Ib Alpha Research Articles

Triflamp, a snake venom metalloproteinase, reduces neutrophil-platelet adhesion through proteolysis of PSGL-1 but not glycoprotein Ib alpha.

Triflamp, a metalloproteinase isolated from Trimeresurus flavo-viridis, inhibits heterotypic adhesion between platelets and neutrophils. Coincubation studies demonstrate that direct interaction of triflamp with neutrophils is sufficient to inhibit the formation of neutrophil-platelet complexes. Its anti-adhesive effect is in a concentration- and incubation time-dependent manner. Triflamp reduces the expression of P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils and glycoprotein (GP) Ibalpha on platelets as probed by flow cytometry and Western blot. Moreover, triflamp disrupts P-selectin-mediated adhesion by cleaving PSGL-1 from the neutrophil surface. There are obvious differences regarding PSGL-1 proteolysis by triflamp and cathepsin G. Besides the NH2-terminus of PSGL-1, other sites are truncated by triflamp. The inhibitory effect of triflamp on PSGL-1 expression was prevented by pretreatment with a metalloproteinase inhibitor, phenanthroline. However, triflamp-treated platelets fully keep the ability for binding to PAF- or fMLP-stimulated neutrophils. Our results indicate that degradation of platelet GPIb alpha by triflamp does not interfere with neutrophil-platelet adhesion. Its effect on neutrophil PSGL-1 appears to be a critical factor for its inhibition on neutrophil-platelet interaction.

Platinum-induced space-group transformation in crystals of the platelet glycoprotein Ib alpha N-terminal domain.

The interaction between platelet glycoprotein (GP) Ib alpha and von Willebrand factor (VWF) is essential for thrombus formation, leading to the arrest of bleeding. The N-terminal domain of GP Ib alpha, which contains the binding sites for VWF and alpha-thrombin, crystallized in the tetragonal space group P4(3) with one molecule in the asymmetric unit. When the crystals were treated with platinum, the crystals changed their symmetry from tetragonal to monoclinic P2(1) with two molecules in the asymmetric unit. The structure of the monoclinic form was solved using two-wavelength platinum anomalous dispersion data. The tetragonal crystal structure was subsequently solved using molecular-replacement techniques using the monoclinic structure as the search model and was refined with 1.7 A resolution data.

Glycoprotein Ib polymorphisms influence platelet plug formation under high shear rates.

Platelet polymorphisms (Kozak, VNTR and HPA-2) within glycoprotein (GP)Ib alpha may be associated with an increased risk of arterial thrombosis. However, the functional role of these polymorphisms has not been clarified. Their influence on platelet plug formation under high shear rates was, therefore, examined in 233 healthy individuals. Collagen-adrenaline-induced closure time was shorter in carriers of the C/D versus C/C VNTR allele and in homozygotes with the (-5)T/T versus (-5)C/T Kozak genotype as determined by novel polymerase chain reaction methods. The HPA-2 genotype had no effects, and the density of GPIb alpha molecules was not influenced by GPIb alpha genotypes.

Cytoplasmic truncation of glycoprotein Ib alpha weakens its interaction with von Willebrand factor and impairs cell adhesion.

The interaction of the platelet glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib-IX-V complex, the recognition of VWF appears to be mediated entirely by GP Ibalpha, the largest of four GP Ib-IX-V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibalpha in the GP Ib-IX-VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibbeta, GP IX, and either wild-type GP Ibalpha or GP Ibalpha mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibalpha polypeptides with truncations of 6-82 amino acids rolled faster than cells expressing wild-type GP Ibalpha. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibalpha with longer truncations. The rolling velocity of cells expressing truncated GP Ibalpha mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibalpha-VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibalpha-VWF bonds.

