Scandura J M, Walsh P N
Sol Sherry Thrombosis Research Center, Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Biochemistry. 1996 Jul 9;35(27):8903-13. doi: 10.1021/bi9525031.
Factor X is a zymogen in the blood coagulation system which is activated by the serine protease, factor IXa, in a reaction that is promoted by the presence of stimulated platelets. We have shown previously that platelets possess a binding site for factor IXa, the occupancy of which is correlated with the rate of factor X activation (Ahmad et al., 1989b,c). Similarly, we have described a different binding site on the surface of activated platelets to which the substrate for this reaction, factor X, can bind (see the accompanying paper). This "zymogen binding site" is of moderate affinity and is relatively nonspecific; apparently shared to some degree by factor X and other vitamin K-dependent proteins, most notably prothrombin. We have found that prothrombin fragment 1 not only is able to displace factor X from this platelet binding site but also possesses the ability to inhibit the platelet-dependent activation of factor X. We have developed two mathematical models for the activation of factor X by platelet-bound factor IXa. The first model assumes that factor X is activated in a manner that is totally unrelated to the presumptive zymogen binding site, whereas the second model requires factor X to first bind to this site before it may interact with platelet-bound factor IXa and become activated. Within the context of each of these models, we have evaluated three mechanisms by which prothrombin fragment 1 may inhibit factor X activation. The data presented herein are most consistent with the precept that platelet-bound factor X is activated by platelet-bound factor IXa (kcat approximately 0.0011 s-1) in an explicitly two-dimensional reaction (Km.2D approximately 230 molecules per platelet). Prothrombin fragment 1 is believed to disrupt this reaction by competing with factor X for the zymogen binding site (Ki approximately 470 nM) and, to a lesser degree, by displacing factor IXa from its binding site (Ki approximately 7 microM). These findings suggest that platelet-bound zymogen factor X represents a kinetically important pool of substrate that is preferentially activated on the surface of activated platelets.
凝血因子X是血液凝固系统中的一种酶原，它在受刺激血小板存在所促进的反应中，被丝氨酸蛋白酶凝血因子IXa激活。我们之前已经表明血小板拥有凝血因子IXa的结合位点，该位点的占据情况与凝血因子X的激活速率相关（艾哈迈德等人，1989b、c）。同样，我们已经描述了活化血小板表面上的另一个结合位点，该反应的底物凝血因子X可以结合到这个位点上（见随附论文）。这个“酶原结合位点”具有中等亲和力且相对非特异性；显然凝血因子X和其他维生素K依赖蛋白在一定程度上共享该位点，最显著的是凝血酶原。我们发现凝血酶原片段1不仅能够从这个血小板结合位点上置换凝血因子X，而且还具有抑制血小板依赖性凝血因子X激活的能力。我们已经开发了两种关于血小板结合的凝血因子IXa激活凝血因子X的数学模型。第一个模型假设凝血因子X以一种与假定的酶原结合位点完全无关的方式被激活，而第二个模型要求凝血因子X在与血小板结合的凝血因子IXa相互作用并被激活之前，首先结合到这个位点上。在这些模型中的每一个背景下，我们评估了凝血酶原片段1可能抑制凝血因子X激活的三种机制。本文给出的数据与这样的观点最为一致，即血小板结合的凝血因子X在一个明确的二维反应中（Km.2D约为每个血小板230个分子）被血小板结合的凝血因子IXa激活（kcat约为0.0011 s-1）。凝血酶原片段1被认为通过与凝血因子X竞争酶原结合位点（Ki约为470 nM）来破坏这个反应，并且在较小程度上通过将凝血因子IXa从其结合位点上置换下来（Ki约为7 microM）。这些发现表明血小板结合的酶原凝血因子X代表了一个动力学上重要的底物池，它优先在活化血小板的表面被激活。
