Estimation of the Possible Recognition Sites for Thrombomodulin, Procoagulant, and Anticoagulant Proteins around the Active Center of α-Thrombin

抄録

α-Thrombin has several characteristic module structures around its active center. Previ-ously we showed that a synthetic peptide, TWTANVGKGQPS, corresponding to the residues Thr¹⁴⁷ to Ser¹⁵⁸ of the B-chain of human thrombin, a possible interaction site of thrombomodulin near the active center of thrombin, specifically blocked the interactions between 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, RIGKHSRTRYER, LEKIYIHP, RYNWREN, DSTRIRI, EGDSGGP, and SWGEGCDRDGK, respectively corresponding to the residues Phe¹⁹ to Leu²⁷, Leu⁴⁵ to Phe⁵⁴, Arg⁶² to Arg⁷³, Leu⁸¹ to Pro⁸⁸, Arg⁸⁹ to Asn⁹⁵, Asp¹⁷⁵ to Ile¹⁸¹, Glu²⁰² to Pro²⁰⁸, and Ser²²⁶ to Lys²³⁶ of the B-chain of human thrombin, which are located around the active center, as well as TWTANVGKGQPS, on the interaction between thrombin and thrombomodulin, protein C, fibrinogen, Factor V, antithrombin III, or hirudin. Thrombin-thrombomodulin interaction was inhibited significantly by RYNWREN as well as TWTANVGKGQPS, and partially by LLYPPWDKNF. The inhibitory effects of the two former peptides were additive and thrombomodulin directly bound to them. RYNWREN and TWTANVGKGQPS also in-creased the K_m values 3-7 times for protein C as compared with the conditions without peptide. Thrombin-induced protein C activation in the absence of thrombomodulin was specifically blocked by EGDSGGP. Thrombin-induced fibrinogen clotting was blocked by FRKSPQELL, RIGKHSRTRYER as well as TWTANVGKGQPS at lower concentrations, and by RYNWREN and DSTRIRI at higher concentrations. Thrombin-induced Factor V activation was blocked by FRKSPQELL, RIGKHSRTRYER as well as TWTANVGKGQPS. Thrombin inhibition by antithrombin III was blocked by RIGKHSRTRYER and RYNWREN. Thrombin inhibition by hirudin was blocked by FRKSPQELL, RIGKH-SRTRYER, and TWTANVGKGQPS. These findings suggest that the module structures around the active center of thrombin serve as recognition sites for several protein substrates playing a role in procoagulation or anticoagulation, and may restrict substrate specificity.