Von Willebrand Factor Receptor Research Articles

Different levels of platelet activation in preeclamptic, normotensive pregnant, and nonpregnant women

ObjectiveThe aims of our study were to determine the basal platelet activation state in women with preeclampsia compared with normotensive pregnant women and nonpregnant women and to investigate the platelet reactivity on in vitro stimulation with adenosine diphosphate or thrombin receptor activation peptide. Study designPlatelet expression of CD61 (fibrinogen receptor), CD42a (von Willebrand factor receptor), CD62P (P-selectin), CD63 (Glycoprotein 53), and PAC-1 binding (activated fibrinogen receptor) were determined in 20 pairs of women with preeclampsia/normotensive pregnant women and in 12 nonpregnant women, with the use of flow cytometry. ResultsBasal platelet expression of CD61, CD42a and CD62P, and adenosine diphosphate–stimulated CD62P expression were increased in women with preeclampsia compared with normotensive pregnant women. Platelets from women with preeclampsia and normotensive pregnant women differed from platelets from nonpregnant women by expressing higher basal CD63 levels and being more responsive to in vitro agonist stimulation, which was demonstrated by increased expression of CD61, CD62P, and CD63. ConclusionThis study supports the notion that platelets are important in the pathophysiologic condition of preeclampsia.

Effect of selective inhibition of the p38 MAP kinase pathway on platelet aggregation

Systemic inflammation has been shown to be a contributing factor to the instability of atherosclerotic plaques in patients with acute coronary syndromes (ACS). VX-702, a novel p38 mitogen-activated protein kinase (MAPK) inhibitor, is currently under investigation in ACS patients with unstable angina to evaluate its safety and efficacy during percutaneous coronary intervention (PCI). The role of p38 MAPK in platelet aggregation of normal individuals was examined using the selective second generation p38 MAPK inhibitor VX-702. Treatment of platelets with thrombin (activates PAR1 and PAR4 thrombin receptors), SFLLRN (PAR1), AYPGKF (PAR4), collagen (alpha2beta1 and GPVI/FCgammaIIR receptors) and U46619 (TXA(2)) resulted in strong activation of p38 MAPK. Activation of the GPIb von Willebrand factor receptor with ristocetin did not stimulate p38 MAPK. Pre-treatment of platelets with 1 microM VX-702 completely inhibited activation of p38 MAPK by thrombin, SFLLRN, AYPGKF, U46619, and collagen. There was no effect of VX-702 on platelet aggregation induced by any of the agonists in the presence or absence of aspirin, heparin or apyrase. It has been postulated that a potential role of p38 MAPK is to activate phospholipase A(2) (cPLA(2)) which catalyses formation of arachidonic acid leading to production of thromboxane. Interestingly, we show contrasting effects of p38 MAPK inhibition as compared to aspirin inhibition on platelet aggregation in response to collagen. Blockade of TXA(2) production by aspirin results in significant inhibition of collagen activation. However,VX-702 has no effect on collagen-mediated platelet aggregation, suggesting that blocking p38 MAPK does not effect thromboxane production in human platelets. Therefore, unlike aspirin blockade of thromboxane production in platelets, p38 MAPK inhibitors such as VX-702 do not significantly affect platelet function and would not be expected to contribute to an elevated risk of bleeding side-effects in treated patients.

Compound heterozygosity for a novel nine-nucleotide deletion and the Asn45Ser missense mutation in the glycoprotein IX gene in a patient with Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder due to quantitative or qualitative abnormalities in the platelet glycoprotein (GP) Ib/IX/V complex, the major von Willebrand factor receptor. The complex comprises four subunits, each encoded by a separate gene. Several mutations have been described for each of the subunits, except for GPV, as a cause of BSS. We describe here the genetic basis of the disorder in a child with BSS. Flow-cytometric analysis of the patient's platelets showed a markedly reduced surface expression of all three glycoproteins of the GPIb/IX/V complex. DNA sequencing analysis showed the patient to be a compound heterozygote for two mutations in the GPIX gene, a novel nine-nucleotide deletion starting at position 1952 of the gene that changes asparagine 86 for alanine and eliminates amino acids 87, 88, and 89 (arginine, threonine, and proline) and a previously reported point mutation that changes the codon asparagine (AAC) for serine (AGC) at residue 45. Her mother was heterozygous for the Asn45Ser mutation, and her father, for the nine-nucleotide deletion. Our findings suggest that the additive effects of both mutations in the GPIX gene are responsible for the BSS phenotype of the patient.

