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

Abstract

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.

Ex vivoanticoagulants affect human blood platelet biomechanics with implications for high-throughput functional mechanophenotyping

“…Platelets collected in K 2 -EDTA have the highest F-actin content under resting conditions in comparison to the other anticoagulants and show the lowest deformation. Our observations are in agreement with previous studies, which have demonstrated K 2 -EDTA induced ultrastructural changes of the surface-bound canal system (narrowing and dilatation of the OCS) and an irreversible dissociation of the αIIbβ3 complexes 43–45 46 . It is possible that the high content of F-actin in non-stimulated and TRAP-6 stimulated platelets and the associated platelet deformation could be explained by the irreversible dissociation of the αIIbβ3 complex and the associated cytoskeletal reorganization.…”

Cell surface-expressed phosphatidylserine as therapeutic target to enhance phagocytosis of apoptotic cells

“…EGTA conditions were chosen such that no dissociation of the a-and b-subunit of the integrin receptor occurred. 17 Binding of RGD-M1234-fluorescein to THP-1 cells (Figure 2d RGD-anxA5 enhances efferocytosis by THP-1 macrophages in vitro. RGD-anxA5 stimulated efferocytosis by THP-1 macrophages with 40%, whereas RGT-anxA5 inhibited it with 33% ( Figure 3a) and lactadherin stimulates efferocytosis with 60% (data not shown) as measured with a recently described efferocytosis assay (Supplementary Figures S2A and B).…”