Structural analysis of human platelet membrane glycoprotein I complex.

Abstract

The glycoprotein I complex, consisting of two polypeptides of Mr 210,000 and 150,000, was isolated from human platelet membranes by wheat germ lectin affinity chromatography. Glycocalicin, a soluble loosely bound membrane glycoprotein of M, 150,000 related to the glycoprotein I system, was also purified. The isolated poly eptides were radioiodinated in sodium dodecyl sulfate/! Iyac lamide gels and digested with trypsin, and the labeled Reptide digest was analyzed by two-dimensional high-voltage electrophoresis and thin-layer chromatography. The two polypeptides of Mr 210,000 and 150,000 in the glycoprotein I complex had essentially identical radioactive peptide maps. Glycocalicin had a completely different t ptic peptide map. These studies shed light on the molecular relationships of some of the components of the platelet membrane glycoprotein I system. The possibility is raised that the receptorlike function of the intrinsic platelet membrane glycoproteins may be related to the polymeric subunit associations of the constituent polypeptides. The external surface glycoproteins of mammalian cells mediate important cellular reactions, including aggregation, adhesion, and surface contact interactions (1). It is therefore not surprising that platelet membrane glycoproteins have been implicated in von Willebrand factor-dependent platelet agglutination induced by ristocetin (2, 3). The glycoprotein I (GPI) complex on the surface of the human platelet may act as a von Willebrand factor receptor (4) and thus directly influence the adhesion of platelets to the subendothelium. Depending on the method of membrane solubilization and the gel system utilized for analysis, the platelet GPI complex has been found to consist of at least two distinct glycoproteins (GPIa, GPIb) (5, 6). GPIc, which is part of the GPI system, does not stain with periodic acid/Schiff reagent and is probably not a glycoprotein (6). Another constituent of the GPI complex, glycocalicin or GPIs, is easily removed from the platelet membrane by homogenization and is ordinarily detected in the soluble subcellular fractions of homogenized preparations (7). The platelets of patients with the Bernard-Soulier syndrome are deficient in GPI as well as in glycocalicin (2,8). These platelets fail to agglutinate in the presence of von Willebrand factor and ristocetin and also do not bind thrombin normally (9). The possibility has been raised that glycocalicin may act as a single receptor for ristocetin-induced and thrombin-induced platelet aggregation (10). We have previously isolated two proteins of the GPI complex from human platelet membranes by using wheat germ lectin affinity columns and demonstrated that these proteins mediate von Willebrand factor-dependent platelet agglutination induced by ristocetin (11). At present, it is not clear what the structural relationships are among the isolated separate constituents of the GPI complex. This study presents a comparison of the radioactive tryptic peptide maps of the isolated polypeptides of the platelet-GPI...

Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

“…George et al (37) have expressed a similar view following the analysis of normal platelets before and after glycocalicin loss. In contrast, Nachman et al (41) have claimed that lb and glycocalicin give different peptide maps following trypsin digestion and are different GP although absolute proof for the identification of Ib was not given.…”

Complex formation of platelet membrane glycoproteins IIb and IIIa with fibrinogen.

“…Platelet GPIb was prepared as described (15 Washed platelets were incubated with thrombin in the presence of 1 mM EDTA without stirring. The platelets were removed by centrifugation at 8370 g for 3 min using a Beckman microfuge (Beckman Instruments, Inc., Fullerton, Calif.).…”

Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib.

“…Alternatively, the recovery of some of the glycoproteins with actin filaments might result from the presence of complexes of these glycoproteins with glycoproteins comprising the true attachment sites. GP Ib, for example, has been purified as a complex with a polypeptide of Mr = 210,000 (44). The latter glycoprotein might represent one ofthe high molecular weight glycoproteins recovered with actin filaments in the present study.…”

“…This indicates that GP lb is associated with an additional polypeptide(s), an interaction that is maintained in the presence of Ca2+-dependent proteolytic activity (40). A candidate for such a polypeptide is the one of Mr = 210,000 reported previously to co-purify with GP lb (44). Another candidate is GP IX, which has been shown to co-purify with GP lb on anti-GP lb monoclonal antibody affinity columns (45).…”