Andree H A, Contino P B, Repke D, Gentry R, Nemerson Y
Department of Biochemistry, Mt. Sinai School of Medicine, City University of New York, New York 10029.
Biochemistry. 1994 Apr 12;33(14):4368-74. doi: 10.1021/bi00180a034.
Blood coagulation is initiated on cells which present a macroscopic surface to the flowing blood stream. We have used a continuous flow enzyme reactor to model this system and to investigate the effects of shear rate and mass transport on the activation of factor X by the complex of the transmembrane protein, tissue factor, and the serine protease, factor VIIa. This initial step of blood coagulation was found to be half-maximal at very low enzyme densities (0.03-0.06%) on the wall of the capillaries. In agreement with hydrodynamic theory, the apparent Km in the flow reactor was correlated with the cube root of the wall shear rate. These data indicate that at high tissue factor densities (> 0.6%) the activation of 150 nM factor X is controlled by the flux of X toward the surface, which is controlled by wall shear rate and substrate concentration. The appearance of the product, Xa, in the effluent was delayed to 8-12 min, which was caused by high-affinity binding of Xa to the phospholipid. This delay was considerably shortened by embedding tissue factor into PC or by coating the PS/PC surface with the phospholipid binding protein, annexin V. At low tissue factor densities, annexin V inhibited X activation by 45%, while no inhibition was observed at high densities. We demonstrate that when the reaction is limited by substrate flux, addition of further enzyme does not increase reaction rates. This contrasts with classical three-dimensional catalysis in which the initial velocity is ordinarily linear with the enzyme concentration.
血液凝固在向流动的血流呈现宏观表面的细胞上启动。我们使用连续流动酶反应器对该系统进行建模，并研究剪切速率和传质对跨膜蛋白组织因子与丝氨酸蛋白酶因子VIIa的复合物激活因子X的影响。发现血液凝固的这一初始步骤在毛细血管壁上的酶密度非常低（0.03 - 0.06%）时达到半最大反应速率。与流体动力学理论一致，流动反应器中的表观米氏常数与壁面剪切速率的立方根相关。这些数据表明，在高组织因子密度（> 0.6%）下，150 nM因子X的激活受X向表面的通量控制，而该通量由壁面剪切速率和底物浓度控制。产物Xa在流出物中的出现延迟至8 - 12分钟，这是由于Xa与磷脂的高亲和力结合所致。通过将组织因子嵌入磷脂酰胆碱（PC）中或用磷脂结合蛋白膜联蛋白V包被磷脂酰丝氨酸/磷脂酰胆碱（PS/PC）表面，可大大缩短这种延迟。在低组织因子密度下，膜联蛋白V抑制X的激活达45%，而在高密度下未观察到抑制作用。我们证明，当反应受底物通量限制时，添加更多酶不会提高反应速率。这与经典的三维催化不同，在经典三维催化中，初始速度通常与酶浓度呈线性关系。