A Short Total Synthesis of Sulfobacin A.

A total synthesis of the von Willebrand factor receptor antagonist sulfobacin A is described. Key steps for this short route to sulfobacin A include ruthenium-catalyzed asymmetric hydrogenation and diastereoselective electrophilic amination for the construction of the three stereogenic centers.

Bernard-Soulier syndrome due to the homozygous Asn-45Ser mutation in GPIX: an unexpected, frequent finding in Germany.

Bernard-Soulier syndrome (BSS) is a rare inherited disorder with giant platelets, thrombocytopenia and a prolonged bleeding time. These abnormalities are caused by genetic defects of the glycoprotein (GP) Ib-IX complex that constitutes the von Willebrand factor receptor on the platelet surface. Here, we describe four unrelated German patients with low platelet counts and mild bleeding tendency. Three patients had been diagnosed with immune thrombocytopenia (ITP) and were treated with steroids without response. Another patient presented with easy bruising. Peripheral blood smears showed giant platelets. Ristocetin-induced platelet aggregation was almost absent, and quantitative flow cytometry and Western blotting disclosed a greatly reduced surface expression of GPIb-IX. Unexpectedly, sequencing the entire coding regions of GPIbalpha, GPIbbeta and GPIX revealed that all four unrelated patients were homozygous for an A to G mutation in position 1826 of the GPIX gene, constituting a Asn-45Ser change. This mutation has been described before and now represents by far the most often identified molecular defect causing BSS in Caucasians. Because BSS patients are likely to be misdiagnosed with ITP, treatment-resistant ITP patients should be re-evaluated thoroughly. Asn-45Ser genotyping may be a helpful tool for differential diagnosis.

Glycosylation restores survival of chilled blood platelets.

Cooling of blood platelets clusters the von Willebrand factor receptor complex. Macrophage alphaMbeta2 integrins bind to the GPIbalpha subunit of the clustered complex, resulting in rapid clearance of transfused, cooled platelets. This precludes refrigeration of platelets for transfusion, but the current practice of room temperature storage has major drawbacks. We document that alphaMbeta2 is a lectin that recognizes exposed beta-N-acetylglucosamine residues of N-linked glycans on GPIbalpha. Enzymatic galactosylation of chilled platelets blocks alphaMbeta2 recognition, prolonging the circulation of functional cooled platelets. Platelet-associated galactosyltransferase produces efficient galactosylation when uridine diphosphate-galactose is added, affording a potentially simple method for storing platelets in the cold.

A short total synthesis of sulfobacin A

A total synthesis of the von Willebrand factor receptor antagonist sulfobacin A is described. Key steps for this short route to sulfobacin A include ruthenium-catalyzed asymmetric hydrogenation and diastereoselective electrophilic amination for the construction of the three stereogenic centers.

Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling.

Bidirectional signaling of integrin αIIbβ3 requires the β3 cytoplasmic domain. To determine the sequence in the β3 cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin αIIb, and mutants of β3 with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncation at Y759 did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of β3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F754, Y747, or T741 completely abolished integrin activation. A point mutation replacing Y759 with alanine also abolished integrin activation. Thus, the T755NITY759 sequence of β3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of β3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.

Signaling events underlying thrombus formation

Recent in vivo studies have highlighted the dynamic and complex nature of platelet thrombus growth and the requirement for multiple adhesive receptor-ligand interactions in this process. In particular, the importance of von Willebrand factor (VWF) in promoting both primary adhesion and aggregation under high shear conditions is now well established. In general, the efficiency with which platelets adhere and aggregate at sites of vessel wall injury is dependent on the synergistic action of various adhesive and soluble agonist receptors, with the contribution of each of the individual receptors dependent on the prevailing blood flow conditions. In this review, we will discuss the major platelet adhesive interactions regulating platelet thrombus formation under high shear, with specific focus on the VWF (GPIb and integrin alphaIIbbeta3) and collagen receptors (GPVI and integrin alpha2beta1). We will also discuss the signaling mechanisms utilized by these receptors to induce platelet activation with specific emphasis on the role of cytosolic calcium flux in regulating platelet adhesion dynamics. The role of soluble agonists in promoting thrombus growth will be highlighted and a model to explain the synergistic requirement for adhesive and soluble stimuli for efficient platelet aggregation will be discussed.

