Different role of platelet glycoprotein GP Ia/IIa in platelet contact and activation induced by type I and type III collagens

The role of glycoprotein Ia/IIa was studied during platelet contact and aggregation induced by type I and type III collagen. The anti-glycoprotein Ia/IIa (6F1) antibody inhibited type I collagen-induced aggregation but did not inhibit the first contact between platelets and collagen. In contrast, it was without effect either on type III collagen-induced contact or platelet interaction with the subendothelium in a static assay. Platelet aggregation induced by type III collagen was only slightly slowed down by 6F1 but pp72 spleen tyrosine kinase phosphorylation was not modified even at concentrations of 6F1 that completely blocked platelet activation induced by type I collagen. Our results indicate that glycoprotein Ia/IIa is not a primary binding site for type I or type III collagen on the platelet membrane. This receptor is more specifically involved in type I collagen-induced platelet spreading and aggregation.     

Cleavage site of calcium-dependent protease in human platelet membrane glycoprotein Ib

Chicken muscle-derived m-type calcium-dependent protease cleaved purified glycoprotein Ib alpha-chain (GPIb alpha, Mr 130,000) from human platelets into two fragments (Mr 100,000 and Mr 38,000) in the presence of 5 mM calcium. With partially purified glycoprotein Ib (alpha beta-dimer), an appearance of a fragment of Mr 100,000 was also demonstrated after treatment with both the m-type and human platelet-derived mu-type protease. These processes in glycoprotein Ib were inhibited by inhibitors of calcium-dependent proteases, 50 muM E-64-C or 0.2 mM leupeptin and by the chelation of calcium. Using two-dimensional gel electrophoresis system, release of glycocalicin in addition to 100 kDa fragment was demonstrated by calcium-dependent proteases. Then surface-labeled platelets were stimulated with A23187 in the presence of 5mM calcium. Under this condition, endogenous calcium-dependent protease is activated. Of the labeled glycoproteins, glycocalicin and glycoprotein V but not 100 kDa fragment were released from the platelet membrane. The released glycocalicin was further digested into a fragment of Mr 100,000 by the addition of m-type calcium-dependent protease. These results showed (i) that GPIb alpha was hydrolyzed by exogenous calcium-dependent proteases in two points and glycocalicin and 100 kDa fragment were produced and (ii) that endogenous protease cleaved GPIb alpha at one point and released glycocalicin.     

Transient expression of recombinant glycoprotein Ib alpha polypeptides in COS cells that inhibit von Willebrand factor binding to the platelet glycoprotein Ib/IX complex.

The cDNA encoding the glycoprotein Ib alpha polypeptide has been expressed in COS cells. Transfection with full-length cDNA and a cDNA truncated at an internal XbaI site produced a recombinant polypeptide doublet with estimated molecular weights of 48 and 46 kDa (rGpIb alpha L318) which could be resolved into a single band of molecular weight 36 kDa following digestion with endoglycosidase F. A portion of the truncated polypeptide was retained in the endoplasmic reticulum of COS cells and slowly released. A second fraction was rapidly secreted into COS cell-conditioned medium and could be used for functional studies. Soluble rGpIb alpha L318 harvested from COS cell-conditioned medium inhibited ristocetin-dependent binding of [125I]-vWF to fixed washed human platelets (IC50 20 nM). Binding was inhibited by reduction and alkylation of "rGp1b alpha L318" suggesting the need for a critical disulfide bond to maintain biological activity of the recombinant polypeptide. We conclude that the recombinant polypeptides produced by transfection of GpIb alpha cDNA into heterologous cells are secreted, soluble, and can inhibit vWF binding to platelet GpIb/IX.     

Polymorphism of platelet glycoprotein Ib in the United States

Platelet glycoprotein (GP) Ib from healthy individuals from the United States was analyzed using SDS-polyacrylamide gel electrophoresis and staining with peanut agglutinin-coupled peroxidase or wheat germ agglutinin-coupled peroxidase after transfer of electrophoresed proteins to nitrocellulose. The GPIb from American Caucasians (109 individuals) was found to be polymorphic, showing types A, B, C, and D of GPIb, similar to what was observed in Japanese. The phenotype distribution pattern, however, was very different from that observed in the Japanese population. GPIb heterogeneity was also observed in American Blacks, Asian Americans, and Americans of Hispanic origin. Thus, it appears that the polymorphism of GPIb is a general phenomenon which should be regarded as an important factor when doing studies on this glycoprotein.     

