As the cells forming the luminal vascular surface, endothelium is strategically located to play a role in the regulation of coagulation. Participation of endothelium in coagulation involves specific receptors on the cell surface functioning at the level of initiation and propagation of hemostatic reactions. In the anticoagulant protein C pathway, for example, the receptor thrombomodulin initiates thrombin-mediated activation of protein C and a binding site for protein S on bovine endothelium promotes assembly of the functional activated protein C/protein S complex. Endothelium also synthesizes, stores and releases functional protein S constitutively and in response to specific stimuli such as norepinephrine.Since activation of protein C requires thrombin formation in proximity to the vessel wall, we have examined procoagulant reactions on the endothelial cell surface. Endothelium provides a receptor for Factor IX/IXa which is relatively selective for the enzyme form and facilitates Factor IXa-VIII-mediated activation of Factor X. Half-maximal Factor Xa formation occurs at a Factor IXa concentration of 0.4nM on endothelium, whereas lOnM is required on liposomes. This concentration of Factor IXa corresponds to that which results in half-maximal occupancy of endothelial cell Factor IXa binding sites in the presence of Factors VIII and X, thus correlating kinetics and binding measurements. Crosslinking and ligand blotting studies have shown that the receptor is a protein with a molecular weight of ∼160,000. The clinical significance of this receptor is suggested by the moderately severe bleeding disorder observed in a patient with hemophilia B due to an abnormal Factor IX molecule, Factor IXalabama (Factor IXala). Although the coagulant activity of Factor IXala is only mildly decreased on phospholipids, it is severely impaired on endothelium. The affinity of Factor IXala for the endothelial cell Factor X activation complex is decreased by 20-fold compared with the normal enzyme and the binding affinity is similarly decreased. Since the molecular defect in Factor IXala has been previously shown to consist of a single point mutation in the growth factor domain, this indicates a role for the growth factor domain in receptor, recognition.The picture of endothelial cell coagulant properties which emerges from these and other studies is one in which endothelium has either an anticoagulant or procoagulant potential, depending on modulation of receptor expression and release of secreted products. In the quiescent state, anticoagulant mechanisms predominate with only limited amounts of procoagulant activity: there is little tissue factor activity and only a basal level of receptors for Factor IX/lXa. Activation of endothelium by Tumor Necrosis Factor (TNF) or Interleukin 1 can shift this balance. Tissue factor synthesis and expression occurs in a dose-dependent manner, being half-maximal at a TNF concentration of about 150pM. TNF also increases the number of Factor IX/lXa binding sites. Concomitant with enhancement of endothelial cell procoagulant properties is a suppression of cell surface cofactor activity for the anticoagulant protein C pathway. Endothelial cell-dependent, thrombin-mediated activated protein C formation is decreased by 70-80% and activated protein C-protein S-mediated Factor Va inactivation decreases by over 90%. Following the in vivo infusion of Interleukin 1, similar changes in endothelial cell coagulant properties were observed on aortic segments with fibrin deposition occurring on the functionally altered, but morphologically intact endothelium. This modulation of endothelial cell coagulant properties could underlie the prothrombotic state associated with inflammatory disorders and could also explain the recently observed selective intravascular thrombosis of tumor vasculature seen in vivo in meth A sarcomas after administration of TNF.Thus, although endothelium was initially felt to be hemostatically inert, this apparent lack of activity actually masks a delicate balance of procoagulant and anticoagulant mechanisms. The balance can be effectively shifted by physiologic mediators, such as monokines, which alter receptor expression on the endothelial cell surface. Changes in endothelial cell hemostatic properties may be an early indicator of vessel wall disease and underlie the pathogenesis of localized thrombotic processes.
Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII
