Release of fibrinopeptides by the slow and fast forms of thrombin.
Release of fibrinopeptides by the slow and fast forms of thrombin.
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通过慢速和快速形式的凝血酶释放纤维蛋白肽。
DOI：
10.1021/bi952834d
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
DiCera,E
中科院分区：
文献类型：
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作者：
Vindigni,A;DiCera,E
在 5 至 45 °C 的温度范围和 100 至 800 mM 的盐浓度范围内研究了慢速和快速形式的凝血酶释放纤维蛋白肽 A 和 B 的情况。发现在所有检查的条件下都遵循谢弗最初提出的纤维蛋白肽释放的顺序机制。纤维蛋白原和纤维蛋白 I 在过渡状态下与快速形式的凝血酶优先结合的起源在于二阶缔合速率常数 k1。就纤维蛋白原而言，25°C 下与快形式和慢形式相互作用的 k1 值为 19 ± 4 和 2.5 ± 0.3 μM-1s-1，两种形式的活化能约为 10 kcal/mol。对于纤维蛋白 I，k1 的类似值为 9.1 ± 0.7 和 2.5 ± 0.2 μM-1s-1，两种形式的活化能约为 4.5 kcal/mol。凝血酶识别纤维蛋白原和纤维蛋白 I 的机制需要一个具有小能垒的扩散控制步骤。对变构转换耦合自由能温度依赖性的分析表明，纤维蛋白原和纤维蛋白 I 与过渡态凝血酶的快速形式的优先相互作用是熵驱动的，表明疏水效应对慢→快转变的贡献。纤维蛋白肽释放的盐依赖性显示出在所检查的浓度范围内的常数系数Гsalt= d ln(kcat/Km)/d ln [盐]。有趣的是， Гsalt 的值与所用的盐（NaCl、ChCl 或 NaF）无关，对于纤维蛋白肽 A 为 -1.5 ± 0.1，对于纤维蛋白肽 B 为 -2.5 ± 0.1。因此，Гsalt 主要反映静电对过渡态形成的贡献，在凝血酶与纤维蛋白 I 的相互作用中看到更大的贡献。 结论是，凝血酶与纤维蛋白原和纤维蛋白 I 的相互作用，导致纤维蛋白肽 A 和 B 的释放，是由静电力驱动的，静电力可能有利于酶和底物的正确预取向，从而在过渡状态下形成生产性复合物。这种静电导向效应也被报道用于凝血酶-水蛭素相互作用，导致与非常小的能垒的扩散控制的相遇。一旦形成复合物，酶就会由于熵因素而转变为快速形式，这可能与从更广泛的识别表面释放水有关。在研究纤维蛋白肽的释放与盐浓度的关系时，对 Cl- 在纤维蛋白凝块形成中的作用进行了重要观察。这种阴离子显着且特异性地降低了纤维蛋白纤维的厚度，这一点可以通过在 NaCl 中形成的凝块的平衡浊度比在 NaF 中形成的凝块的浊度降低 10 倍来判断。因此，正如 Ferry 首先描述的那样，由离子强度增加引起的从“粗”凝块到“细”凝块的转变是由于随后聚合中 Cl- 与中间体的特异性结合所致。事实上，当 NaF 改变离子强度时，没有观察到凝血曲线的变化。
The release of fibrinopeptides A and B by the slow and fast forms of thrombin was studied over the temperature range from 5 to 45 °C and the salt concentration range from 100 to 800 mM. The sequential mechanism for the release of fibrinopeptides originally proposed by Shafer was found to be obeyed under all conditions examined. The origin of preferential binding of fibrinogen and fibrin I to the fast form of thrombin in the transition state is in the second-order rate constant for association,k1. In the case of fibrinogen, the values ofk1for interaction with the fast and slow forms at 25 °C are 19 ± 4 and 2.5 ± 0.3 μM-1s-1, with an activation energy of about 10 kcal/mol in both forms. In the case of fibrin I, the analogous values ofk1are 9.1 ± 0.7 and 2.5 ± 0.2 μM-1s-1, and the activation energy is about 4.5 kcal/mol in both forms. The mechanism of recognition of fibrinogen and fibrin I by thrombin entails a diffusion-controlled step with a small energy barrier. Analysis of the temperature dependence of the coupling free energy for allosteric switching indicates that the preferential interaction of fibrinogen and fibrin I with the fast form of thrombin in the transition state is entropy-driven, signaling a contribution of the hydrophobic effect to the slow → fast transition. The salt dependence of the release of fibrinopeptides shows a constant coefficient Γsalt= d ln(kcat/Km)/d ln [salt] in the concentration range examined. Interestingly, the value of Γsaltis independent of the salt used (NaCl, ChCl, or NaF) and is −1.5 ± 0.1 for fibrinopeptide A and −2.5 ± 0.1 for fibrinopeptide B. Hence, Γsaltreflects predominantly the electrostatic contribution to the formation of the transition state, with a larger contribution seen in the interaction of thrombin with fibrin I. It is concluded that the interaction of thrombin with fibrinogen and fibrin I, leading to the release of fibrinopeptides A and B, is driven by electrostatic forces that presumably favor the correct preorientation of the enzyme and the substrate to form a productive complex in the transition state. This electrostatic-steering effect, also reported for thrombin−hirudin interaction, leads to a diffusion-controlled encounter with a very small energy barrier. Once the complex is formed, the enzyme switches to the fast form as a result of entropic factors presumably linked to water release from a more extended surface of recognition. While the release of fibrinopeptides as a function of salt concentration was being studied, an important observation was made on the role of Cl-in the formation of the fibrin clot. This anion drastically and specifically reduces the thickness of fibrin fibers, as judged by the 10-fold decrease in the equilibrium turbidity of clots developed in NaCl as compared to the turbidity of clots developed in NaF. Hence, the transition from a “coarse” to a “fine” clot induced by an increase in ionic strength as first described by Ferry is, instead, due to the specific binding of Cl-to intermediates in the ensuing polymerization. In fact, no change in the clotting curve is observed when the ionic strength is changed with NaF.