Nesheim M E, Taswell J B, Mann K G
J Biol Chem. 1979 Nov 10;254(21):10952-62.
The rates of prothrombin activation under initial conditions of invariant concentrations of prothrombin and Factor Xa were studied in the presence of various combinations of Ca2+, homogeneous bovine Factor V, Factor Va, phosphatidylcholine-phosphatidylserine vesicles, and activated bovine platelets. Reactions were monitored continuously through the enhanced fluorescence accompanying the interaction of newly formed thrombin with dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide. The complete prothrombinase (Factor Xa, Ca2+, phospholipid, and Factor Va) behaved as a "typical" enzyme and catalyzed the activation of prothrombin with an apparent Vmax of 2100 mol of thrombin/min/mol of Factor Va or Factor Xa, whichever was the rate-limiting component. Regardless of whether the enzymatic complex was composed of Factor Xa, Ca2+, and plasma Factor Va plus phospholipid vesicles, or activated platelets in the place of the latter components, similar specific activity values were observed. The combination of Factor Va, Ca2+, and phospholipid enhanced the rate of the Factor Xa-catalyzed activation of prothrombin by a factor of 278,000. Factor Va itself when added to Factor Xa, Ca2+, and phospholipid, enhanced the rate of prothrombin activation by a factor of 13,000. Unactivated Factor V appears to possess 0.27% of the procoagulant activity of thrombin-activated Factor Va. From the kinetics of prothrombinase activity, an interaction between Factor Xa and both Factor V and Factor Va was observed, with apparent 1:1 stoichiometries and dissociation constants of 7.3 x 10(-10) M for Factor Va and 2.7 x 10(-9) M for Factor V. The present data, combined with data on the equilibrium binding of prothrombinase components to phospholipid, indicate that the model prothrombinase described in this paper consists of a phospholipid-bound, stoichiometric complex of Factor Va and Factor Xa, with bound Factor Va serving as the "binding site" for Factor Xa, in concert with its proposed role in platelets.
在凝血酶原和因子Xa浓度恒定的初始条件下，研究了在各种Ca2+、均一的牛因子V、因子Va、磷脂酰胆碱 - 磷脂酰丝氨酸囊泡和活化牛血小板组合存在时凝血酶原的活化速率。通过新形成的凝血酶与丹磺酰精氨酸 - N -（3 - 乙基 - 1,5 - 戊二胺）酰胺相互作用所伴随的荧光增强来连续监测反应。完整的凝血酶原酶（因子Xa、Ca2+、磷脂和因子Va）表现为一种“典型”酶，催化凝血酶原的活化，以因子Va或因子Xa（以限速成分计）的摩尔数计，表观Vmax为2100摩尔凝血酶/分钟/摩尔。无论酶复合物是由因子Xa、Ca2+和血浆因子Va加磷脂囊泡组成，还是由活化血小板替代后一组分组成，都观察到相似的比活性值。因子Va、Ca2+和磷脂的组合使因子Xa催化的凝血酶原活化速率提高了278,000倍。当将因子Va本身添加到因子Xa、Ca2+和磷脂中时，凝血酶原活化速率提高了13,000倍。未活化的因子V似乎具有凝血酶活化的因子Va促凝活性的0.27%。从凝血酶原酶活性的动力学来看，观察到因子Xa与因子V和因子Va之间存在相互作用，因子Va的表观化学计量比为1:1，解离常数为7.3×10^(-10) M，因子V的解离常数为2.7×10^(-9) M。本文的数据与凝血酶原酶成分与磷脂平衡结合的数据相结合，表明本文描述的模型凝血酶原酶由因子Va和因子Xa的磷脂结合化学计量复合物组成，结合的因子Va作为因子Xa的“结合位点”，与其在血小板中的假定作用一致。