Integrin alphaIIbbeta3 and its antagonism.

AlphaIIbbeta3, the major membrane protein on the surface of platelets, is a member of the integrin family of heterodimeric adhesion receptors. The alphaIIb and beta3 subunits are each composed of a short cytoplasmic tail, a single transmembrane domain, and a large, extracellular region that consists of a series of linked domains. Recent structural analyses have provided insights into the organization of this and other integrins and how a signal is initiated at its cytoplasmic tail to transform the extracellular domain of alphaIIbbeta3 into a functional receptor for fibrinogen or von Willebrand factor to support platelet aggregation and thrombus formation. These functions of alphaIIbbeta3 have been targeted for antithrombotic therapy, and intravenous alphaIIbbeta3 antagonists have been remarkably effective in the setting of percutaneous coronary interventions, showing both short-term and long-term mortality benefits. However, the development of oral antagonists has been abandoned on the basis of excess of mortality in clinical trials, and the extension of therapy with existing alphaIIbbeta3 antagonists to broadly treat acute coronary syndromes has not fully met expectations. An in-depth understanding of how antagonists engage and influence the function of alphaIIbbeta3 and platelets in the context of the new structural insights may explain its salutary and potential deleterious effects.

Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibβ

The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates initial platelet adhesion and activation. It is known that the cytoplasmic domain of GPIbbeta is phosphorylated at Ser(166) by cAMP-dependent protein kinase (PKA). To understand the physiological role of GPIbbeta phosphorylation, a GPIb-IX mutant replacing Ser(166) of GPIbbeta with alanine (S166A) and a deletion mutant lacking residues 166-181 of GPIbbeta (Delta165) were constructed. These mutants, expressed in Chinese hamster ovary (CHO) cells, showed an enhanced VWF-binding function compared with wild type GPIb-IX. Treatment of CHO cells expressing wild type GPIb-IX with a PKA inhibitor, PKI, reduced Ser(166) phosphorylation and also enhanced VWF binding to GPIb-IX. Furthermore, cells expressing S166A or Delta165 mutants showed a significantly enhanced adhesion to immobilized VWF under flow conditions. Consistent with the studies in CHO cells, treatment of platelets with PKI enhanced VWF binding to platelets. In contrast, a PKA stimulator, forskolin, reduced VWF binding and VWF-induced platelet agglutination, which was reversed by PKI. Thus, PKA-mediated phosphorylation of GPIbbeta at Ser(166) negatively regulates VWF binding to GPIb-IX and is one of the mechanisms by which PKA mediates platelet inhibition.

Sepsis- and endotoxemia-generated cytokines do not trigger activation of human platelets.

To analyze the effect of cytokines generated in sepsis and endotoxemia (tumor necrosis factor [TNF]-alpha and interleukins [IL]-1beta, -6, and -8) on activation of human platelets and to study the effect of cytokines on platelet activation in the presence of alpha-thrombin, a potent inducer of coagulation and platelet activation generated in sepsis and endotoxemia. flow cytometric study of platelet activation induced by cytokines and/or thrombin in the whole blood and platelet-rich plasma (PRP) of healthy volunteers. Research laboratory in a Canadian hospital. Nine healthy volunteers recruited from laboratory staff. Venous blood samples were obtained into acid-citrate-dextrose anticoagulant. Whole blood and PRP were diluted with appropriate buffer optimized for analyzing platelet activation by flow cytometry. TNF-alpha, IL-1beta, IL-6, and IL-8 were added to blood or PRP in concentrations ranging from 1 to 100 ng/mL and incubated for 15 mins at 37 degrees C in the presence or absence of a submaximal concentration of human alpha-thrombin (0.025 units/mL). Samples were stained with fluorescent antibodies against markers of platelet activation (P-selectin [CD62], lysosomal protein [CD63], and fibrinogen and von Willebrand factor receptors [CD41 and CD42b, respectively]) and analyzed by flow cytometry. The data obtained show that none of these cytokines trigger activation of resting platelets in whole blood or PRP and do not modulate the effect of thrombin on platelet activation as measured by quantitation of CD62, CD63, and CD42b markers on the platelet surface. Cytokines TNF-alpha, IL-1beta, IL-6, and IL-8, which are extensively produced in sepsis and endotoxemia, do not trigger activation of resting human platelets directly or indirectly by mediating processes in white or red blood cells. The cytokines did not affect thrombin-induced platelet activation.

