CA2+-INDUCED STRUCTURAL TRANSITIONS OF THE PLATELET GP IIb-IIIa COMPLEX

Abstract

Previous studies have shown that the membrane glycoprotein (GP) IIb-IIIa complex can be reversibly dissociated by incubating platelets for 5 min at 37°C in an EDTA-containing buffer. Prolonged incubations (30 min) with EDTA, however, result in the formation of high molecular weight aggregates of GP IIb and GP IIIa. These aggregates of individual GP's neither bind fibrinogen nor support platelet aggregation, indicating that chelation of Ca2+ can affect the functional activity of GP IIb-IIIa. The present study was designed to identify conditions for the generation of functionally active GP IIb and GP IIIa. Functionally active subunits were defined as those which reformed GP IIb-IIIa complexes. The complexes were quantified by sucrose gradient sedimentation (complexed, dissociated and aggregated GP’s have different sedimentation coefficients) and thrombin hydrolysis (dissociated and aggregated GP lib are susceptible to hydrolysis by thrombin while GP lib in the GP IIb-IIIa complex is thrombin resistant). Purified GP IIb-IIIa could be dissociated by a 5 min incubation at 37°C with ≤ 10−5 M Ca2+. When the complexes were dissociated in the presence of Ca2+ concentrations below 10−6 m, the monomeric GP IIIa was converted to a slower sedimenting form; this change in structure caused it to become functionally inactive. In the presence of very low Ca2+ concentrations 10−6 M) both dissociated subunits subsequently formed high molecular weight aggregates. However, these changes in structure and loss in function could be prevented by dissociating the complexes in 10−6 M Ca2+ and immediately readding raM Ca2+ at 4°C. When this solution was warmed to 20°C, almost 70% of the dissociated subunits reformed heterodimeric complexes. Storage at 4°C for as long as 6 h did not alter the functional activity of these subunits. Octylglucoside, but not Triton X-100, completely inhibited reassociation. Experiments performed in the presence of various H+ and salt concentrations showed that the interactive forces between GP IIb and GP IIIa are both electrostatic and hydrophobic. Thus, conditions have been obtained for the preparation of functionally active GP IIb and GP IIIa which can reform the native heterodimeric complex. Various Ca2+ concentrations can have multiple effects on the structure of the dissociated subunits.