Pospisil Caroline H, Stafford Alan R, Fredenburgh James C, Weitz Jeffrey I
Department of Medicine, McMaster University and the Henderson Research Centre, Hamilton, Ontario L8V 1C3, Canada.
J Biol Chem. 2003 Jun 13;278(24):21584-91. doi: 10.1074/jbc.M300545200. Epub 2003 Apr 7.
Exosite 1 on thrombin mediates low affinity binding to sites on the NH2 termini of the alpha- and beta-chains of fibrin. A subpopulation of fibrin molecules (gammaA/gamma'-fibrin) has an alternate COOH terminus of the normal gamma-chain (gammaA/gammaA-fibrin) that binds thrombin with high affinity. To determine the roles of exosites 1 and 2 in the high affinity interaction of thrombin with gammaA/gamma'-fibrin, binding studies were done with thrombin variants and exosite 1- or 2-directed ligands. alpha-Thrombin bound gammaA/gamma'-fibrin via high and low affinity binding sites. A peptide analog of the COOH terminus of the gamma'-chain that binds alpha-thrombin via exosite 2 blocked the high affinity binding of alpha-thrombin to gammaA/gamma'-fibrin, suggesting that the interaction of alpha-thrombin with the gamma'-chain is exosite 2-mediated. In support of this concept, (a) gamma-thrombin, which lacks a functional exosite 1, bound to gammaA/gamma'-fibrin, but not to gammaA/gammaA-fibrin; (b) thrombin R93A/R97A/R101A, an exosite 2-defective variant, bound only to gammaA/gamma'-fibrin via low affinity sites; and (c) exosite 2-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. However, several lines of evidence indicate that exosite 1 contributes to the high affinity interaction of thrombin with gammaA/gamma'-fibrin. First, the affinity of gamma-thrombin for gammaA/gamma'-fibrin was lower than that of alpha-thrombin. Second, removal of a low affinity binding site on the beta-chain of gammaA/gamma'-fibrin reduced its affinity for alpha-thrombin. Third, exosite 1-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. Taken together, these data suggest that, although exosite 2 mediates the interaction of thrombin with the gamma'-chain of gammaA/gamma'-fibrin, simultaneous ligation of exosite 1 by low affinity binding sites is essential for the high affinity interaction of thrombin with gammaA/gamma'-fibrin.
凝血酶上的外位点1介导其与纤维蛋白α链和β链NH2末端位点的低亲和力结合。纤维蛋白分子的一个亚群（γA/γ'-纤维蛋白）具有正常γ链（γA/γA-纤维蛋白）的另一个COOH末端，它与凝血酶具有高亲和力结合。为了确定外位点1和2在凝血酶与γA/γ'-纤维蛋白的高亲和力相互作用中的作用，使用凝血酶变体和外位点1或2定向配体进行了结合研究。α-凝血酶通过高亲和力和低亲和力结合位点结合γA/γ'-纤维蛋白。通过外位点2与α-凝血酶结合的γ'-链COOH末端的肽类似物阻断了α-凝血酶与γA/γ'-纤维蛋白的高亲和力结合，表明α-凝血酶与γ'-链的相互作用是由外位点2介导的。支持这一概念的证据有：（a）缺乏功能性外位点1的γ-凝血酶与γA/γ'-纤维蛋白结合，但不与γA/γA-纤维蛋白结合；（b）外位点2缺陷变体凝血酶R93A/R97A/R101A仅通过低亲和力位点与γA/γ'-纤维蛋白结合；（c）外位点2定向配体减少α-凝血酶与γA/γ'-纤维蛋白的结合。然而，几条证据表明外位点1有助于凝血酶与γA/γ'-纤维蛋白的高亲和力相互作用。首先，γ-凝血酶对γA/γ'-纤维蛋白的亲和力低于α-凝血酶。其次，去除γA/γ'-纤维蛋白β链上的低亲和力结合位点会降低其对α-凝血酶的亲和力。第三，外位点1定向配体减少α-凝血酶与γA/γ'-纤维蛋白的结合。综上所述，这些数据表明，尽管外位点2介导凝血酶与γA/γ'-纤维蛋白的γ'-链相互作用，但外位点1被低亲和力结合位点同时连接对于凝血酶与γA/γ'-纤维蛋白的高亲和力相互作用至关重要。
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