Molecular modeling of the seven tandem leucine-rich repeats within the ligand-binding region of platelet glycoprotein Ib alpha

Platelet glycoprotein (GP)Ib-IX-V mediates von Willebrand Factor (vWF)-dependent adhesion to vascular subendothelium at high shear in (patho)physiological thrombus formation. The ligand-binding domain of GPIb-IX-V is within the N-terminal 282 residues of GPIb alpha, that contains seven tandem leucine-rich repeats (Leu36-Ala200). Repeats 2-4 are critical for vWF binding. In this study, we have built molecular models of the seven leucine-rich repeats of human, canine and mouse GPIb alpha, providing novel insights into the species-specific interaction between human vWF and its receptor. Interestingly, a major difference between the models was a large negatively charged patch on the concave face of human, but not canine, repeats 2-4. In addition, five individual mutations within the leucine-rich repeats of GPIb alpha associated with the bleeding disorder Bernard-Soulier syndrome, that result in dysfunctional vWF binding, were mapped to the model of human GPIb alpha. This provides the basis for relating these genetic lesions to abnormal function of the receptor.     

Localization of a thrombin-binding site on human platelet membrane glycoprotein Ib determined by a monoclonal antibody

To determine a thrombin-binding site on GPIb alpha on platelet membrane, we have examined the binding activities of tryptic or chymotryptic fragments of purified GPIb alpha to a monoclonal antibody against GPIb (TM60) and thrombin using (immuno)affinity chromatography. When purified GPIb alpha was digested with trypsin, two fragments (94-kDa, and 43-kDa) were obtained. The 43-kDa fragment was shown to bind to both affinity columns of TM60- and thrombin-Affi-Gel, while the 94-kDa fragment did not bind to either Affi-Gel columns. When trypsin fragments were incubated with TM60 and then applied to the column of thrombin-Affi-Gel, neither fragments were bound to the column. When the same experiment was performed using chymotrypsin, three fragments (94-kDa, 45-kDa and 39-kDa) were observed. On TM60- and thrombin-Affi-Gel columns, the smaller fragments (45-kDa and 39-kDa) were bound to the column. After incubation of these fragments with TM60, neither bound to the thrombin column. These results indicate (i) that the epitope for TM60 is located near, or on the thrombin-binding site of GPIb alpha, and (ii) that the thrombin-binding site is located on the tail portion of GPIb alpha, especially on a chymotrypsin cleavage site.     

Unravelling the mechanism and significance of thrombin binding to platelet glycoprotein Ib

The main question concerning the mechanism of a-thrombin binding to platelet membrane glycoprotein (GP)Ib is whether it involves both thrombin exosite I and exosite II. The solution of two independent crystal structures suggests alternative explanations that may actually reflect different modes of binding with distinct pathophysiological significance. With respect to function, it is still unclear whether thrombin binding to GPIb promotes procoagulant and prothrombotic pathways of response to vascular injury or limits such responses by sequestering, at least temporarily, the active enzyme. We review here published information on these topics and touch upon ongoing studies aimed at finding definitive answers to outstanding questions relevant for a better understanding of thrombosis and haemostasis.     

Importance of fibrinogen and platelet membrane glycoprotein IIb/IIIa in shear-induced platelet aggregation

The mechanism of shear-induced platelet aggregation was investigated using a polycarbonate cone and plate viscometer. After exposed to shear stress of 54-90 dyne/cm2 for 2 min. at 37 degrees C, platelets aggregated without a significant amount of serotonin release and lactic dehydrogenase leakage from platelets. Under this conditions, platelets from 2 patients with thrombasthenia and a patient with congenital afibrinogenemia failed to aggregate. When fibrinogen was added to platelet rich plasma from a patient with afibrinogenemia, shear-induced platelet aggregation occurred at the same extent of aggregation as observed in normal platelets. Shear-induced platelet aggregation was inhibited by monoclonal antibody to GPIIb/IIIa (1 microgram/ml) and synthetic peptide, Arg-Gly-Asp-Ser (RGDS) (1 mM). Apyrase and hirudin showed no effect on this aggregation. Indomethacin (100 microM) and thromboxane A2 synthetase inhibitor, OKY-046 (100 microM) markedly inhibited aggregation, while thromboxane A2 competitive inhibitor, ONO-3708 (100 microM) exhibited only partial inhibition. These results indicate that fibrinogen and GPIIb/IIIa are important for shear-induced platelet aggregation and that the induction of fibrinogen receptor on GPIIb/IIIa may partially depend upon thromboxane A2 synthesis in platelets.     

