Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane

Abstract

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...

The glycoprotein Ib complex of human blood platelets

“…Earlier reports describing GPIb purifications were mostly done with labeled platelet proteins as starting material [21] or the purity of the isolated components was established by gel electrophoresis followed by staining with relatively insensitive reagents such as Coomassie brilliant blue or periodic acid/Schiff s reagent [15]. Therefore, associated proteins within the plasma membrane or cytoplasmic proteins, or even membrane proteins that are partially external, which are poorly labeled, could not be detected.…”

“…The 'GPIb complex' as we define it here consists of those proteins which are reproducibly found associated with GPIb when the latter is isolated in the presence of non-denaturing detergents. GP17, also called GP22 or GPIX, is apparently present as a 1 : l complex with GPIb [15] and it partitions exclusively into the hydrophilic phase in Triton X-114 separations along with GPIb, although it also has the properties of an integral protein. Purified GP17 can only be kept in solution in the presence of detergent, suggesting that it must contain one or more hydrophobic domains.…”

“…Purified GP17 can only be kept in solution in the presence of detergent, suggesting that it must contain one or more hydrophobic domains. Berndt et al [15] recently demonstrated that monoclonal antibodies against GPIb and GP17 recognize the same number of binding sites per platelet. GP17, together with GPIb, is also absent on platelets of patients with Bernard-Soulier syndrome [17].…”

“…Fox et al [20] reported that actin-binding protein is linked to GPIb and that the linkage can be cleaved by calcium-activated protease. Solum et al [18] and Berndt et al [15] have recently demonstrated that when platelets are solubilized, for analysis by crossed immunoelectrophoresis, in the presence of either leupeptin or EDTA as inhibitors of the calcium-activated protease, the precipitation peak corresponding to GPIb is decreased and another slower migrating GPIb-related precipitation arc appears. This shift in mobility seems to be due to association with actin-binding protein.…”

“…It also changes to a slightly higher molecular mass under reducing conditions. Berndt et al [15] reported contaminations of 43-kDa and 100-kDa proteins in their GPIb complex preparations and described the 43-kDa protein as actin. In our isolations this protein had a different isoelectric point from actin.…”