ESTIMATION OF THE POSSIBLE RECOGNITION SITES FOR THROMBOMODULIN, PROCOAGULANT, AND ANTICOAGULANT PROTEINS AROUND THE ACTIVE-CENTER OF ALPHA-THROMBIN

Alpha-thrombin has several characteristic module structures around its active center. Previously we showed that a synthetic peptide, TWTANVG KGQPS, corresponding to the residues Thr147 to Ser158 of the B-chain o f human thrombin, a possible interaction site of thrombomodulin near t he active center of thrombin, specifically blocked the interactions be tween thrombin and thrombomodulin, fibrinogen, Factor V, or platelets [Suzuki, K. & Nishioka, J. (1991) J. Biol. Chem. 266, 18498-18501]. To elucidate further the role of the other module structures, we studied the effects of several synthetic peptides; FRKSPQELL, LLYPPWDKNF, RIG KHSRTRYER, LEKIYIHP, RYNWREN, DSTRIRI, EGDSGGP, and SWGEGCDRDGK, respe ctively corresponding to the residues Phe19 to Leu27, Leu45 to Phe54, Arg62 to Arg73, Leu81 to Pro88, Arg89 to Asn95, Asp175 to Ile181, Glu2 02 to Pro208, and Ser226 to Lys236 of the B-chain of human thrombin, w hich are located around the active center, as well as TWTANVGKGQPS, on the interaction between thrombin and thrombomodulin, protein C, fibri nogen, Factor V, antithrombin III, or hirudin. Thrombin-thrombomodulin interaction was inhibited significantly by RYNWREN as well as TWTANVG KGQPS, and partially by LLYPPWDKNF. The inhibitory effects of the two former peptides were additive and thrombomodulin directly bound to the m. RYNWREN and TWTANVGKGQPS also increased the K(m) values 3-7 times f or protein C as compared with the conditions without peptide. Thrombin -induced protein C activation in the absence of thrombomodulin was spe cifically blocked by EGDSGGP. Thrombin-induced fibrinogen clotting was blocked by FRKSPQELL, RIGKHSRTRYER as well as TWTANVGKGQPS at lower c oncentrations, and by RYNWREN and DSTRIRI at higher concentrations. Th rombin-induced Factor V activation was blocked by FRKSPQELL, RIGKHSRTR YER as well as TWTANVGKGQPS. Thrombin inhibition by antithrombin III w as blocked by RIGKHSRTRYER and RYNWREN. Thrombin inhibition by hirudin was blocked by FRKSPQELL, RIGKHSRTRYER, and TWTANVGKGQPS. These findi ngs suggest that the module structures around the active center of thr ombin serve as recognition sites for several protein substrates playin g a role in procoagulation or anticoagulation, and may restrict substr ate specificity.