Localization of von Willebrand factor and thrombin-interactive domains on human platelet glycoprotein Ib

Platelet membrane glycoprotein Ib (GPIb) functions as receptors for thrombin and von Willebrand factor (vWF) in the presence of ristocetin. To precisely locate the domains on GPIb interacting with vWF and thrombin, we prepared several peptides that have amino acid sequences analogous to that of the GPIb alpha-chain and examined their effects on ristocetin-induced (vWF-dependent) and thrombin-induced platelet aggregations. A peptide extending from residues Asp235 to Lys262 showed the strongest inhibitory effect on ristocetin-induced platelet agglutination, and a group of overlapping peptides composed of 24-28 amino acid residues representing sequences extending from Phe216 to Asp274 was found to inhibit platelet aggregation induced by thrombin. Other peptides did not inhibit platelet aggregations. Moreover, the binding to platelets of the monoclonal anti-GPIb antibody (TM60) which had been shown to inhibit both ristocetin- and thrombin-induced platelet aggregations was strongly inhibited by a peptide extending from Asp249 to Asp274. These data demonstrate that the vWF-binding domain exists in a small region between residues Asp235 and Lys262; the thrombin-interacting domain, in contrast, is located between residues Phe216 and Ala274, with a possible center of interaction in the sequence from Phe216 to Thr240 on the GPIb alpha-chain, and thrombin binding requires a relatively strict conformation in this domain.     

血小板膜糖蛋白受体基因多态性在缺血性心脑血管病中的作用

心、脑血管病的许多危险因素已被确定,如吸烟、高血压、高血脂、高血糖等,控制这些危险因素可以降低其发病率。在所有心、脑血管病发病因素中只有2/3可以归为可控制的危险因素,其它的遗传变异如血小板和凝血因子的多态性可从几     

Ristocetin and botrocetin involve two distinct domains of von Willebrand factor for binding to platelet membrane glycoprotein Ib.

We have evidence that ristocetin and botrocetin mediate binding of von Willebrand Factor (vWF) to platelet glycoprotein Ib (GPIb) through two distinct domains on the vWF molecule. This was established by using monoclonal antibodies (MAbs) to vWF and synthetic peptides derived from the sequence of vWF. MAb 322 and MAb NMC/vW4 both recognize native vWF as well as fragments containing the GPIb-binding domain of vWF, obtained with the following enzymes: trypsin (116 kDa), V-8 protease (SpIII, 320 kDa) and V-8 protease plus subtilisin (33-28 kDa). Nevertheless, the lack of reciprocal displacement between the two MAbs in experiments of competitive inhibition for binding to vWF demonstrate that their respective epitopes are separate. Both MAbs inhibit 125I-vWF binding to platelet membrane GPIb and vWF-dependent platelet agglutination induced by ristocetin. However, only MAb NMC/vW4 inhibits these functions in the presence of botrocetin and when ristocetin-induced platelet agglutination is inhibited by MAb 322, botrocetin is still able to restore the agglutination. The involvement of two distinct domains of vWF for binding to GPIb in the presence of ristocetin or botrocetin was confirmed in experiments of binding of 125I-vWF to platelets using a competitor synthetic peptides corresponding to the GPIb binding domain of vWF (Cys 474 to Pro 488 and Ser 692 to Pro 708). At a final concentration of 2.5 mM both peptides inhibit more than 90% of the binding of vWF to ristocetin-treated platelets but are unable to modify this binding in the presence of botrocetin. In conclusion our data suggest that botrocetin and ristocetin involve distinct sites on vWF for binding to GPIb.     

