Bezeaud A, Elion J, Guillin M C
Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
Blood. 1988 Mar;71(3):556-61.
The genetic variant prothrombin Salakta has been described in a patient presenting with a normal level of prothrombin antigen but reduced prothrombin activity. Initial studies indicated that factor Xa-catalyzed cleavages proceed normally but lead to the production of a thrombin molecule with an altered enzymatic activity. To characterize the functional abnormality of thrombin Salakta more precisely, it was purified by chromatography on heparin-Sepharose and diethylaminoethyl-Sephadex. The purified variant does not differ from normal thrombin by size, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and is 93.1% +/- 7.6% active by titration with p-nitrophenyl-p'-guanidinobenzoate. Its activity, however, is altered to various extents toward the following substrates: H-D-phenylalanyl-L-pipecolyl-L-arginine paranitroanilide (S 2238), fibrinogen, factor V, protein C, and antithrombin III. The Michaelis constant (Km) of thrombin Salakta for S 2238 is higher (12.2 +/- 3.3 mumol/L) than normal (2.8 +/- 0.7 mumol/L), whereas the turnover number (Kcat) is normal (84.4 +/- 6.6 s-1 v 85.9 +/- 14.0 s-1 for normal thrombin). The interaction of thrombin Salakta with benzamidine is also altered as evidenced by an increased inhibition constant (Ki = 3.5 mmol/L v 0.28 mmol/L for normal thrombin). The inability of fibrinogen to act as a competitor in the inactivation of thrombin Salakta by diisopropylfluorophosphate clearly indicates that fibrinogen binding to the fibrinopeptide groove is drastically impaired. In contrast, interactions involving sites remote from the active site such as those with fibrin and thrombomodulin are only slightly impaired. These results indicate that thrombin Salakta exhibits a specific pattern of functional alterations different from those reported for other variants. The structural defect seems to affect essentially the primary substrate binding site and to a lesser extent recognition site(s) remote from the catalytic site such as those for fibrin and thrombomodulin.
在一名凝血酶原抗原水平正常但凝血酶原活性降低的患者中，已发现了遗传变异体凝血酶Salakta。初步研究表明，因子Xa催化的裂解过程正常，但会产生一种酶活性改变的凝血酶分子。为了更精确地表征凝血酶Salakta的功能异常，通过肝素-琼脂糖凝胶和二乙氨基乙基-琼脂糖凝胶柱色谱法对其进行了纯化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断，纯化后的变异体在大小上与正常凝血酶无异，用对硝基苯基-p'-胍基苯甲酸酯滴定显示其活性为93.1%±7.6%。然而，其对以下底物的活性有不同程度的改变：H-D-苯丙氨酰-L-哌啶甲酰-L-精氨酸对硝基苯胺（S 2238）、纤维蛋白原、因子V、蛋白C和抗凝血酶III。凝血酶Salakta对S 2238的米氏常数（Km）高于正常水平（12.2±3.3μmol/L对比2.8±0.7μmol/L），而转换数（Kcat）正常（84.4±6.6 s-1对比正常凝血酶的85.9±14.0 s-1）。凝血酶Salakta与苯甲脒的相互作用也发生了改变，抑制常数增加即为证明（Ki = 3.5 mmol/L对比正常凝血酶的0.28 mmol/L）。纤维蛋白原无法在二异丙基氟磷酸酯灭活凝血酶Salakta的过程中起到竞争作用，这清楚地表明纤维蛋白原与纤维蛋白肽凹槽的结合严重受损。相比之下，涉及远离活性位点的位点（如与纤维蛋白和血栓调节蛋白的位点）的相互作用仅略有受损。这些结果表明，凝血酶Salakta表现出一种与其他变异体报道不同的特定功能改变模式。结构缺陷似乎主要影响主要底物结合位点，对远离催化位点的识别位点（如纤维蛋白和血栓调节蛋白的识别位点）的影响较小。
