ADHESION OF ACTIVATED PLATELETS TO VENOUS ENDOTHELIAL-CELLS IS MEDIATED VIA GPIIB IIIA/

Normal circulating platelets do not adhere to intact, undisturbed endo thelium. Studies have shown, however, that platelets will adhere to vi rally infected or thrombin-stimulated human umbilical vein endothelial cells. Using a novel platelet/endothelial cell adhesion assay we stud ied the interaction of thrombin-activated platelets to human saphenous vein endothelial cells (HSVEC), and its mechanism(s). Biotinylated pl atelets 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 ex posed to thrombin-stimulated monolayer HSVEC. Phycoerythrin-streptavid in was added to the wells to fluorescently label the platelets, follow ed by formaldehyde fixation and washing to remove nonadherent platelet s. Adhesion of platelets to HSVEC was assessed using a fluorescent mul tiwell plate reader. Antibodies which blocked the GPIIb-IIIa receptor and agents which competitively bound the receptor all significantly in hibited activated platelet adhesion to the activated HSVEC. We have fo und that thrombin significantly increases platelet/HSVEC adhesion, and this event is mediated via the integrin GPIIb-IIIa (fibrinogen recept or). These GPIIb-IIIa receptor blocking Mabs and RGDS may be useful ad juncts for improving patency following angiographic intervention and/o r vein grafting in patients with high risk of thrombosis. The assay we have developed is a valuable and relatively simple method for assessi ng platelet/endothelial cell adhesion and activation. (C) 1996 Academi c Press, Inc.