Site-directed mutagenesis of platelet glycoprotein Ibα demonstrating residues involved in the sulfation of tyrosines 276, 278, and 279

The interaction between platelet glycoprotein (GP) Ib alpha and von Willebrand factor (VWF) is essential for initiation of hemostasis. The sulfation of the 3 tyrosine residues 276, 278, and 279 in GPIb alpha is an important posttranslational modification that seems to promote the interaction with VWF. The environment where sulfation of tyrosines occurs has been proposed to contain highly acidic residues. This investigation has examined the highly acidic region from Asp249 to Asp287 in the mature GPIb alpha protein. Changes to most of the carboxylic acids in this region resulted in decreased reactivity to VWF. Only 3 mutants (Glu270Gln, Asp283Asn, Asp283Asn/Glu285Gln/Asp287Asn) resulted in the abolition of sulfation. Two novel mutations were also created. First, a deletion of the 7 amino acids from Tyr276 to Glu282 led to a loss of sulfation and totally abolished VWF binding in the presence of botrocetin. This confirms that it is these 3 tyrosines that undergo sulfation and that this region is crucial for botrocetin-mediated VWF binding. The second mutation involves changing the lysine residues at 253, 258, and 262 to alanine. This also led to distinct changes in VWF binding and abolition of sulfation.

Platelet glycoprotein Ib alpha receptor polymorphisms and recurrent ischaemic events in acute coronary syndrome patients.

To examine the relationship between polymorphisms in the platelet receptor glycoprotein (GP) Ib(alpha) and recurrent ischaemic events, and assess their impact on response to anti-platelet treatment. 1014 patients presenting with unstable coronary syndrome were recruited from the OPUS-TIMI 16 clinical trial of the platelet GPIIb/IIIa antagonist, orbofiban. The subjects were genotyped for two polymorphisms in the gene for GPIb(alpha). These were a T-5C polymorphism in the 5' untranslated Kozak region of the GPIb(alpha) gene, and the variable number of tandem repeats (VNTR) in the macroglycopeptide region.165 patients had events (recurrent ischaemia, urgent revascularisation, myocardial infarction (MI), stroke and death). There was no effect of the number of -5C alleles on composite endpoint frequency among Caucasian subjects (test for trend, p = 0.47). However, MI risk increased with the number of -5C alleles carried, with MI occurring in 2.3% of patients with the -5T/-5T genotype, 5.0% of -5T/-5C, and 16.7% of -5C/-5C (p < 0.01). The effect of treatment on MI outcome was not significantly modified by genotype (test for interaction, p = 0.10). The overall risk of bleeding was not strongly influenced by either the -5C or the VNTR polymorphisms. In an unstable coronary syndrome population the T-5C polymorphism in GPIb(alpha) influences risk of subsequent MI.

Pathological shear stress stimulates the tyrosine phosphorylation of alpha-actinin associated with the glycoprotein Ib-IX complex.

Shear-induced platelet responses are triggered by VWF binding to the platelet GpIb-IX complex, and there is evidence that this ligand-receptor coupling stimulates transmembranous signaling through the cytoplasmic tail of glycoprotein (Gp) Ib alpha. To investigate the mechanism by which signaling is effected, new molecular interactions involving GpIb-IX that develop in response to pathological shearing stress were examined in intact human platelets. Exposure to shear, but not alpha-thrombin, results in the co-immunoprecipitation of the actin cross-linking protein alpha-actinin with the GpIb-IX complex. Blockers of VWF binding to GpIb alpha or actin polymerization inhibit the association of alpha-actinin with the GpIb-IX complex, but the association of alpha-actinin with the GpIb-IX complex is not affected by inhibiting VWF binding to platelet integrin alpha IIb beta 3 (GpIIb-IIIa). alpha-Actinin becomes tyrosine phosphorylated in response to pathological shear stress, and phosphorylated alpha-actinin associates with GpIb-IX. In resting platelets, class IA heterodimeric phosphatidylinositol 3-kinase (PI 3-K) and protein kinase N (PKN) associate with nonphosphorylated alpha-actinin. Shear stress causes PI 3-K to disassociate from alpha-actinin, while it stimulates PKN binding to alpha-actinin. These results demonstrate that shear-induced VWF binding to GpIb alpha causes enhanced binding of cytoskeletal alpha-actinin to GpIb-IX and suggest that alpha-actinin, perhaps through tyrosine phosphorylation, serves as an adapter for a signaling complex that could regulate VWF-induced platelet aggregation.

