Cellular requirements for tissue factor generation by bovine aortic endothelial cells in culture

Cultured bovine aortic endothelial cells acquired the ability to initiate coagulation after treatment with endotoxin or phorbol ester. The acquired procoagulant activity was identified as tissue factor since cells treated with endotoxin or phorbol ester activated factor X only in the presence of factor VIIa, and factor X activation could be completely blocked by a specific antibody to bovine tissue factor apoprotein. The generation of tissue factor activity was evident after 6 hours of incubation and was dependent on RNA and protein synthesis, as indicated by the inhibitory effects of actinomycin D and cycloheximide. Endotoxin and phorbol ester are toxic to cultured endothelial cells as evidenced by release of LDH and detachment from the culture dish. Surviving endothelial cells lose their stress fibers and assume a cytoskeletal organization characteristic of mobility or radial extension. Because these changes in cell shape occurred parallel with the acquisition of procoagulant activity, the effects of drugs interfering with organization of the cytoskeleton were tested. Cytochalasins B and D, vinblastine, and cotchicine, each decreased the generation of tissue factor activity when cells were exposed to endotoxin or phorbol ester. Trifluoperazine, a calmodulin antagonist, also prevented the generation of tissue factor activity in a dose-dependent fashion. Thus, perturbation of endothelial cells by treatment with phorbol ester or endotoxin induces potent tissue factor procoagulant activity. This cellular response appears to require protein and RNA synthesis, normal cytoskeletal functions, and the Ca⁺⁺-calmodulin system.