoke a bleeding syndrome and that they
allowed normal GPIb The purpose of the present work was to evaluate in more detail
possible functional defects of GPV null platelets, particularly regarding their interactions with components of the subendothelial matrix. Studies in an intravital thrombosis model indicated a decreased
tendency of GPV null platelets to form occlusive thrombi, and in vitro
adhesion tests indicated a defective initial attachment to collagen.
These platelets had decreased reactivity to collagen, as demonstrated
by a prolonged lag phase and reduced amplitude of aggregation
especially at lower agonist concentrations. The collagen aggregation
defect was also observed in normal human and rat platelets incubated
with a monoclonal antibody (mAb) against the extracellular domain of
GPV. Finally, recombinant soluble GPV inhibited collagen-induced
platelet aggregation and bound to a collagen-coated matrix.
Reagents
Insoluble bovine collagen type I, soluble human and rat collagen type
I, soluble human collagen type III, ADP, the thromboxane analog U46619,
PGI2, arachidonic acid, and mouse MOPC21 IgG1 were from
Sigma Chemicals (St Louis, MO). Equine tendon collagen (a mixture of
types I and III) were obtained from Nycomed (Münster, Germany), and human fibrinogen was obtained from Kabi (Stockholm, Sweden). Human serum albumin (HSA) and human Mouse strains
In vivo murine thrombosis model Thrombosis studies were performed according to a published method.22,23 Male mice of both genotypes (3-5 weeks old) were injected with genotype-matched calcein-labeled washed platelets (108 platelets/10 g body weight) and allowed to recover for at least 12 hours. The mice were then anesthetized, and the mesentery was exposed by abdominal incision. Arterioles (60-80-µm diameter) with shear flow rates estimated at 1400 second 1 were
observed22,23 under an inverted fluorescence microscope (Leica DMIRB; Leica Microsystems SA, Wetzlar, Germany) coupled to a
video camera (DAGE MTI, Michigan City, MI), and images were recorded on
DVD-Ram tapes (WDR 200; Matsushita Electric Industrial, Osaka, Japan).
After 30-µL application of a 250-mM FeCl3 solution, the
arterioles were monitored for 20 minutes or until blood flow stopped.
The operator was unaware of the mouse genotype while performing experiments.
Platelet preparation Washed mouse and rat platelets were prepared from blood (9 vol) drawn from the abdominal aorta of anesthetized animals into a plastic syringe containing acid-citrate-dextrose (1 vol) and centrifuged at 1570g (10 seconds per milliliter blood). Platelet-rich plasma (PRP) was removed and centrifuged at 1570g for 10 minutes. The platelet pellet was washed twice at 1100g for 3-8 minutes in Tyrode buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO3, 0.3 mM NaH2PO4, 1 mM MgCl2, 5.5 mM glucose, 5 mM HEPES, pH 7.3) containing 0.35% HSA and 7.5 nM PGI2 and resuspended at a density of 3 × 105 platelets per microliter in the same buffer in the presence of 0.02 U/mL ADP scavenger apyrase (adenosine 5'-triphosphate diphosphohydrolase, EC 3.6.1.5) a concentration
sufficient to prevent desensitization of platelet ADP receptors during
storage. Washed human platelets were prepared from
acid-citrate-dextrose anticoagulated blood obtained from aspirin-free
healthy volunteers by sequential centrifugation as previously
described.24 Platelets were kept at 37°C throughout all
experiments. Citrated mouse and rat PRP was obtained by collecting blood into 3.15% sodium citrate followed by centrifugation at 1570g (10 seconds per milliliter blood) and adjusted to
3.105 platelets per microliter by the addition of plasma.
Platelet adhesion assays Platelet adhesion under flow was studied using a modification of the method of Cranmer et al.25 Glass microcapillary tubes (microslides of 100-mm length and 0.2 × 2-mm cross-section; VitroCom, Mountain Lakes, NJ) were coated with 2.5 mg/mL insoluble bovine type I collagen for 2 hours at room temperature or with 50 µg/mL bovine vWF overnight at 4°C and then blocked with 0.1% HSA for 1 hour at room temperature. Citrate-anticoagulated whole blood was labeled with 5 µM DiOC6 for 10 minutes before perfusion with a syringe pump at 1500 seconds1 (collagen studies) or 3000 seconds1 (vWF studies) through the coated capillaries.
The interaction of platelets with the matrix was viewed in real time
under a fluorescence microscope and was saved for off-line analysis.