Blood platelet membrane fluidity and the exposition of membrane protein receptors in Alzheimer disease (AD) patients--preliminary Study.

We estimated membrane fluidity by the (ESR) spectroscopy and the expression of membrane P-selectin and glycoproteins GP Ib alpha and GP IIb/IIIa by flow cytometry in platelets (plts) from 12 AD sufferers. In AD patients membrane fluidity was significantly increased at two different depths (p < 0.05 or less). Platelet reactivity was significantly decreased, as reflected by reduced expression of GP Ib alpha in the resting (p < 0.0001) and activated (p < 0.005) platelets, as well as the expression of P-selectin and beta 3 subunit of GP IIb/IIIa in activated platelets (p < 0.0001; p < 0.04).

Genetic linkage of Kozak sequence polymorphism of the platelet glycoprotein Ib alpha with human platelet antigen-2 and variable number of tandem repeats polymorphism, and its relationship with coronary artery disease.

The -5 C/T polymorphism of platelet glycoprotein (GP) Ib alpha is a major determinant of the level of GP Ib/V/IX complex surface expression. We investigated the frequency of this polymorphism among Asian populations. The gene frequencies of cytosine (C) in this polymorphism were 0.283 and 0.219 in Japanese and Korean populations respectively. The C allele is linked with human platelet antigen (HPA)-2a and smaller types of variable number of tandem repeats (VNTR). A novel allele, C-HPA-2a-D of VNTR, was found. No association was observed between these alleles and coronary artery disease in this case-control study. The clinical relevance of this polymorphism in the thrombotic status remains undetermined.

Necessity of Conserved Asparagine Residues in the Leucine-Rich Repeats of Platelet Glycoprotein Ibα for the Proper Conformation and Function of the Ligand-Binding Region

The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator-dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ib alpha antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ib alpha for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction.

Bernard-Soulier syndrome: common ancestry in two African American families with the GP Ib alpha Leu129Pro mutation.

Bernard-Soulier syndrome (BSs) is a rare bleeding disorder characterized by circulating giant platelets, thrombocytopenia, and a prolonged bleeding time. BSs usually has an autosomal recessive inheritance pattern, with a preponderance of Caucasian and Japanese ancestry when the ethnic background has been reported. Underlying this disorder of platelet function is a defect in the platelet glycoprotein (GP) Ib-IX-V complex, composed of four polypeptides, GP Ib alpha, GP Ib beta, GP IX, and GP V. Molecular characterization of individuals with BSs has identified mutations in the GP Ib alpha, GP Ib beta, and GP IX genes responsible for the expressed phenotype. In this study, we report a family of African-American descent, with autosomal recessive BSs showing a point mutation in codon 129 of the GP Ib alpha gene. This mutation, CTC:wild-type to CCC:mutant, is similar to that of another African American family where the resulting leucine to proline substitution in the 5(th) leucine-rich repeat of GP Ib alpha is responsible for the observed BSs phenotype. Comparison of the intragenic polymorphisms of GP Ib alpha, as well as microsatellite markers in a 17.5 cM region of chromosome 17p12 that contains the GP Ib alpha gene, suggests that, although socially unrelated, the Leu129Pro mutation in these two families has a common founder.

Total inhibition of high shear stress induced platelet aggregation by homodimeric von Willebrand factor A1-loop fragments.

