Bezeaud A, Guillin M C
Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
J Biol Chem. 1988 Mar 15;263(8):3576-81.
Autolysis or tryptic hydrolysis converts human alpha-thrombin to its beta-derivative and subsequently to gamma-thrombin. Human beta-thrombin was obtained by tryptic digestion of alpha-thrombin and isolated by BioRex chromatography. The kinetic parameters for human alpha- and beta-thrombins with H-D-phenylalanyl-L-pipecolyl-L-arginine-para-nitroanilide were similar, as well as the rate of inactivation by tosyl-lysine chloromethyl ketone. By contrast, the rate of inactivation by diisopropyl fluorophosphate was reduced by half, and the inhibition constant for benzamidine was increased 2.5-fold. Moreover, the beta cleavages induced a drastic reduction in reactivity toward protein C, affinity for thrombomodulin, and fibrinogen clotting activity. Unlike alpha-thrombin, beta-thrombin was not protected from inhibition by diisopropyl fluorophosphate in the presence of fibrinogen and failed to bind to fibrin-Sepharose. Our results indicate that the beta cleavages induce multiple defects in the functions of human thrombin. Although the three catalytic residues remain in an active configuration, subtle changes are induced in the microenvironment of the active serine. However, the drastic reduction of fibrinogen clotting activity should rather be ascribed to major alterations observed in both the fibrinopeptide groove and the fibrin recognition site. These observations provide further evidence for a double-site mechanism in the interaction of fibrinogen with thrombin.
自溶或胰蛋白酶水解可将人α-凝血酶转化为其β衍生物，随后再转化为γ-凝血酶。人β-凝血酶是通过对α-凝血酶进行胰蛋白酶消化获得的，并通过BioRex色谱法进行分离。人α-和β-凝血酶对H-D-苯丙氨酰-L-哌啶基-L-精氨酸-对硝基苯胺的动力学参数相似，对甲苯磺酰赖氨酸氯甲基酮的失活速率也相似。相比之下，二异丙基氟磷酸酯的失活速率降低了一半，而苯甲脒的抑制常数增加了2.5倍。此外，β裂解导致对蛋白C的反应性、对血栓调节蛋白的亲和力以及纤维蛋白原凝血活性急剧降低。与α-凝血酶不同，在纤维蛋白原存在的情况下，β-凝血酶不能免受二异丙基氟磷酸酯的抑制，并且不能与纤维蛋白-琼脂糖结合。我们的结果表明，β裂解会导致人凝血酶功能出现多种缺陷。尽管三个催化残基保持活性构型，但活性丝氨酸的微环境中会发生细微变化。然而，纤维蛋白原凝血活性的急剧降低应更多地归因于在纤维蛋白肽凹槽和纤维蛋白识别位点观察到的主要变化。这些观察结果为纤维蛋白原与凝血酶相互作用中的双位点机制提供了进一步的证据。
