Naski M C, Lorand L, Shafer J A
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Biochemistry. 1991 Jan 29;30(4):934-41. doi: 10.1021/bi00218a008.
Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.
动力学和热力学研究表明，纤维蛋白I（聚合的去A纤维蛋白原）在α-凝血酶催化的血浆因子XIII激活肽（AP）蛋白水解中的辅因子活性可归因于纤维蛋白I-血浆因子XIII复合物的形成（解离常数Kd = 65 nM），该复合物被α-凝血酶处理的效率更高（催化常数与米氏常数的比值kcat/Km = 1.2×10⁷ M⁻¹ s⁻¹），高于游离的、未复合的血浆因子XIII（kcat/Km = 1.4×10⁵ M⁻¹ s⁻¹）。特异性常数（kcat/Km）的增加很大程度上是由于α-凝血酶对血浆因子XIII和纤维蛋白I复合物的表观亲和力增加，这体现在与未复合的血浆因子XIII相比，与纤维蛋白I结合的血浆因子XIII的米氏常数下降了30倍。在血浆因子XIII存在的情况下，对α-凝血酶催化纤维蛋白I聚合物中纤维蛋白肽B（FPB）水解的初始速率分析表明，在α-凝血酶、血浆因子XIII和纤维蛋白I聚合物的三元复合物中与纤维蛋白I结合的α-凝血酶能够催化从纤维蛋白I中裂解FPB以及从血浆因子XIII中裂解AP。这一观察结果与以下观点一致：三元复合物中的α-凝血酶通过一个不同于活性位点（一个外位点）的结合位点锚定在纤维蛋白I聚合物上，并且活性位点可与血浆因子XIII的AP部分或纤维蛋白I的FPB部分形成复合物。这一结论得到以下观察结果的支持：一种12个残基的肽，它与α-凝血酶的一个外位点结合并阻断α-凝血酶与纤维蛋白原和纤维蛋白的相互作用，竞争性抑制α-凝血酶催化从纤维蛋白I - 血浆因子XIII复合物中释放FPB和AP。