Under high shear stress, the binding of von Willebrand factor (VWF) A1-loop domain to platelet glycoprotein (GP) Ib alpha occurs as the earliest event in thrombus formation. Therefore recombinant VWF A1-loop fragments could be of therapeutic use in blocking this interaction as competing ligands. We have prepared three homodimeric VWF A1-loop fragments [315 kD Fr III (a homodimer of amino acid [aa] residues 1-1365 of the subunit), 220 kD Fr (a homodimer of aa residues 1-708 of the subunit), and 116 kD Fr (a homodimer of aa residues 449-728 of the subunit) and two monomeric fragments [39/34 kD Fr (a monomer of aa residues 480/481-718 of the subunit] and His-rVWF465-728 (a monomer of aa residues 465-728 of the subunit)], and assessed their potency as inhibitors of botrocetin-induced VWF binding to GPIb alpha and high shear stress induced platelet aggregation mediated by intact VWE All these fragments completely inhibited botrocetin-induced VWF binding to GPIb alpha at a final concentration of 40-200 microM. The homodimeric A1-loop fragments also totally inhibited high shear stress induced platelet aggregation at a final concentration of 0.45-2.0 microM in the following order: 315 kD Fr > or = 220 kD Fr >> 116 kD Fr. In contrast, the monomeric A1-loop fragments were only partial inhibitors of high shear stress induced platelet aggregation even at a final concentration of 20 microM, and their IC50s were 13-39 times higher than those of the homodimers. These results indicate that the homodimeric structure of the A1-loop fragment is important for optimal molecular interaction with GPIb alpha under high shear stress.

Arterial shear stress stimulates surface expression of the endothelial glycoprotein Ib complex

Exposure to shear stress has been shown to alter the expression of a number of surface components of cultured endothelial cells (EC). However, relatively few studies have examined the status of human EC surface proteins after prolonged flow, more closely corresponding to the steady state in vivo. Since the promoter region of glycoprotein (Gp) Ib alpha contains several copies of a putative shear stress response element, 5'-GAGACC-3', we investigated the response of cultured human umbilical vein EC (HUVEC) GpIb alpha to shear stress over a 72 h time period. In response to 30 dynes/cm2 of shear stress, total cell content of GpIb alpha protein was markedly increased above static levels at 7 and 24 h, as determined immunohistochemically. Western blot analysis of whole cell lysates after 24, 48, and 72 h of shear treatment demonstrated a 2.4-, 4.1-, and 3.2-fold increase in total GpIb alpha protein, respectively. Cell surface protein expression of GpIb alpha increased 2.5-fold at 7 h, as measured by quantitative immunofluorescence, and remained at that level at 24 h. After 48 h of shear stress, cell surface GpIb alpha, GpIX, and GpV, analyzed by flow cytometric analysis, were further increased over the levels observed at 24 h. The increase in cell surface membrane expression of GPIb alpha at 24, 48, and 72 h was confirmed by immunoprecipitation of biotinylated surface proteins. No upregulation of GpIb alpha was noted after exposure to shear stress of 1-3 dynes/cm2. These observations imply that under steady-state arterial shear conditions endothelial expression of the GpIb complex is significantly greater than observed in static EC cultures, and raise the possibility of a more important role for this complex under flow, rather than static conditions.

Genetic polymorphism in human platelet glycoprotein GP Ib/IX/V complex is enriched in GP V (CD42d).