Platelet glycoprotein Ia C807T, Ib C3550T, and IIIa Pl(A1/A2) polymorphisms and ischemic stroke in young Taiwanese

Platelet plays a pivotal role in the pathogenesis of thrombotic cardiovascular diseases. Recently, the polymorphism of platelet glycoprotein (GP) genes has been reported to be associated with an increased risk for ischemic stroke. The purpose of this study is to evaluate the association between platelet GP genetic variants and ischemic stroke in young Taiwanese. We conducted a case-control study in 157 young ischemic stroke patients recruited between September 2001 and March 2003 and 157 age- and sex-matched controls. The genotypes of platelet GP Ia C807T, GP Ib C3550T, and GP IIIa Pl(A1/A2) polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Student's t-test, chi-square test, and logistic regression modeling were used for data analyses. The GP Ia C807T CC, CT and TT genotype frequencies were similar between patients (50.3%, 43.9%, 5.7%) and controls (53.5%, 38.9%, 7.6%; p=0.58). There were no significant differences in GP Ib C3550T CC and CT genotype distributions between patients (91.1%, 8.9%) and controls (91.7%, 8.3%; p=0.84). Of all subjects, none carries GP IIIa Pl(A2) mutation. In conclusion, platelet GP Ia C807T and GP Ib C3550T polymorphisms in our population are less common compared with Caucasians, and GP IIIa Pl(A1/A2) genetic mutation is not found, and all of them are not associated with ischemic stroke in young Taiwanese.     

血小板膜糖蛋白受体ⅠbαKozak序列基因多态性与缺血性脑血管病相关性的研究

目的 研究GPⅠbα Kozak序列基因多态性与缺血性脑血管病的相关性.方法 本研究采用聚合链式反应对正常对照组228例和缺血性脑血管病组232例进行研究,对所得结果进行统计学处理,得出GP Ⅰ bαKozak序列基因多态性与缺血性脑血管病的相关性.结果 缺血性脑血管病组Kozak序列C等位基因(TC 33.4%,CC 4.3%)的比例达到37.7%.对照组C等位基因(TC 21.2%,CC 21.8%)的比例为23%,经卡方检验,两组比较,χ2=17.378,df=1,P=0.03,具有显著性差异.Kozak序列C等位基因脑栓塞组21.6%;腔梗组28.1%;大面积梗塞组36.3%;对照组与脑栓塞组比较,χ2=3.086,df=1,P=0.079,没有显著性差异.与腔梗组比较χ2=1.854,df=1,P=0.173,没有显著性差异.与大面积梗塞组比较χ2=4.293,df=1,P=0.038,有显著性差异.结论 血小板膜糖蛋白受体Ⅰ b α Kozak序列基因多态性与缺血性脑血管病明显相关.     

Evidence for the involvement of platelet glycoproteins other than GP Ib in heparin-associated thrombocytopenia and thrombosis

Heparin-associated thrombocytopenia and thrombosis (HATT) is a potentially fatal complication of heparin therapy which is characterized by a progressive fall in the platelet count associated with arterial or venous thrombosis. The etiology is consistent with the development an antibody which, in the presence of heparin, induces intravascular platelet aggregation. Biochemical analysis has demonstrated that the platelet membrane glycoprotein (GP) Ib binds heparin. Recent studies have shown that polyclonal antisera or monoclonal antibodies to GP Ib can inhibit platelet aggregation induced by HATT plasma in the presence of heparin implicating GP Ib as the site of heparin-antibody interaction. The Bernard-Soulier syndrome (BSS) is an inherited bleeding disorder in which the platelets fail to adhere to exposed subendothelium due to a deficiency of GP Ib. We have used the platelets from a patient with documented BSS to further investigate the role of GP Ib in the heparin- dependent platelet aggregation induced by the plasma of three patients with HATT. We have shown that platelet aggregation by HATT plasma in the presence of heparin occurred as readily with BSS platelets as with normal donor platelets. These results indicate that glycoprotein Ib cannot be the only site of platelet-heparin-antibody interactions.     

