The glycoprotein Ib complex of human blood platelets

Abstract

Human glycoprotein Ib (GPIb) is a major integral membrane protein on human blood platelets responsible for the initial attachment of platelets to the exposed vascular subendothelium. In this report we describe an isolation method for a 'GPIb complex' as well as for its individual components. A three-step procedure involving Triton X-114 phase-partition, affinity chromatography on wheat germ agglutinin and ion-exchange chromatography on DEAE-Sephacel yielded milligram quantities of purified GPIb complex. The single components of the complex were further purified by gel filtration on AcA34 in 0.1 YO sodium dodecyl sulfate. As well as GPIb, the complex contains GP17, actin-binding protein, actin and a series of unidentified proteins with different molecular masses. In contrast to GPIba, which is very rich in 0-linked oligosaccharides, sugar analysis revealed that GPIbg and GP17 seem to have only N-linked chains of the lactosamine type. The C-terminal a-chain remnant, which probably spans the plasma membrane, was identified and isolated for the first time. Western blotting with polyclonal rabbit anti-GPIb antibodies and silver-staining of one-or two-dimensional dodecyl sulfate/polyacrylamide gels revealed that it has an apparent molecular mass of 20 kDa and is linked to GPIbP by a disulfide bridge close to the membrane. The thrombin-binding site on GPIb is located near the N-terminus on a 40-kDa fragment of GPTba. A disulfide bridge in the N-terminal region is not essential for thrombin binding to GPIb.Blood platelets play a crucial role in the initial event of hemostasis by adhering to exposed vascular subendothelium [l, 21. This complex process is mediated by binding of a plasma component, von Willebrand factor, first to exposed subendothelium and then to a receptor on platelets, the membrane glycoprotein Ib [3, 41. Glycoprotein Ib is also involved in activation of platelets by thrombin [5-71 and in binding of quininelquinidine-dependent antibodies [8 -101. Glycoprotein Ib has an apparent molecular mass of 165 kDa and consists of two subunits, GPIba and GPIbB, with molecular masses of 135 kDa and 25 kDa, linked by a disulfide bridge [Ill. Most of the information about GPIb is derived from studies with glycocalicin, a major proteolytic fragment [12, 131. Glycocalicin can itself be split by various proteases into two pieces, a carbohydrate-rich 90-kDa fragment and a 45-kDa fragment which contains the receptor sites for thrombin and von Willebrand factor [6, 241. GPIb has all the characteristics of a typical receptor and probably functions as part of a complex containing several types of molecules.

The α1 helix-β13 strand spanning Leu214 to Val229 of platelet glycoprotein Ibα facilitates the interaction with von Willebrand factor: evidence from characterization of the epitope of monoclonal antibody AP1

“…Of interest, AP1, VM16d, and TM60 all also inhibit thrombin-induced binding to GP Ib␣, [20][21][22][23] suggesting that this region contains an important thrombin binding site. This had been suggested previously by Katagiri et al,12 who showed that the peptide Phe216 to Thr240 inhibited high-affinity thrombin binding and by Wicki and Clemetson,24 who showed that an important thrombin binding site on GP Ib␣ lies N-terminal to Lys237. 21 This presumption has now been confirmed in 2 crystal structures of the GP Ib␣ N-terminus in complex with thrombin, which show this region of GP Ib␣ bound by thrombin exosite I.…”

Identification of a tetrasialylated monofucosylated tetraantennary N‐linked carbohydrate chain in human platelet glycocalicin

“…Glycocalicin can be split into a 90 kDa fragment and a 45 kDa fragment [18]. Carbohydrate analysis of these fragments (not shown) shows that the 90 kDa fragment contains almost exclusively O-linked and the 45 kDa fragment N-linked carbohydrate chains.…”

Antithrombotic Effect of Platelet Glycoprotein Ib–Blocking Monoclonal Antibody Fab Fragments in Nonhuman Primates

“…GPIb was purified from outdated platelet concentrates collected by the Belgian Red Cross in Leuven, Belgium, according to the method described by Wicki and Clemetson. 24 BALB/c mice were immunized by intraperitoneal injection with the purified GPIb. Murine antibodies were prepared by conventional hybridoma technology as previously described, 25 and generated hybridoma cells were screened for antibody production in an ELISA by using purified GPIb.…”