Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism

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Abstract
The endothelial cell surface provides a receptor for thrombin-designated thrombomodulin (TM) which regulates thrombin formation and the activity of the enzyme at the vessel wall surface by serving as a potent cofactor for the activation of protein C by thrombin. Heparin-like structures of the vessel wall have been proposed as another regulatory mechanism catalyzing the inhibition of thrombin by antithrombin III. In the present study, the interaction of antithrombin III with the thrombin-TM complex and its interference with heparin and polycations were investigated by using human components and TM isolated from the microvasculature of rabbit lung. Purified TM bound thrombin and acted as a cofactor for protein C activation. The addition of heparin (0.5 unit/mL) to the reaction mixture interfered neither with the binding of thrombin to TM nor with the activation of protein C. However, the polycations protamine (1 unit/mL) as well as polybrene (0.1 mg/mL) affected the thrombin-TM interaction. This was documented by an increase in the Michaelis constant from 8.3 .mu.M for thrombin alone to 19.5 .mu.M for thrombin-TM with the chromogenic substrate compound S-2238 in the presence of 1 unit/mL protamine. When the inhibition of thrombin by antithrombin III was determined, the second-order rate constant k2 = 8.4 .times. 103 M-1 s-1 increased about 8-fold in the presence of TM, implying an accelerative function of TM in this reaction. Although purified TM did not bind to antithrombin III-Sepharose, suggesting the absence of heparin-like structures within the receptor molecule, protamine reversed the accelerative effect of TM in the inhibition reaction. Furthermore, TM was insensitive to heparinase, heparitinase, or periodic acid treatment, indicating that the accelerative effect of TM was not mediated by heparin-like structures of TM. In addition, the presence of TM neither led to the inhibition of thrombin by heparin cofactor II nor did TM accelerate the inhibition of factor Xa by antithrombin III. However, reduction and carboxymethylation of TM, which destroyed its known cofactor activities, also abrogated the accelerative effect of TM in the thrombin-antithrombin III reaction. Thus, the function of TM to alter the specificity of thrombin for macromolecular substrates is not limited to the activation of protein C and to the inhibition of thrombin''s procoagulant activity but also includes the increased sensitivity of the enzyme for inactivation by antithrombin III. Although all the activities of the thrombin-TM complex were equally affected by polycations, the interaction between enzyme and receptor was not mediated by heparin-like structures.