Platelet glycoproteins Ib beta (CD42c), IX (CD42a), and V (CD42d), together with GP Ib alpha (CD42b), form a receptor whose interaction with the von Willebrand factor is essential in the initial stages of haemostasis. Genetic variation in these proteins can cause alloimmunization leading to neonatal alloimmune thrombocytopenia and platelet transfusion refractoriness. Defective mutations cause a rare bleeding disorder, Bernard-Soulier syndrome. Only two antigenic polymorphisms have thus far been established in these proteins: the HPA-2 in GP Ib alpha and the rare Iy variant in GP Ib beta. Recently, we reported that only a limited degree of polymorphism can be found in the GP Ib alpha gene; the level of variation in the other components is not known. We therefore systematically screened polymorphism in the GP Ib beta, GP IX, and GP V genes in 50 unrelated Finnish blood donors. Nine polymorphic sites were found in the GP V gene, of which four changed the amino acid code and five were silent. The gene frequencies for substitutions Asp114Tyr, Met273Ile, Gly341Arg, and Leu397Arg were 1%, 1%, 2%, and 1% respectively. The five silent polymorphisms also had low frequencies, 1-4%. No polymorphism was found in the GP Ib beta gene and only one mutation was found in the 3' untranslated region of the GP IX gene. Our results indicate that genetic variation in the GP Ib/IX/V complex is mostly tolerated in the GP V protein--whose function in the complex is not clear whereas the other components have only very limited genetic polymorphism.

Crystal structure of the von Willebrand factor A1 domain in complex with the function blocking NMC-4 Fab.

The presence of one or more copies of von Willebrand factor type A domains identifies a superfamily of proteins usually involved in biological processes controlled by specific molecular interactions, often adhesive in nature. We have solved the crystal structure of the prototypic von Willebrand factor A1 domain, essential for the antihemorrhagic activity of platelets, in complex with the function blocking antibody, NMC-4, at 2.2 A resolution. This has led to the recognition of a putative binding groove for the platelet receptor, glycoprotein Ib alpha, formed by two adjacent alpha-helices and a beta-strand. The structure also shows a contact interface between A1 domain pairs, suggesting a hypothetical mechanism for the regulation of protein assembly and heterologous ligand binding mediated by homophilic interactions of type A domains.

Characterization of the Initial α-Thrombin Interaction with Glycoprotein Ibα in Relation to Platelet Activation

We have evaluated the properties of alpha-thrombin interaction with platelets within 1 min from exposure to the agonist, a time frame during which most induced activation responses are initiated and completed. Binding at 37 degrees C was rapidly reversible and completely blocked by a monoclonal antibody, LJ-Ib10, previously shown to be directed against the alpha-thrombin interaction site on glycoprotein (GP) Ib alpha. By 2-5 min, however, binding was no longer fully reversible and was only partially inhibited by the anti-GP Ib alpha antibody. Results were similar at room temperature (22-25 degrees C), whereas the initial characteristics of alpha-thrombin interaction with platelets were preserved for at least 20 min at 4 degrees C. Equilibrium binding isotherms obtained at the latter temperature were compatible with a two-site model, but the component ascribed to GP Ib alpha, completely inhibited by LJ-Ib10, had "moderate" affinity (kd on the order of 10(-8) M) and relatively high capacity, rather than "high" affinity (kd on the order of 10(-10) M) and low capacity as currently thought. The parameters of alpha-thrombin binding to intact GP Ib alpha on platelets at 4 degrees C corresponded closely to those measured with isolated GP Ib alpha fragments regardless of temperature. Blocking the alpha-thrombin-GP Ib alpha interaction caused partial inhibition of ATP release and prevented the association with platelets of measurable proteolytic activity. These results support the concept that GP Ib alpha contributes to the thrombogenic potential of alpha-thrombin.

Loss of high-affinity thrombin receptors during platelet concentrate storage impairs the reactivity of platelets to thrombin.

