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

Abstract

SummaryIntact 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.

Structure-Function Relationships of the Thrombin-Thrombomodulin Interaction

“…Rabbit thrombomo dulin is, however, not particularly potent: ~ 200 ng of rabbit thrombomodulin (contain ing ~1 5 -2 0 n g chondroitin sulfate) had ac tivity similar to that of only 0.3 ng heparin [51], Furthermore, in the presence of heparin both rabbit [57] and bovine [ 17,58] thrombo modulin decrease the rate of thrombin inhibi6191 by antithrombin III; purified human thrombomodulin has little or no effect on the thrombin-antilhrombin III reaction [10,31,59], A model can be proposed to explain the effects of the chondroitin sulfate moiety on the direct anticoagulant and antithrombin Illdependent anticoagulant activities of throm bomodulin. EGF-like domains 5 and 6 of thrombomodulin bind to anion-binding exo site I on thrombin, and the chondroitin sul fate moiety may bind to the heparin binding site on thrombin.…”

Physiological Role of Vessel Wall Related Antithrombotic Mechanisms: Contribution of Endogenous and Exogenous Heparin-Like Components to the Anticoagulant Potential of the Endothelium

“…The loss of anticoagulant activity fol lowing treatment with Flavobacterium heparinase further supported the heparin-like na ture of these preparations [49,51]. However, these observations contrasted with other re ports in which a heparin-like activity for the rate enhancement of thrombin inhibition by AT-III could not be identified on the intact endothelium [52,53], rather the surface of cultured endothelial cells appeared to pro tect thrombin against fast inactivation by AT-III.…”

“…In a system with cultured bovine aortic endo thelial cells, no acceleration of the inhibition of cell-bound thrombin by AT-I1I was noted, and, consequently, appreciable amounts of activated protein C could be generated in the presence of excess AT-III ( fig. 2) [53]. Like wise, human thrombomodulin acted as a weak competitive inhibitor of thrombin inactivation by AT-III [91, 92], such that thrombin inhibition appeared to be partly protected by the endothelial cell receptor.…”

Pathophysiologic and Biochemical Events in Disseminated Intravascular Coagulation: Dysregulation of Procoagulant and Anticoagulant Pathways

“…232 Also, the more pronounced inactivation of thrombin by antithrombin III mediated by heparan sulfate could not be confirmed by other investi gators. [237][238][239] Finally, thrombin may be inhibited by heparin cofactor II, which facilitates its action when dermatan sulfate is present. 240 Finally, thrombin can be inactivated by the en dothelial cell membrane receptor thrombomodu lin.…”