Persson E, Hogg P J, Stenflo J
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
J Biol Chem. 1993 Oct 25;268(30):22531-9.
The assembly of macromolecular complexes containing factors Xa and Va on suitable phospholipid surfaces is crucial for rapid activation of prothrombin. We have used quantitative affinity chromatography to characterize the interaction between factor Va and intact factor Xa on the one hand and between factor Va and factor Xa lacking the gamma-carboxyglutamic acid (Gla)-containing module on the other. The dissociation constants were found to be 1.0 +/- 0.1 and 9.5 +/- 1.8 microM, respectively. There was good agreement between these dissociation constants and the concentrations of active site-inhibited factor Xa and Gla-domainless factor Xa that caused half-maximal inhibition of prothrombin activation. To investigate whether the noncatalytic modules of factor Xa interacted directly with factor Va, intact modules were isolated from proteolytic digests of factor X and used as inhibitors of prothrombin activation. The inhibitory effect observed with the isolated Gla module in the absence of phospholipid was due to inhibition of the amidolytic activity of factor Xa rather than to an interaction with factor Va. The epidermal growth factor-like modules did not inhibit prothrombin activation. Using antibodies specific for calcium-dependent epitopes in the serine protease module of factor Xa we demonstrated that Ca2+ binding to the Gla module alters the conformation of the catalytic module. Half-maximal binding was observed at approximately 0.8 mM Ca2+. Evidence was also obtained for the presence of two Gla-independent Ca(2+)-binding sites in factor Xa. One of these sites, located in the NH2-terminal epidermal growth factor-like module, was half-saturated at approximately 60 microM Ca2+ in intact factor Xa and at approximately 1.2 mM Ca2+ in Gla-domainless factor Xa. This site appeared not to influence the conformation of the protease module. The second site, which was half-saturated at approximately 0.16 mM Ca2+, appeared to reside in the serine protease module and to alter its conformation as judged by binding of antibodies specific for calcium-dependent epitopes.
在合适的磷脂表面组装含有因子Xa和因子Va的大分子复合物对于凝血酶原的快速激活至关重要。我们使用定量亲和色谱法来表征一方面因子Va与完整因子Xa之间的相互作用，另一方面因子Va与缺乏含γ-羧基谷氨酸（Gla）模块的因子Xa之间的相互作用。发现解离常数分别为1.0±0.1和9.5±1.8微摩尔。这些解离常数与导致凝血酶原激活受到半数抑制的活性位点抑制的因子Xa和无Gla结构域的因子Xa的浓度之间具有良好的一致性。为了研究因子Xa的非催化模块是否直接与因子Va相互作用，从因子X的蛋白水解消化物中分离出完整的模块并用作凝血酶原激活的抑制剂。在没有磷脂的情况下，分离出的Gla模块观察到的抑制作用是由于对因子Xa的酰胺水解活性的抑制，而不是与因子Va的相互作用。表皮生长因子样模块不抑制凝血酶原激活。使用针对因子Xa的丝氨酸蛋白酶模块中钙依赖性表位的特异性抗体，我们证明Ca2 +与Gla模块的结合改变了催化模块的构象。在约0.8 mM Ca2 +处观察到半数最大结合。还获得了因子Xa中存在两个不依赖Gla的Ca（2 +）结合位点的证据。这些位点之一位于NH2末端表皮生长因子样模块中，在完整因子Xa中约60 microM Ca2 +时半饱和，在无Gla结构域的因子Xa中约1.2 mM Ca2 +时半饱和。该位点似乎不影响蛋白酶模块的构象。第二个位点在约0.16 mM Ca2 +时半饱和，似乎位于丝氨酸蛋白酶模块中，并通过结合针对钙依赖性表位的特异性抗体来判断其改变了构象。
