Adhesion of activated platelets to venous endothelial cells is mediated via GPIIb/IIIa.
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.
Li
J M
JM
Division of Vascular Surgery, University of Massachusetts Medical School, Worcester 01655, USA.
Podolsky
R S
RS
Rohrer
M J
MJ
Cutler
B S
BS
Massie
M T
MT
Barnard
M R
MR
Michelson
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004492:Edetic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D015539:Platelet Activation; D010973:Platelet Adhesiveness; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000502:physiology*; D012501:Saphenous Vein
D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D004492:Edetic Acid; C046617:arginyl-glycyl-aspartyl-serine