The number of individual platelets adhering to the surface
(6400-µm2 field) was analyzed frame by frame (24 frames/s) over the first 20 seconds.
Platelet adhesion under static conditions was studied on 8-well Super Teflon slides (HTC Super cured (R); Polylabo, Strasbourg, France) coated with 2.5 mg/mL collagen for 90 minutes or 20 µg/mL bovine vWF for 2 hours at room temperature and blocked with 0.1% HSA in phosphate-buffered saline (PBS) for 1 hour at room temperature. Washed platelets (30 000 platelets/well) in Tyrode-albumin buffer (collagen studies) or the same buffer containing 10 mM EDTA (vWF studies) were incubated in the wells at room temperature for 5 or 20 minutes. Slides were then washed 3 times with PBS, fixed with 4% 1-paraformaldehyde in PBS for 20 minutes, washed 3 times with PBS, and labeled with a 1/500 dilution of phalloidin-TRITC (Sigma) for 20 minutes in the dark. After 5 more washes in PBS, adherent platelets were covered with 5 µL Mowiol 4-88 solution (France Biochem, Meudon, France) and mounted for examination under a Leica DMR fluorescence microscope. Platelets were counted in 5 random fields (26 140 µm2/field). Platelet aggregation Aggregation was measured at 37°C by a turbidimetric method in a dual-channel Payton aggregometer (Payton Associates, Scarborough, Ontario, Canada). A 450-µL aliquot of platelet suspension was stirred at 1100 rpm and activated by the addition of different agonists, with or without antagonists, in the presence of human fibrinogen (0.05 mg/mL) and in a final volume of 500 µL. The extent of aggregation was estimated quantitatively by measuring the maximum curve height above the baseline. In studies with collagen, the lag phase was taken to be the time from agonist addition to the onset of aggregation.Ex vivo aggregation of rat platelets Anesthetized male Wistar rats (400 g) were given a bolus injection of V.1 or V.3 IgG1 (1 mg/kg) in the dorsal penile vein. After 20 minutes, 7 mL blood was withdrawn from the jugular vein into 3.15% sodium citrate. PRP was prepared, and aggregation studies were performed as described above.Flow cytometry Washed mouse platelets (3 × 105 platelets in 50 µL) were incubated with 20 µg/mL purified IgG or 50 µL hybridoma culture supernatant at 22°C for 30 minutes, washed in PBS, and incubated in the dark at 22°C for 30 minutes with 10 µg/mL DATF-conjugated goat anti-rat F(ab)'2 or FITC-conjugated goat anti-hamster immunoglobulin antibodies. Cells were then resuspended in PBS and analyzed on a FACScalibur fluorescence cytometer (Becton Dickinson, San Jose, CA) using Cell Quest software. The light scattering and fluorescence intensity from 10 000 platelets were collected using a logarithmic gain.Surface plasmon resonance analysis Experiments were performed using a Biacore 2000 apparatus (Biacore, Uppsala, Sweden). Activation of a CM5 sensor chip with the Biacore amine coupling kit was followed by the injection of 70 µL soluble or insoluble type I or type III collagen of variable origin diluted to 100 µg/mL in 10 mM sodium acetate, pH 4.8. Excess reactive amine groups were blocked with 1 M ethanolamine, pH 8.5. Parallel experiments were performed with sensor chips coupled with HSA or human vWF, and the total immobilized proteins corresponded to 8000 to 12 000 resonance units. Recombinant soluble GPV (59 µg/mL) in Hepes buffer saline containing EDTA and polysorbate (HBS-EP) buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% [vol/vol], polysorbate 20, pH 7.4) was injected at 25°C at a flow rate of 5 µL/min using HBS as a running buffer. Responses (in resonance units) obtained on chips coupled with collagen or vWF were subtracted from those obtained with immobilized HSA using Biaevaluation 3.0 software. The surface was regenerated by 10-µL injection of 2 M NaCl.
GPV null mice form less occlusive thrombi in an intravital arterial thrombosis model To evaluate the consequences of a lack of GPV in thrombotic situations, we used an established intravital microscopy model of thrombosis. Injury of the mesenteric arterioles of mice with FeCl3 leads to rapid platelet attachment to the vessel wall and progressive thrombus formation that eventually occludes the artery. The average time to occlusion was significantly longer in GPV null mice than in wild-type (WT) littermates (1027 ± 105 seconds [n = 10] vs 556 ± 123 seconds [n = 12]; P = .01) (Figure 1A). Vessel lumen closure occurred in less than 20 minutes in 90% of WT mice, whereas half the arteries were still permeable at this time in GPV/ mice. Analysis of
the dynamics of platelet interaction at early times indicated that most
subsequently detached from the matrix. The time interval between
initial tethering and detachment of single attached platelets was
significantly shorter in GPV null mice (0.47 ± 0.05 seconds,
n = 24) than in GPV WT mice (2.97 ± 0.48 seconds, n = 34)
(Figure 1B).
