Reassociation and translocation of glycoprotein IIB-IIIA in EDTA-treated human platelets

Platelet membrane glycoproteins IIb and IIIa form a calcium-dependent heterodimer that plays a key role in platelet adhesion and aggregation. The present objective was to measure the dissociation and reassociation of GPIIb-IIIa by flow cytometric analysis of platelets labelled with mAbs specific for the glycoprotein complex or each monomer. In agreement with previous studies, EDTA chelation of extracellular calcium, [Ca2+]o, dissociated the heterodimer in a time and temperature dependent manner. Agonist stimulation of EDTA-treated platelets induced subunits to reassociate with the following order of potency: thrombin > collagen > ADP. Two-fold increases in GPIIb-IIIa and GPIIb indicate that thrombin caused reassociation of surface subunits and concurrent translocation of complexes from intracellular pools. The latter was partially inhibited by cytochalasin B thus indicating that a subpopulation of GPIIb-IIIa required cytoskeletal remodelling for translocation. Surface GPIIIa as reported by anti-CD61 declined more and upregulated less compared with GPIIb-IIIa or GPIIb. Results suggest that EDTA incubation might have altered the conformation of this epitope and decreased mAb binding. Collagen induced GPIIb-IIIa reassociation but not translocation of cryptic complexes. BAPTA suppression of rises in cytosolic calcium concentration or low [Ca2+]o inhibited GPIIb-IIIa reassociation, thus indicating that this reaction was driven by signal transduction. Thrombin and collagen induced a comparable level of aggregation of EDTA-treated platelets despite a 3-fold difference in cell surface GPIIb-IIIa. It is concluded that the effects of EDTA on GPIIb-IIIa dissociation and loss of adhesive functions are largely but not completely reversible.