人α-凝血酶与因子XIII A链上相互结合位点的表征

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

作者信息

Achyuthan K E

机构信息

ZymeTx Inc., Oklahoma City, OK 73104, USA.

出版信息

Mol Cell Biochem. 1998 Jan;178(1-2):289-97. doi: 10.1023/a:1006807312772.

DOI:10.1023/a:1006807312772

PMID:9546612

Abstract

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.

摘要

采用溶液和固相技术研究因子 XIII A 链与α-凝血酶的相互作用。α-凝血酶激活因子 XIII 的效率高于β-凝血酶（Km = 0.83 ± 0.08×10⁻⁷ M；V/K = 14.90 ± 3.20×10⁻³ min⁻¹）或γ-凝血酶（Km = 6.14 ± 1.26×10⁻⁷ M；V/K = 3.30 ± 1.00×10⁻³ min⁻¹）。固定化的FPR-α-凝血酶与血浆因子 XIII（Kd = 0.17 ± 0.04×10⁻⁷ M）的结合力大于因子 XIIIa（Kd = 0.69 ± 0.18×10⁻⁷ M）、肝转谷氨酰胺酶（Kd = 4.73 ± 1.01×10⁻⁷ M）和因子 XIII A 链（Kd = 49.00 ± 9.40×10⁻⁷ M）。FPR-α-凝血酶和α-凝血酶也以与蛋白酶活性成反比的亲和力结合固定化的因子 XIII A 链：最大结合浓度分别为 1.36×10⁻⁷ M 和 13.6×10⁻⁷ M。血浆因子 XIII、转谷氨酰胺酶和二硫苏糖醇竞争性抑制因子 XIII A 链与 FPR-α-凝血酶的结合：IC50 分别为 1.0×10⁻⁷ M、3.0×10⁻⁶ M 和 1.52×10⁻⁴ M。转谷氨酰胺酶也抑制因子 XIII 与α-凝血酶的结合（IC50 = 2.0×10⁻⁶ M）。凝血酶结合位点定位于因子 XIII A 链的 G38 - M731 片段，可能在转谷氨酰胺酶的同源区域（N72 - A493）内。α-凝血酶的 R320 - E579 是因子 XIII A 链的结合位点。α-凝血酶 B 链内的二硫键对于结合至关重要，但催化性的 H363 或残基 R382 - N394 和 R443 - G475 并非如此重要。这些研究为因子 XIII 的激活提出了结构基础，为因子 XIIIa 的产生提供了调节机制，并最终可能有助于开发基于结构的新型凝血酶和因子 XIIIa 抑制剂。