Defective adhesion of GPV null platelets to a collagen-coated surface Defective interaction with components of the subendothelial matrix, including collagen, could contribute to the GPV knockout phenotype. Adhesion of GPV WT and GPV null platelets was, therefore, evaluated under flow conditions using microcapillaries coated with bovine type I collagen and citrated whole blood perfused at 1500 second1. Initial attachment and adhesion of single
platelets took place quickly after blood injection and was followed by
the formation of stable aggregates progressively covering the adhesive
surface. At early times (10-20 seconds), there was less attachment of
individual platelets in blood from mice lacking GPV (Figure
2A). This effect was also observed when
hirudin was added to the citrated blood to completely inactivate
thrombin (data not shown). In separate static adhesion tests using
washed platelets, approximately 42% fewer GPV/
platelets attached to collagen compared with WT cells (Figure 2B). A
bovine vWF matrix showed comparable interaction with platelets of the 2 phenotypes under dynamic (3000 seconds1, Figure 2A) and
static (Figure 2B) conditions. Mouse platelets interacted weakly, if at
all, with human vWF (data not shown).
Diminished collagen-induced aggregation of GPV null platelets Washed WT mouse platelets activated with 2.5 µg/mL collagen in the presence of fibrinogen aggregated irreversibly after a typical 10- to 20-second lag phase before the onset of aggregation. At this agonist concentration, WT and GPV null platelets displayed no difference in the extent of aggregation at 3 minutes (Figure 3A). However, close analysis of the tracings revealed a slight but reproducible increase in lag time in the GPV-deficient cells. At progressively lower doses of collagen, the diminished response of GPV null platelets became more evident, and at 0.625 µg/mL collagena concentration that induced 60% aggregation
of WT plateletsno aggregation was detected using GPV-deficient
platelets. The estimated concentration of collagen that induced
half-maximal aggregation was 0.43 µg/mL for WT and 0.94 µg/mL for
GPV null platelets. This decreased reactivity was limited to collagen
because normal aggregation was observed in response to ADP, arachidonic
acid, and the thromboxane analog U46619 (Figure 3B). Aggregation was
also normal in response to nanomolar concentrations of thrombin (data
not shown) but increased in response to lower (less than 0.1 nM)
concentrations (Figure 3C). The collagen aggregation defect was further
confirmed using citrated PRP instead of washed platelets (data
not shown).
Platelet activation induced by collagen is typically accompanied by
thromboxane A2 (TXA2) production, which in turn
amplifies the aggregation response.26 After treatment with
aspirin, both WT and GPV null platelets displayed a decreased response
to collagen. Lower reactivity was nonetheless observed in GPV-deficient
platelets (Figure 4), suggesting that the
lower collagen reactivity of these cells was not due to a defect of the
cyclo-oxygenase pathway. Moreover, responses to arachidonic acid and
the TXA2 analog U46619 were normal in GPV null platelets
(Figure 3B).
GPVI-dependent activation is affected in platelets lacking GPV GPVI plays a major role in triggering the activation of platelets exposed to insoluble fibrillar collagen.27,28 At a 20-µg/mL concentration, JAQ1, a rat mAb specific for mouse GPVI,29 completely inhibits the aggregation induced by collagen concentrations below approximately 7 µg/mL.30 When JAQ1 was used at lower doses (2.5 and 5 µg/mL), only partial inhibition, leading to a slightly increased lag phase, was observed in WT platelets, with no change in the amplitude of aggregation (Figure 5). At these concentrations of JAQ1, GPV null platelets were more sensitive to inhibition than WT cells, displayed an extended lag phase, and decreased levels of aggregation. Contrary to WT platelets, the shape change induced by collagen was abolished in GPV null platelets treated with 10 µg/mL JAQ1. This increased sensitivity to JAQ1 was not attributed to lower expression of GPVI because identical levels were found on WT and GPV-deficient platelets by flow cytometry (Figure 6) and Western blot analysis (data not shown). Flow cytometry further revealed normal amounts of the 2 1 integrin (Figure 6), the
GPIb-IX complex, and the IIb3 integrin (data not shown) on GPV
null platelets.
