Formation of Activated Protein C and Inactivation of Cell-Bound Thrombin by Antithrombin III at the Surface of Cultured Vascular Endothelial Cells - A Comparative Study of Two Anticoagulant Mechanisms^[*]

Summary

Intact vascular endothelium provides several anticoagulant mechanisms for the maintenance of blood fluidity and the prevention of thrombosis. High-affinity binding of proteolytic active thrombin to thrombomodulin at the cell surface effectively facilitates the activation of the potent anticoagulant protein C (PC). Rapid inactivation of cell-bound thrombin by antithrombin III (AT III) accelerated by heparin-like structures represents another anticoagulant mechanism. In the present investigation the interference of these two events has been studied. Inhibition of thrombin bound to cultured bovine aortic endothelial cells (BAEC) by AT III and the effect of the inhibitor on the activation of PC has been studied using purified components of bovine origin. Exposure of thrombin (45 nM) with prewashed confluent BAEC-monolayers for 10 min resulted in the binding of 12% thrombin. The subsequent incubation with various concentrations (0.3-2.4 μM) of ATIII revealed no acceleration of the inhibition of thrombin by ATIII at the endothelial cell surface when compared with the uncatalyzed fluid phase reaction. However, heparin added to the reaction mixture substantially increased the inactivation of cell-bound thrombin. Modified ATIII that did not possess heparin cofactor activity presented a comparable inactivation pattern for endothelial cell bound-thrombin as native ATIII indicating that heparin-like structures did not accelerate the interaction. When PC (32 nM) and ATIII (1.8 μM) competed for thrombin bound to BAEC, activation of PC was demonstrated within the initial 6 min of the incubation amounting to 62% of the activated PC formation in the absence of ATIII. Preincubation of BAEC with blocking antibodies against ATIII excluded a possible influence of BAEC-bound ATIII on the capacity of cell-bound thrombin to activate PC. The results suggest that an enhancing mechanism for the inactivation of thrombin by ATIII was not operative at the surfaces of cultured bovine aortic endothelial cells, but instead, appreciable amounts of activated PC become generated in the presence of an excess of ATIII.

Keywords

Endothelial cells - Protein C - Antithrombin III - Thrombin - Heparin-like proteoglycans

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