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.
Ma
Yongqiang
Y
The Canadian Blood Services, Edmonton Centre, Edmonton, Alberta, Canada.
Wong
Kenneth
K
D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology*; D006801:Humans; D010973:Platelet Adhesiveness; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport; D013917:Thrombin / Q000494:pharmacology
D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D003094:Collagen; D004492:Edetic Acid; D013917:Thrombin; D002118:Calcium