In vitro and ex vivo inhibition of collagen-induced platelet aggregation by an mAb against GPV In view of the results with GPV null mice, we next tested the effects on normal human platelets of mAbs against the extracellular domain of GPV.14 One mAb (V.3) decreased the collagen responses of human platelets in washed platelet suspensions (Figure 7) and citrated PRP (data not shown). The inhibition profile was similar to that observed in GPV null mice, with an increased lag phase at all agonist concentrations tested and with reduced amplitude of aggregation in response to low concentrations of collagen (Figure 7). V.3 had no influence on botrocetin- or ristocetin-induced agglutination or on aggregation induced by ADP, U46619, arachidonic acid, or high- or low-dose thrombin (data not shown). Other mAbs directed against distinct epitopes on GPVf1 (V.1) or against GPIX (ALMA.16) did not affect platelet aggregation in response to collagen or any other agonist (Figure 7).
V.3 also reacted with rat GPV11 and demonstrated identical
inhibition of collagen-induced aggregation of rat platelets (data not
shown). In vivo effects of V.3 were tested by giving a bolus intravenous injection of the mAb (1 mg/kg) to Wistar rats and performing ex vivo aggregation studies on citrated PRP. Treatment with
V.3 resulted in a decreased response to collagen but did not alter the
ADP response (Figure 8). GPV occupancy
was confirmed by flow cytometry with FITC-conjugated anti-mouse
immunoglobulin F(ab)'2 (data not shown), and there was no
significant change in platelet count after V.3 injection.
Soluble recombinant GPV binds to collagen and decreases collagen-induced platelet aggregation To test for a possible interaction of GPV with collagen, studies were performed using recombinant soluble forms of GPV consisting of the entire extracellular domain or the f1 fragment produced by thrombin. These proteins were purified from the culture supernatants of CHO cells transfected with the human GPV gene.14 Soluble GPV (4 µg/mL) inhibited the aggregation response induced by collagen (Figure 9A) but had no effect on the ADP response (data not shown). This inhibition was more effective when soluble GPV was preincubated with the collagen than when it was added to the platelet suspension before collagen. To look for a direct interaction of GPV with collagen, the binding of soluble GPV to collagen coupled to a dextran-coated chip was analyzed by surface plasmon resonance (Figure 9B-C). In the presence of bovine or equine fibrillar insoluble type I collagen, the sensor-grams displayed a time-dependent increase in resonance units indicating the binding of GPV. Binding was also observed on surfaces coupled to human- or rat-soluble type I or human-soluble type III collagen. No binding to albumin or vWF matrices and no binding of control IgG to the collagen matrix were detected.
Questions regarding the exact function of platelet GPV have been a longstanding topic of research but have never been satisfactorily answered. The hypotheses raised to date mainly favored a role of GPV in platelet thrombin responses on the grounds of its selective cleavage by thrombin.6,9,11 Our present study establishes a novel role of GPV in platelet responses to collagen based on the following findings: (1) GPV knockout mice had a lower tendency to form occlusive thrombi in an intravital arterial thrombosis model, (2) GPV-deficient platelets displayed collagen adhesion and aggregation defects, (3) antibody against GPV blocked platelet aggregation induced by collagen in vitro and ex vivo, and (4) recombinant soluble human GPV bound to collagen. The availability of a GPV knockout mouse strain provided a valuable
tool to explore the functions of GPV. Initial reports indicated no
major defect of primary hemostasis in GPV-deficient mice17,18 but gave no information on a possible effect in
thrombotic situations. The present study provided indication of such a
role in an intravital thrombosis model adapted to small animals such as
rats and mice.22,23 In this model, ferric chloride injury of the mesenteric arteries provokes de-endothelialization and rapid
platelet adhesion to the exposed subendothelium. Progressive platelet
recruitment from the circulation generally leads to vessel closure. GPV
deficiency did not prevent platelet interaction with the vessel wall;
rather, it appeared to delay stable thrombus formation and, when a
thrombus formed, to render it less stable and less prone to occlude the
artery. There is now ample evidence that platelet GPIb Analysis of the dynamics of single platelet adhesion revealed an
increased tendency to detachment, suggestive of weaker interaction with
components of the subendothelium. This led us to explore the role of
GPV in platelet adhesion in vitro by examining the interaction of GPV
null platelets with a collagen-coated surface. Platelets lacking GPV
displayed a decreased collagen interaction in flowing anticoagulated
whole blood and static washed platelet suspensions, thus providing
evidence for the involvement of GPV in platelet adhesive properties.
