Effects of activation peptide bond cleavage and fragment 2 interactions on the pathway of exosite I expression during activation of human prethrombin 1 to thrombin
Effects of activation peptide bond cleavage and fragment 2 interactions on the pathway of exosite I expression during activation of human prethrombin 1 to thrombin
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DOI：
10.1074/jbc.m306917200
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发表时间：
2003-11-07
影响因子：
4.8
通讯作者：
Bock, PE
中科院分区：
文献类型：
--
作者：
Anderson, PJ;Nesset, A;Bock, PE
Xa因子激活凝血酶原（Pro）形成凝血酶发生在Arg(271)-Thr（272）和Arg(320)-Ile（321）的蛋白水解过程中，导致调控外源位点I和II的表达。凝血酶裂解Pro片段1，产生酶原类似物凝血酶1 （Pre - 1）。利用荧光素标记探针水蛭素（54-65）([5F]Hir(54-65)-(SO3-))，在光谱和平衡结合研究中表征了外源物I对Pre - 1及其因子Xa活化中间体的性质。凝血酶前2 （Pre - 2）是由Xa因子在Arg(271)-Thr（272）处切割Pre - 1形成的，在缺少或存在近饱和片段2的情况下，Pre - 1与水蛭素（54-65）肽具有相同的亲和力（F2）。Pre - 2和凝血酶对F2也有难以区分的亲和力。相比之下，Pre - 1在Arg(320)- ile（321）处裂解，形成活性减数凝血酶碎片1 MzT(- f1)，对水蛭素的亲和力增加了11- 20倍（54-65），与Pre - 2转化为凝血酶的亲和力增加了13- 20倍没有区别。因此，因子Xa在Arg(271)-Thr（272）处切割Pre - 1并不影响外源位点I的表达，而在Arg(320)-Ile（321）处切割会导致催化位点和外源位点I的同时激活。此外，在Pre - 1活化中间体上，外源位点I的表达不受F2的调节，外源位点II也不会被构象激活。exosite I在Pre - 1激活中间体上的亲和力差异表达以及（pro） exosite I在因子va依赖性底物识别中的作用表明，exosite I表达的变化可能调节Pre - 1激活途径的速率和方向。
Activation of prothrombin (Pro) by factor Xa to form thrombin occurs by proteolysis of Arg(271)-Thr(272) and Arg(320)-Ile(321), resulting in expression of regulatory exosites I and II. Cleavage of Pro by thrombin liberates fragment 1 and generates the zymogen analog, prethrombin 1 (Pre 1). The properties of exosite I on Pre 1 and its factor Xa activation intermediates were characterized in spectroscopic and equilibrium binding studies using the fluorescein-labeled probe, hirudin(54-65) ([5F]Hir(54-65)-(SO3-)). Prethrombin 2 (Pre 2), formed by factor Xa cleavage of Pre 1 at Arg(271)-Thr(272), had the same affinity for hirudin(54-65) peptides as Pre 1 in the absence or presence of near-saturating fragment 2 (F2). Pre 2 and thrombin also had indistinguishable affinities for F2. By contrast, cleavage of Pre 1 at Arg(320)-Ile(321), to form active meizothrombin des-fragment 1 MzT(-F1), showed a 11- to 20-fold increase in affinity for hirudin(54-65), indistinguishable from the 13- to 20-fold increase seen for conversion of Pre 2 to thrombin. Thus, factor Xa cleavage of Pre 1 at Arg(271)-Thr(272) does not effect exosite I expression, whereas cleavage at Arg(320)-Ile(321) results in concomitant activation of the catalytic site and exosite I. Furthermore, expression of exosite I on the Pre 1 activation intermediates is not modulated by F2, and exosite II is not activated conformationally. The differential expression of exosite I affinity on the Pre 1 activation intermediates and the previously demonstrated role of (pro) exosite I in factor Va-dependent substrate recognition suggest that changes in exosite I expression may regulate the rate and direction of the Pre 1 activation pathway.