Concanavalin A induces patching/capping of the platelet membrane glycoprotein IIb/IIIa complex

Two recent reports have shown platelet patching/capping by concanavalin A (Con A). In these studies, Con A receptors were shown to mobilize from pseudopodia and lamellipodia to the central cell parts during platelet attachment and spreading. The molecular mechanism underlying Con A receptor capping was not examined in either study. Con a binds maximally to human platelet membrane glycoproteins IIb and IIIa. In order to test whether Con A-induced capping caused the capping of this membrane glycoprotein complex, we treated normal human platelets with unlabeled Con A. After fixation, platelets were further treated with mouse monoclonal antibodies against the membrane glycoprotein IIb/IIIa complex and stained with fluorescein isothiocyanate (FITC) tagged anti-mouse IgG. An average of 16% platelets manifested capping with one monoclonal antibody preparation (N = 2) and 12% with a second preparation (N = 2). Control studies showed that only 18% of normal human fresh platelets exhibit capping with FITC-Con A (N = 17). If platelets were first incubated with unlabeled Con A, followed by staining with FITC-labeled anti-Con A antibody, an average of 15% platelets manifested caps (N = 17). Capping was inhibited by methyl-alpha-D-mannopyranoside (a known inhibitor of Con A), at cold temperature and by pre-treatment of platelets with colchicine. Our studies confirm the earlier findings on Con A induced capping. Also, our findings suggest that the molecular mechanism for Con A receptor capping involves patching and capping of the platelet membrane glycoprotein IIb/IIIa complex. It is possible that glycoprotein IIb/IIIa redistribution might be intimately involved during platelet attachment and spreading.     

Lack of association between the 807 C/T polymorphism of glycoprotein Ia gene and post-treatment platelet reactivity after aspirin and clopidogrel in patients with acute coronary syndrome

Variability in platelet response to antiplatelet therapy and its clinical relevance have been well described. However, the underlying mechanisms remain unclear. It was the aim of the present study to assess whether the response to aspirin and clopidogrel may be influenced by the 807 C/T polymorphism of the glycoprotein Ia (GpIa) gene in patients with non-ST elevation acute coronary syndrome (NSTE ACS). Six hundred one NSTE ACS patients were included in our study and were divided into three groups: CC homozygotes, CT heterozygotes ad TT homozygotes. All patients received loading doses of 600 mg clopidogrel and 250 mg aspirin at least 12 hours before blood samples were drawn. Post-treatment platelet reactivity was assessed by post treatment ADP 10 microM-induced platelet aggregation (ADP-Ag), VASP phosphorylation (PRI VASP) and P-selectin expression. Non-response to dual antiplatelet therapy was defined by high post-treatment platelet reactivity (HPPR=ADP-Ag > 70%). Significant variability in the distribution of platelet parameters was observed in the overall study population. No significant difference in platelet parameters profiles was observed within patients having the same genotype, for ADP-Ag (p=0.33), PRIVASP (p=0.72) and P-selectin expression (p=0.37). The genotype frequencies of the 807 C/T polymorphism of the GpIa gene were similar in responders and non-responders defined by persistent HPPR (p=0.104). In conclusion, our study did not show any influence of 807 C/T polymorphism of GpIa gene on post-treatment platelet reactivity assessed by ADP-Ag, PRI VASP or P-selectin expression in 601 NSTE ACS patients.     

807C/T polymorphism of platelet glycoprotein Ia gene is associated with cerebral hemorrhage in a Chinese population

Platelet glycoprotein (GP) mediated the role of platelet in coagulation. Platelet GP Ia 807C/T is the only GP polymorphism associated with the expression levels of GP Ia/IIa (the platelet collagen receptor). Recently, the GP Ia 807C/T polymorphism has been reported to have no association with cerebral hemorrhage (CH) in two studies pertained to Caucasian populations. The purpose of this study is to evaluate the association between platelet GP Ia 807C/T polymorphism and CH in a Han Chinese population. We performed genotype analysis for platelet GP Ia 807C/T polymorphism in a case-control study involving 195 patients with CH and 116 age- and sex-matched controls. In contrast to previous reports, we found that the frequencies of GP Ia 807C/T T allele, CT and TT genotype were much higher in CH patients than in controls (33.9% vs. 22.8%, p = 0.004; 45.5% and 11.1% vs. 40.4% and 2.6%, p = 0.022). Logistic regression analysis revealed that the presence of GP Ia 807C/T C allele and CC genotype were both associated with a decreased risk of CH compared with T allele, CT and TT genotypes, respectively (adjusted odds ratio [OR] = 0.565, 95% CI: 0.384–0.887, p = 0.005; adjusted OR = 0.172, 95% CI: 0.043–0.639, p = 0.009; adjusted OR = 0.254, 95% CI: 0.085–0.961, p = 0.041, respectively). These findings indicated that platelet GP Ia 807C/T polymorphism could be a protective factor of CH in the Chinese population.