This involvement of GPV in platelet adhesion to collagen was further
confirmed by the defective collagen adhesion of human platelets treated
with the V.3 GPV blocking mAb (data not shown). The adhesion defect
appeared to be independent of vWF because it was observed with washed
platelets, whereas GPV null platelets adhered normally to a vWF-coated
surface. This agrees with results showing that the adhesion of CHO
cells expressing optimal amounts of GPIb-IX to a vWF matrix under flow
was not modified by the presence of GPV.25 Previous flow
studies using whole blood at similar shear rates (1500 seconds In accordance with the known importance of attachment to collagen
fibers as a prerequisite for platelet activation and
aggregation,27-29,36 GPV null or normal platelets
incubated with the mAb V.3 displayed an increased lag phase at all
collagen concentrations. At lower agonist concentrations, the defective
adhesion led to reduced levels of aggregation. The current view of
collagen-induced platelet activation is that the cells initially
interact with the Surface plasmon resonance binding studies, using a recombinant soluble
form of GPV, provided evidence for a direct interaction of GPV with
collagen, supporting the contention that GPV could serve as a collagen
receptor. Lack of soluble GPV binding to other immobilized proteins,
such as albumin, IgG, or vWF, and similar interaction with different
types of collagen (soluble and insoluble, types I and III collagen)
strongly suggest that binding was not caused by nonspecific capture.
Analysis of the tracings indicates a high rate of dissociation of GPV
from collagen and, hence, low-affinity interaction. To our knowledge,
the collagen affinities of other purified receptors, such as The only in vitro defect previously observed in GPV null platelets was
an increased aggregation response in the presence of low concentrations
of thrombin,18 confirmed in the present work. Early
studies in human platelets using blocking antibodies or GPV removal
suggested that GPV was not absolutely required to support
thrombin-induced aggregation,15,16 and experiments in
transfected cells have indicated that the association of GPV with
GPIb Interaction of platelets with collagen of the subendothelium plays an
important role in normal hemostasis, as illustrated by the bleeding
disorders encountered in patients with deficiencies of the 2 main
collagen receptors, GPVI and the The lower thrombotic tendency in mice lacking GPV and the diminished
platelet collagen responses of rats treated with an anti-GPV antibody
promote GPV as a new potential target for antithrombotics. Interestingly, the administration of GPV antibodies to rats or mice
does not significantly modify the number of circulating platelets, contrary to the severe and prolonged thrombocytopenia with most antibodies directed against GPIb In conclusion, this study has revealed an unsuspected role of GPV as a modulator of collagen-dependent platelet adhesion and activation. Evidence was provided that GPV is a binding site for collagen on the platelet surface and that the decreased collagen-binding properties of GPV null platelets is the likely explanation for their lower tendency, observed in vitro and in vivo, to adhere and to aggregate. The role of GPV in platelet collagen responses was further confirmed by the effect of a blocking antibody against GPV, pointing out new ways for pharmacologic inhibition of platelet function.
We thank Shaun Coughlin for providing the GPV null mouse strain, Shaun Jackson for pertinent comments, Sacha Dopheide for introducing us to the flow technique, Juliette Mulvihill for correcting the English of the manuscript, and Catherine Schwartz and Michèle Finck for animal care.
Submitted December 4, 2000; accepted April 9, 2001.
Supported by grants from ARMESA (P.M.) and GEHT (C.S.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: François Lanza, INSERM U.311, Etablissement Français du Sang-Alsace, 10 rue Spielmann, BP 36, 67065 Strasbourg Cédex, France; e-mail: francois.lanza{at}efs-alsace.fr.
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T. J. Kunicki The Influence of Platelet Collagen Receptor Polymorphisms in Hemostasis and Thrombotic Disease Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 14 - 20. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
/
.015, 2-tailed t test) in GPV null
platelets than in WT platelets for 2.5 and 5 µg/mL JAQ1. The lag time
was significantly increased (P 
), or V.3 (
) against human GPV, or mAb
ALMA.16 against human GPIX (
) (all 20 µg/mL) were activated by the
addition of collagen (2.5, 1.2, 0.8, or 0.6 µg/mL) in the presence of
fibrinogen. The amplitude of aggregation at 3 minutes (A) and lag phase
(B) were expressed as the percentages of the values for MOPC-treated
platelets. Treatment with V.3 decreased the amplitude of aggregation
for 0.6 µg/mL collagen (P = .02; 2-tailed t
test, A) and increased the lag phase for all collagen concentrations
(P 
chain, leading to the activation of
syk and PLC
signaling protein.