Characterization of the reciprocal binding sites on human alpha-thrombin and factor XIII A-chain.

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-alpha-thrombin interactions. Alpha-thrombin activated Factor XIII more efficiently (Km = 0.83 +/- 0.08 x 10(-7) M; V/K = 14.90 +/- 3.20 x 10(-3) min(-1)) than beta-thrombin (Km = 6.14 +/- 1.26 x 10(-7) M; V/K = 3.30 +/- 1.00 x 10(-3) min(-1)) or gamma-thrombin (Km = 6.25 +/- 1.15 x 10(-7) M; V/K = 3.00 +/- 0.80 x 10(-3) min(-1)). Immobilized FPR-alpha-thrombin bound plasma Factor XIII (Kd = 0.17 +/- 0.04 x 10(-7) M) > Factor XIIIa (Kd = 0.69 +/- 0.18 x 10(-7) M) > liver transglutaminase (Kd = 4.73 +/- 1.01 x 10(-7) M) > Factor XIII A-chain (Kd = 49.00 +/- 9.40 x 10(-7) M). FPR-alpha-thrombin and alpha-thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 x 10(-7) M and 13.6 x 10(-7) M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR-alpha-thrombin: IC50 = 1.0 x 10(-7) M, 3.0 x 10(-6) M and 1.52 x 10(-4) M, respectively. Transglutaminase also inhibited Factor XIII binding to alpha-thrombin (IC50 = 2.0 x 10(-6) M). Thrombin-binding site was localized to G38-M731 fragment of Factor XIII A-chain, probably within homologous regions (N72-A493) of transglutaminase. R320-E579 of alpha-thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in alpha-thrombin were essential for binding but not catalytic H363 or residues R382-N394 and R443-G475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.