Coagulation Factor V(a) binds to human umbilical vein endothelial cells and accelerates protein C activation

Abstract

In vitro the rate of protein C activation by thrombin is significantly accelerated by two distinct cofactors (a) the endothelial cell surface protein, thrombomodulin, (b) human coagulation Factor V(a). We have recently reported that the activity of Factor V(a) is contained in the 78,000-D light chain. In this study we have investigated the effects of Factor V(a) and its light chain on the activation of protein C in the presence of cultured endothelial cells. Thrombin-catalyzed protein C activation on human umbilical vein endothelial cells was enhanced by Factor V(a). The ability of Factor V(a) to stimulate protein C activation on these cells was saturated at 50 nM Factor V(a) and was observed at several protein C concentrations. Isolated Factor V(a) light chain in concentrations up to 50 nM also accelerated protein C activation on endothelial cells, but higher concentrations inhibited the reaction. The effects of Factor V(a) or its light chain on protein C activation were also shown on a mouse hemangioma cell line but not on human fibroblasts nor on a human amelanotic melanoma cell line. Protein C activation on endothelial cells was partially inhibited by a goat anti-thrombomodulin antibody and further addition of a polyclonal rabbit anti-Factor V(V(A)) antibody resulted in additional inhibition. Endothelial cells grown in medium supplemented with human serum, devoid of Factor V coagulant activity, contained cell surface Factor V(V(a))(~15,000 molecules/cell) as measured by the binding of a monoclonal IgG directed against Factor V(V(a)). These cells also bound an additional 6,000-10,000 molecules Factor V(a) per cell as determined by direct binding studies using 125I-Factor V(a). We suggest that thrombomodulin and Factor V(a) act in concert to regulate protein C activation on the surface of endothelial cells.