A coagulation pathway on bovine aortic segments leading to generation of factor X(a) and thrombin

Abstract

Previous studies have demonstrated the binding of Factors IX and IX(a) to cultured bovine aortic endothelial cells. The present study examines the interaction of Factors IX, IX(a), and X(a) with the luminal surface of calf aortas, shown by microscopic examination to have a continuous layer of endothelium. Radioimmunoassay of Factor IX showed that 74 fmol/106 cells of Factor IX could be eluted from freshly prepared aortic segments. Binding of 3H-Factors IX and IX(a) to aortic segments was saturable, and comparable to binding in previous studies using cultured endothelial cells. Preincubation of aortic segments with 3H-Factor IX(a) and von Willebrand factor (VWF)/Factor VIII, followed by washing and addition of Factor X, resulted in formation of Factor X(a). The addition of prothrombin to these activation mixtures resulted in formation of thrombin. Exogenous phospholipid and Factor V were not required for Factor X and prothrombin activation on the intact native endothelium. Incubation of 125I-Factor X(a) with the vessel segments resulted in most of the tracer being complexed with antithrombin III originally present on the aortic segment (3.8 pmol antithrombin III/106 cells). The Factor X(a)-antithrombin III complex was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis exclusively in the supernatants. 125I-Factor X(a) not complexed with antithrombin III bound specifically to the vessel segment. The time course of binding was biphasic, consisting of an initial more rapid reversible phase followed by a slower irreversible phase. The latter phase correlated with the formation of a covalent complex (M(r), 76,000) between 125I-Factor X(a) and a vessel-localized protein presumably distinct from antithrombin III. The activation of prothrombin by vessel-bound Factor X(a) was inhibited by anti-bovine Factor V IgG, suggesting that there is interaction of Factor X(a) with a Factor V-like molecule provided by the endothelial cell surface. Addition of antibody to antithrombin III prevented formation of Factor X(a)-antithrombin III and thrombin-antithrombin III complexes in the supernatant and increased apparent thrombin activity 30-50-fold. These studies demonstrate that freshly obtained vessels with a continuous layer of native endothelium can support activation of Factor X and prothrombin: vessel-bound Factor IX(a) can activate Factor X in the presence of VWF/Factor VIII. Factor X(a) can also bind to the vessel and participate in the activation of prothrombin. The apparent efficiency of prothrombin activation, however, is dampened by the presence of functional antithrombin III on the vessel wall.