Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism.
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 microM for thrombin alone to 19.5 microM 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 X 10(3) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D011479:Protamines / Q000494:pharmacology; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism*
D011479:Protamines; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D000990:Antithrombin III; D005170:Factor X; D005337:Fibrin; D006493:Heparin; D013917:Thrombin