Activation of factor IX bound to cultured bovine aortic endothelial cells

Previous studies have shown that factor IX and its activated form, factor IXa, bind to cultured vascular endothelial cells and that cell-bound factor IXa retains its procoagulant activity. The present studies provide evidence that factor IX bound to cultured bovine aortic endothelial cells can be activated. Factor IX activation was assessed by finding cleavage of the factor IX molecule on NaDodSO4/polyacrylamide gel electrophoresis and by the generation of procoagulant activity as assessed by thrombin-treated factor VIII-dependent generation of factor Xa activity. Cell-bound factor IX (0.8 micrograms per 4 X 10(8) cells per ml) could be activated by factor XIa (5 micrograms/ml) or by factor VIIa (0.1 micrograms/ml) without exogenous tissue factor when endothelial cells were treated with phorbol ester and acquired tissue factor-like procoagulant activity. Regardless of how factor IX was activated, the cell-bound factor IXa required thrombin-treated factor VIII and calcium, but not exogenous phospholipid, to activate factor X. In further experiments, factor X bound to endothelial cells specifically and reversibly with a dependence on calcium and with a lower affinity (half-maximal at 480 nM) than factor IX. At saturation, 9.1 X 10(6) factor X molecules were bound per cell. After activation of factor X by factor IXa, approximately 50% of the factor Xa formed could be eluted from the cells by 10 mM EDTA, suggesting that the factor Xa was cell associated. These observations indicate that endothelial cells can bind and promote the activation of factors IX and X in the absence of platelets or exogenous phospholipid.