SPECIAL ARTICLE: Adhesion of Activated Platelets to Venous Endothelial Cells Is Mediated via GPIIb/IIIa

Abstract

Normal circulating platelets do not adhere to intact, undisturbed endothelium. Studies have shown, however, that platelets will adhere to virally infected or thrombin-stimulated human umbilical vein endothelial cells. Using a novel platelet/endothelial cell adhesion assay we studied the interaction of thrombin-activated platelets to human saphenous vein endothelial cells (HSVEC), and its mechanism(s). Biotinylated platelets were exposed to Hepes–Tyrode buffer, 10E5 or PAC-1 [monoclonal antibodies (Mabs) blocking GPIIb–IIIa], AK4 (Mab blocking P-selectin), 6D1 (Mab blocking vWf binding to GPIb), RGDS (small peptide blocking the fibrinogen binding site), or EDTA (dissociates GPIIb–IIIa complex) and then activated with thrombin. The platelets were subsequently exposed to thrombin-stimulated monolayer HSVEC. Phycoerythrin–streptavidin was added to the wells to fluorescently label the platelets, followed by formaldehyde fixation and washing to remove nonadherent platelets. Adhesion of platelets to HSVEC was assessed using a fluorescent multiwell plate reader. Antibodies which blocked the GPIIb–IIIa receptor and agents which competitively bound the receptor all significantly inhibited activated platelet adhesion to the activated HSVEC. We have found that thrombin significantly increases platelet/HSVEC adhesion, and this event is mediated via the integrin GPIIb–IIIa (fibrinogen receptor). These GPIIb–IIIa receptor blocking Mabs and RGDS may be useful adjuncts for improving patency following angiographic intervention and/or vein grafting in patients with high risk of thrombosis. The assay we have developed is a valuable and relatively simple method for assessing platelet/endothelial cell adhesion and activation.