Human platelet glycoprotein Ib (GP Ib) i s a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that G P Ib is normally coinplexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GPIb complex (GPIb plus GPIX) was purified to homogeneity in = 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. G P Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/ polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170000 under nonreducing conditions and 135000 (cr subunit) and 25000 (p subunit) under reducing conditions. GP IX had an apparent M , of 22000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-Factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. G P Ib, had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib, and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib, and G P 1X were blocked; G P Ib, had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (2 I: SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ib, (22000 I: 2700, n = 3), WM 23, epitope on G P Ib, (21 000 3400, n = 3), FMC 25, epitope on GP IX (20100 f 2700, n = 3), and FMC 25 (Fab')2 (27100 800, n = 2). The combined evidence suggests that GP Ib is normally bound to GP IX and that the stoichioinetry of this complex is 1 : 1.The initial event in hemostasis in response to vascular injury involves the adhesion of platelets to the exposed vascular subendothelium. The adhesion reaction appears to be dependent on three components [I]: a receptor on platelets which on the basis of a variety of evidence has been defined as the platelet membrane protein, glycoprotein Ib (GP Ib); a plasma component, factor VIII/von Willebrand fac...
We describe a new variant of Glanzmann's thrombasthenia (variant Strasbourg I). The patient (M.S.) showed an absence of platelet aggregation to ADP, thrombin, and collagen, and a decreased clot retraction. Platelet fibrinogen was -20% of normal levels. ADP-stimulated platelets bound markedly reduced amounts of soluble fibrinogen and platelet adhesion to surface-bound fibrinogen was defective. Normal to subnormal amounts of glycoprotein (GP) Ilb-Illa (am,83) complexes, the platelet fibrinogen receptor, were revealed by SDS-PAGE, crossed immunoelectrophoresis, and antibody binding. However, the complexes were unusually sensitive to dissociation with EDTA at room temperature. Furthermore, flow cytometry showed that the platelets failed to bind the activation-dependent monoclonal antibody, PAC-1, after stimulation. In contrast, an RGDS-containing peptide induced significant binding of the anti-ligand-induced binding site antibody, D3GP3, suggesting the presence ofa functional RGD binding domain on the patient's GPIIb-IIIa complex. Sequence analysis was performed after polymerase chain reaction amplification of selected patient's GPIIIa exons, and of the patient's platelet GPIIb and GPIIIa mRNAs. A point mutation (C to T) was localized in exon D (iv) of GPIIIa that resulted in an 214Arg to 214Trp amino acid substitution. The defect has been inherited from the parents who are heterozygous for the same mutation. This substitution points to an essential amino acid in a region of GPIIIa involved in the binding of fibrinogen and influencing the Ca2+-dependent stability of the GPIIb-IIIa complex. (J. Clin.
Patient C.M. presented platelet function defects symptomatic of Glanzmann's thrombasthenia. However, analysis of surface-labeled platelets by SDS-polyacrylamide gel electrophoresis revealed the usual presence of the major glycoproteins, including GP IIb and GP i1la. Platelet fibrinogen was not detected. Analysis of Triton X-100 extracts of Ca2"-washed C.M. platelets by crossed immunoelectrophoresis (CIE) showed normal amounts of GP IIb-IlIa complexes. However, when samples were electrophoresed through an agarose gel containing "2I-fibrinogen, the usual binding of fibrinogen to GP fib-IIIa did not occur. Furthermore, the GP IIb-IIIa complexes showed an increased sensitivity to dissociation with EDTA, either after Triton X-100 solubilization or in the intact platelet membrane. For example, after incubation with EDTA at room temperature, the patient's platelets bound little of the monoclonal antibodies AP-2 or T10 (anti-GP IIb-IIIa complex) although normally binding Tab (anti-GP fib). Patient C.M. appears to represent a subgroup of thrombasthenia where platelets contain unstable GP Hib-HlIa complexes unable to support fibrinogen binding.
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