Genetic abnormalities of Bernard-Soulier syndrome.

Bernard-Soulier Syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. BSS is characterized by giant platelets, thrombocytopenia, and prolonged bleeding time, and the hallmark of this disorder is the absence of ristocetin-induced platelet agglutination. In the last 10 years, the molecular and genetic bases of many GPIb/IX/V defects have been elucidated, providing a better understanding of primary hemostasis and structure-function relations of the complex. Thus far, more than 30 mutations of the GPIbalpha, GPIbbeta, or GPIX genes have been described in BSS. Recent studies also have shown that the phenotypes caused by mutations in the subunits of the GPIb/IX/V span a wide spectrum, from the normal phenotype, to isolated giant platelet disorders/macrothrombocytopenia, to full-blown BSS and platelet-type von Willebrand disease. Although recent progress in molecular biology has clarified the genotype-phenotype relationships of the GPIb/IX/V disorders, a close examination of platelet morphology on blood smears is still indispensable for a proper diagnosis. In this review, we summarize recent advances in the molecular basis of BSS with special emphasis on giant platelets and the genetic characteristics of Japanese BSS.

Disruption of the long-range GPIIIa Cys5-Cys435 disulfide bond results in the production of constitutively active GPIIb-IIIa (αIIbβ3) integrin complexes

AbstractThe major platelet integrin αIIbβ3, also known as the platelet glycoprotein (GP) IIb-IIIa complex, mediates platelet aggregation by serving as the receptor for fibrinogen and von Willebrand factor. In addition to its physiologic role, GPIIb-IIIa also bears a number of clinically important alloantigenic determinants. Previous studies have shown that disruption of the long-range Cys5-Cys435 disulfide bond of the β3 subunit results in the production of isoforms that bind some, but not all, anti-PlA1 alloantibodies, suggesting that mutations in this so-called long-range disulfide bond can alter the conformation of GPIIIa. The purpose of this study was to examine the effects of either the Cys5Ala or Cys435Ala substitution of GPIIIa on the adhesive properties of the GPIIb-IIIa complex. We found that both Ala5GPIIIa and Ala435GPIIIa were capable of associating with GPIIb and were expressed normally on the cell surface when cotransfected into Chinese hamster ovary (CHO) cells. CHO cells expressing GPIIb-Ala5GPIIIa or GPIIb-Ala435IIIa bound well-characterized, conformationally sensitive ligand-induced binding site (LIBS) antibodies, and were capable of constitutively binding the fibrinogen-mimetic monoclonal antibodies Pl-55 and PAC-1, as well as soluble fibrinogen. Both GPIIb-Ala5IIIa– and GPIIb-Ala435IIIa–transfected CHO cells also bound more avidly to immobilized fibrinogen and were capable of mediating the tyrosine phosphorylation of pp125FAK on cell adhesion. These data are consistent with the notion that these regions of GPIIIa participate in the conformational change associated with receptor activation. Additionally, these studies may provide a molecular explanation for the previously reported ability of mild reducing agents to activate the GPIIb-IIIa complex and promote platelet aggregation.

The Use of Snake Venom Toxins as Tools to Study Platelet Receptors for Collagen and von Willebrand Factor

A large proportion of the biologically active proteins and peptides present within snake venoms interact with components of the haemostatic system to promote or inhibit the normal sequence of events that lead to clot formation. The venom proteins achieve their effects through interaction with various components of the coagulation cascade, endothelial matrix and platelets. Within the latter group, a number of venom proteins target the interaction of platelets with the major adhesive proteins, von Willebrand factor and collagen. The venom proteins bind either the adhesive protein itself or their receptors on the platelet surface, notably GP-Ib-IX-V and GPVI. This review discusses the substantial contribution that venom proteins have made to our understanding of the role of these two adhesive proteins and their receptors (excluding GPIIb-IIIa) in platelet regulation.

Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ibalpha is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway.

It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) gamma-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR gamma-chain, Syk and PLCgamma2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCgamma2 nor functional responses, such as Ca(2+) elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbalpha in washed platelets does not promote functional responses.

Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ibα is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway

It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) γ-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR γ-chain, Syk and PLCγ2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCγ2 nor functional responses, such as Ca2+ elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbα in washed platelets does not promote functional responses.

Platelet integrin alphaIIbbeta3-ligand interactions: what can we learn from the structure?

Upon vascular injury, platelets initiate interaction with exposed subendothelial matrices through various receptors such as glycoprotein (GP) Ib/IX/V complex, alpha2beta1 integrin, and GPVI/FcRgamma. Although these interactions cannot sustain stable platelet thrombus formation by themselves, they ultimately lead to the activation of alphaIIbbeta3 integrin (GPIIb-IIIa complex [GPIIb-IIIa]), the most abundant receptor in platelets. The alphaIIbbeta3 integrin plays a central role in primary hemostasis by serving as a receptor for fibrinogen and von Willebrand factor (vWf). It establishes a stable interaction with vWf bound to the extracellular matrices and uses fibrinogen as a bridging molecule in platelet aggregate formation. The alphaIIbbeta3 integrin also plays an important role in the pathogenesis of thrombosis. Over the past decades, a tremendous amount of effort has been made to elucidate the ligand-binding mechanisms of alphaIIbbeta3, in part because of its clinical significance. Most of the studies have relied on biochemical analyses of purified alphaIIbbeta3 or recombinant proteins generated in vitro. With the lack of actual 3-dimensional structure, molecular modeling has provided a useful framework for interpreting such experimental data on structure-function correlation of integrin molecules. However, it has also generated disagreement between different models. The aim of this minireview is to summarize the past efforts as well as the recent accomplishments in elucidating the structure/function of alphaIIbbeta3. Finally, we will try to explain all those experimental data using the recently published crystal structure of the extracellular domains of the alphaVbeta3 heterodimeric complex.

Platelet glycoprotein Ibalpha HPA-2 Met/VNTR B haplotype as a genetic predictor of myocardial infarction and sudden cardiac death.

Sudden cardiac death (SCD) is one of the leading manifestations of coronary heart disease in early middle age. Platelet glycoprotein (GP) Ib-IX-V receptor complexes play a key role in the initial adhesion of platelets to collagen during the formation of a coronary thrombus. The HPA-2 (Thr145 Met) and VNTR polymorphisms of the gene for GP Ibalpha have been studied previously in hospitalized patients with acute coronary syndromes. The significance of these polymorphisms in victims of sudden cardiac death is not known. The association of these 2 polymorphisms with coronary atherosclerosis, coronary artery stenosis, coronary thrombosis, myocardial infarction (MI), and SCD was studied in the Helsinki Sudden Death Study, which comprised 2 large autopsy series, collected 10 years apart during 1981 to 1982 and 1991 to 1992, of 700 middle-aged white Finnish men who suffered sudden or violent out-of-hospital death. The 2 polymorphisms showed an almost complete linkage disequilibrium. Men with acute MI (n=80) and coronary thrombosis (n=65) were more likely to be carriers of the HPA-2 Met allele (OR 2.0 and 2.6, respectively, P<0.005 for both) than were control subjects who died of noncardiac causes (n=367). In men <55 years old, the Met allele was overrepresented (OR 2.2) among victims of SCD (n=98) compared with control subjects (n=249). In men <55 years old, 17 of 29 men with acute MI (58.6%) and 16 of 23 men with coronary thrombosis (69.6%) were carriers of the HPA-2 Met allele compared with the 49 of 249 (19.7%) who had died of noncardiac causes (ORs 5.6 and 9.2, respectively). Similar associations were observed in the separate analyses of both autopsy series. Our results suggest that the HPA-2 Met/VNTR B haplotype of the platelet von Willebrand factor and thrombin receptor protein GP Ib-V-IX may be considered to be a major risk factor of coronary thrombosis, fatal MI, and SCD in early middle age.

Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation

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)