The objectives of this proposal are to characterize the physiological and pathological effects of vascular cells on activation of coagulation. I have determined that intrabovine aortic endothelial (ABAE) cells lack the ability to initiate coagulation by either the intrinsic or extrinsic pathway. However, ABAE cells, but not non-vascular cells (fibroblasts, fetal lung cells), catalyze activation of prothrombin by Factor X and calcium. Although exogenous Factor V has no effect on this reaction, anti-FactoraV IgG inhibits prothrombin activation. Factor V activity was detected in ABAE cells grown in serum-free media for 3 months, indicating that these cells probably synthesize Factor V. Comparison of prothrombin activation by aortic endothelial cells and platelets suggests that endothelial cells generate more thrombin than do an equivalent number of thrombin-activated platelets. These data suggest that endothelial cells may be an important catalyst in hemostasis. I propose: 1) to further characterize the role of aortic endothelium in prothrombin activation by studying the binding of 125I-Factor Xa to these cells as well as determine how Factor V is activated in this reaction; 2) to determine whether cultured vascular cells or vascular cells in situ catalyze the activation of (a) Factor IX by Factor VII, and b) the activation of Factor X by Factors IXa and VIIIa. If vascular cells catalyze either of these reactions, I will characterize them. In addition, I plan to develop experimental models of vascular injury that permit study of pathological interactions between vascular cells and the coagulation system, including in vitro models of cell unjury induced by lipopolysaccharide, low density lipoprotein and activated granulocytes. The methods to be used for this research include purification of coagulation factors involved in these reactions, establishment of vascular and nonvascular cells in culture; development of methods to study vascular cells in situ; and electrophoretic and/or functional assays to measure activation of coagulation factors by vascular cells. These studies may idenfity previously uncharacterized normal and pathological interactions between vascular cells and the coagulation system. Additionally, they may provide information which would enhance our understanding of the mechanisms involved in thrombosis.
