Lawson J H, Butenas S, Mann K G
Department of Biochemistry, University of Vermont, Burlington 05405.
J Biol Chem. 1992 Mar 5;267(7):4834-43.
Factor VIIa is a plasma glycoprotein which, when bound to the integral membrane glycoprotein tissue factor, forms an enzymatic complex that is essential for normal hemostasis. We have developed a fluorescent substrate (6-(Mes-D-Leu-Gly-Arg)amino-1-naphthalenediethylsulfamide) which can be used to directly measure the enzymatic activity of factor VIIa in the presence and absence of tissue factor and phospholipid. The sensitivity of this substrate allows for detection of factor VIIa at concentrations below 10(-9) M. The kinetics of substrate hydrolysis by factor VIIa were evaluated and it was observed that the binding of factor VIIa to tissue factor increases the catalytic efficiency (kcat/Km) of factor VIIa substrate hydrolysis greater than 100-fold. The increase in enzymatic efficiency of factor VIIa, when complexed to tissue factor, is mediated primarily by an increase in kcat. These data suggest that tissue factor induces an alteration in the catalytic site of factor VIIa, which allows for more efficient hydrolysis of the small fluorescent substrate. Measurements conducted using various phospholipids and detergents demonstrated that the increase in catalytic efficiency of factor VIIa, when complexed to tissue factor, is independent of the supporting surface. The differential rate of substrate hydrolysis when factor VIIa is complexed to tissue factor was used to estimate the binding of factor VIIa to tissue factor. From these data an apparent dissociation constant for factor VIIa binding to tissue factor was calculated to be between 1.1 and 2.1 nM with a binding stoichiometry of 1.04:1 (factor VIIa:tissue factor). When the reactivity of this small fluorescent substrate toward single-chain factor VII was investigated, both in the presence and absence of tissue factor, no substrate hydrolysis was observed.
凝血因子VIIa是一种血浆糖蛋白，当它与整合膜糖蛋白组织因子结合时，会形成一种对正常止血至关重要的酶复合物。我们开发了一种荧光底物（6-（甲基磺酰-D-亮氨酸-甘氨酸-精氨酸）氨基-1-萘二乙磺酰胺），可用于在有或没有组织因子和磷脂存在的情况下直接测量凝血因子VIIa的酶活性。这种底物的灵敏度使得能够检测浓度低于10^(-9) M的凝血因子VIIa。评估了凝血因子VIIa对底物水解的动力学，观察到凝血因子VIIa与组织因子的结合使凝血因子VIIa底物水解的催化效率（kcat/Km）提高了100倍以上。当凝血因子VIIa与组织因子复合时，其酶促效率的提高主要是由kcat的增加介导的。这些数据表明，组织因子诱导凝血因子VIIa催化位点的改变，从而允许更有效地水解小荧光底物。使用各种磷脂和去污剂进行的测量表明，当凝血因子VIIa与组织因子复合时，其催化效率的提高与支持表面无关。当凝血因子VIIa与组织因子复合时底物水解的差异速率用于估计凝血因子VIIa与组织因子的结合。根据这些数据，计算出凝血因子VIIa与组织因子结合的表观解离常数在1.1至2.1 nM之间，结合化学计量比为1.04:1（凝血因子VIIa：组织因子）。当研究这种小荧光底物对单链凝血因子VII的反应性时，无论有无组织因子，均未观察到底物水解。
