凝血酶原和中凝血酶中的自催化肽键裂解。

Autocatalytic peptide bond cleavages in prothrombin and meizothrombin.

作者信息

Petrovan R J, Govers-Riemslag J W, Nowak G, Hemker H C, Tans G, Rosing J

机构信息

Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.

出版信息

Biochemistry. 1998 Feb 3;37(5):1185-91. doi: 10.1021/bi971948h.

DOI:10.1021/bi971948h

PMID:9477942

Abstract

During factor Xa-catalyzed prothrombin activation, several other reaction products accumulate as a result of proteolysis of prothrombin and its activation products by thrombin and meizothrombin. Gel electrophoretic analysis and N-terminal sequencing of reaction products showed that in the absence of Ca2+ ions thrombin cleaved the following peptide bonds: Arg51-Thr52/Arg54-Asp55 in the fragment 1 (F1) domain (k = 0.4 x 10(4) M-1 s-1), Arg155-Ser156 in prothrombin (k = 2 x 10(4) M-1 s-1), and Arg284-Thr285 in prethrombin 1 (k = 0.02 x 10(4) M-1 s-1). In the presence of 2.5 mM CaCl2, cleavage in fragment 1 (Arg51-Thr52/Arg54-Asp55) was not detectable, whereas cleavage at Arg155-Ser156 (i.e., removal of F1) was inhibited 25-fold. Cleavage at Arg284-Thr285 (formation of prethrombin 2 des-1-13) was not affected by the presence of Ca2+ ions. Meizothrombin rapidly converted itself into meizothrombin des-F1. The half-life (t1/2 = approximately 30 s) of this reaction was independent of the meizothrombin concentration (0.1-1 microM meizothrombin), which is indicative for intramolecular autocatalysis (k = 0.02 s-1 in the presence of 2.5 mM Ca2+ ions). Since the rapid removal of fragment 1 precludes investigations of the cleavage at Arg284-Thr285 in intact meizothrombin, we analyzed the cleavage of this peptide bond in R155A-meizothrombin, a recombinant product that is resistant to autocatalytic removal of the fragment 1 domain. In the absence of phospholipids, R155A-meizothrombin converted itself into thrombin des-1-13 by a combination of intramolecular (k = 0.8 x 10(-4) s-1) and intermolecular autocatalysis (k = 0.2 x 10(3) M-1 s-1). Intramolecular autocatalytic conversion of R155A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 x 10(-4) s-1), whereas intermolecular autocatalysis was accelerated 25-fold (k = 5.6 x 10(3) M-1 s-1) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 x 10(8) M-1 s-1, we conclude that in reaction systems containing purified proteins autocatalysis of meizothrombin hardly contributes to thrombin formation during factor Xa-catalyzed prothrombin activation.

摘要

在因子Xa催化的凝血酶原激活过程中，由于凝血酶和中凝血酶对凝血酶原及其激活产物的蛋白水解作用，还会积累其他几种反应产物。对反应产物进行凝胶电泳分析和N端测序表明，在没有Ca2+离子的情况下，凝血酶会切割以下肽键：片段1（F1）结构域中的Arg51-Thr52/Arg54-Asp55（k = 0.4×10⁴ M⁻¹ s⁻¹）、凝血酶原中的Arg155-Ser156（k = 2×10⁴ M⁻¹ s⁻¹）以及凝血酶原1中的Arg284-Thr285（k = 0.02×10⁴ M⁻¹ s⁻¹）。在存在2.5 mM CaCl2的情况下，片段1中的切割（Arg51-Thr52/Arg54-Asp55）无法检测到，而Arg155-Ser156处的切割（即F1的去除）受到25倍的抑制。Arg284-Thr285处的切割（凝血酶原2 des-1-13的形成）不受Ca2+离子存在的影响。中凝血酶迅速将自身转化为中凝血酶des-F1。该反应的半衰期（t1/2≈30 s）与中凝血酶浓度（0.1 - 1 μM中凝血酶）无关，这表明是分子内自催化作用（在存在2.5 mM Ca2+离子的情况下k = 0.02 s⁻¹）所致。由于片段1的快速去除使得无法研究完整中凝血酶中Arg284-Thr285处的切割情况，我们分析了R155A-中凝血酶中该肽键的切割情况，R155A-中凝血酶是一种对片段1结构域的自催化去除具有抗性的重组产物。在没有磷脂的情况下，R155A-中凝血酶通过分子内（k = 0.8×10⁻⁴ s⁻¹）和分子间自催化作用（k = 0.2×10³ M⁻¹ s⁻¹）的组合将自身转化为凝血酶des-1-13。R155A-中凝血酶向凝血酶的分子内自催化转化不受磷脂存在的影响（k = 0.8×10⁻⁴ s⁻¹），而分子间自催化作用则被磷脂囊泡加速了25倍（k = 5.6×10³ M⁻¹ s⁻¹）。由于因子Xa/Va催化中凝血酶转化为凝血酶的反应速率常数k = 5.5×10⁸ M⁻¹ s⁻¹，我们得出结论，在含有纯化蛋白质的反应体系中，中凝血酶的自催化作用在因子Xa催化的凝血酶原激活过程中对凝血酶形成的贡献很小。