The storage of platelet concentrates (PCs) induces a reduction in the platelet surface expression of glycoprotein (GP) Ib alpha. The location of the platelets' high-affinity binding site for thrombin has been postulated as being located on GPIb alpha. This study attempts to determine whether loss or alteration of GPIb alpha during storage of PCs is related to impairment in the reactivity of platelets to thrombin. In this study, platelet surface expression of GPIb alpha was monitored by means of flow cytometry, throughout standard storage of PCs for up to 10 days. Two thrombin-induced platelet responses, the binding of radiolabeled fibrinogen and the platelet surface expression of P-selectin, were evaluated. Thrombin-binding assays were also performed to assess the number of thrombin receptors in platelets. The surface expression of the GPIb/IX complex declines during storage of PCs. The thrombin-induced maximal binding of fibrinogen in platelets stored for 3, 7, and 10 days was 77 +/- 7 percent, 60 +/- 20 percent, and 34 +/- 25 percent, respectively, of that found in fresh platelets. Moreover, the concentration of thrombin needed for 50 percent of platelets to express the CD62 antigen P-selectin at the surface increased from 0.05 U per mL in fresh platelets to 0.11, 0.56, and 1.2 U per mL in platelets stored for 3, 7, and 10 days, respectively. Thrombin-binding experiments demonstrated a significant reduction in the number of high-affinity binding sites throughout storage of PCs (55 +/- 21 sites/platelet in 10-day-stored platelets vs. 73 +/- 25 in fresh platelets). A significant correlation was also observed between the number of high-affinity thrombin-binding sites and surface expression of GPIb alpha. Selective blockage of the thrombin-binding site on GPIb alpha with monoclonal antibody LJ-Ib10 also inhibited the response of fresh platelets to thrombin, up to a level equivalent to that found in 3-day-stored platelets. The loss of the GPIb alpha-located high-affinity thrombin-binding site may impair the ability of platelets to become activated by thrombin as storage time increases.

StyI polymorphism at nucleotide 1610 in the human platelet glycoprotein Ib alpha gene.

StyI polymorphism at nucleotide 1610 in the human platelet glycoprotein Ib alpha gene.

Frequency and functional relevance of genetic threonine145/methionine145 dimorphism in platelet glycoprotein Ib alpha in an Italian population.

Threonine145/methionine145 dimorphism in platelet glycoprotein (GP) Ib alpha defines the human platelet antigen (HPA)-2 system that has been implicated in refractoriness to HLA-matched platelet transfusion and in neonatal immune thrombocytopenic purpura. The occurrence of this amino acid dimorphism was investigated in 379 Italian blood donors by studying their genomic DNA. Two oligonucleotide primers, Ib alpha-3 (5'-GGACGTCTCCTTCAACCGGC-3') and Ib alpha-4 (5'-GCTTTGGTGGGGAACTTGAC-3'), were used in a polymerase chain reaction to generate a 591-base pair fragment that was digested with the restriction enzyme Acy I. To investigate whether this dimorphism is involved in the binding of von Willebrand factor (vWF) to GPlb, the binding of vWF to the GPlb/IX complex was measured in two Met145/Met145 and two Thr145/Thr145 subjects. The genotypic frequencies are 78.9% for Thr/Thr, 19.8% for Thr/Met), and 1.3% for Met/Met; the allelic frequencies are 88.8% for Thr145 and 11.2% for Met145. Estimates for binding of subunit molecules per platelet at saturation and inhibition constant in mol per L, respectively, follow. In the presence of ristocetin (0.5 mg/mL), they are 11,460 +/- 2,040 and 1.26 +/- 0.44 x 10(-8) for normals and 11,230 +/- 2,330 and 1.29 +/- 0.48 x 10(-8) for patients. In the presence of botrocetin (2.5 micrograms/mL), they are 64,260 +/- 7,760 and 2.99 +/- 0.96 x 10(-8) for normals and 65,770 +/- 11,570 and 2.47 +/- 0.22 x 10(-8) for patients. Platelet aggregation responses obtained using platelet-rich plasma from donors with Met145/Met145 or Thr145/Thr145 genotype were within normal limits. Genotypic and phenotypic frequencies in the HPA-2 system in this population are consistent with those reported among the white population. Furthermore, the HPA-2 system is not involved in the binding of vWF to GPlb.

Systematic screening for genetic polymorphism in human platelet glycoprotein Ib alpha

Systematic screening for genetic polymorphism in human platelet glycoprotein Ib alpha