Tracy P B, Eide L L, Mann K G
J Biol Chem. 1985 Feb 25;260(4):2119-24.
A membrane-bound Ca2+-dependent complex of the cofactor Factor Va and the enzyme Factor Xa comprises the prothrombinase coagulation complex which catalyzes the proteolytic conversion of prothrombin to thrombin. Analyses of the kinetics of prothrombin activation permit calculation of the stoichiometry and binding parameters governing the functional interactions of Factor Va and Factor Xa with isolated thrombin-activated human platelets and isolated leukocyte subpopulations. Our kinetic approach indicates that Factor Xa binds to approximately 2700 +/- 1000 (n = 8) functional sites on the surface of thrombin-activated platelets with an apparent dissociation constant (Kd) equal to 1.18 +/- 0.53 X 10(-10) M and kcat equal to 19 +/- 7 mol of thrombin/s/mol of Factor Xa bound. The store of Factor V in normal platelets prevents an analogous determination of the functional Factor Va platelet binding sites. Factor Va and Factor Xa titrations performed using platelets from a Factor V antigen-deficient individual indicate that Factor Va and Factor Xa form a 1:1 stoichiometric complex on the surface of thrombin-activated platelets. Both binding isotherms are governed by the same apparent Kd (approximately equal to 10(-10) M) and expressed the same kcat/site (14-17 s-1. Factor Xa-platelet binding parameters are not altered by the use of different platelet agonists, the choice of anticoagulant, or platelet washing procedure. Kinetics of prothrombin activation indicate also that monocytes, lymphocytes, and neutrophils possess, respectively, 16,000, 45,000, and 8,000 Factor Va-Factor Xa receptor sites/cell, which are all governed by apparent KdS approximately equal to 10(-10) M. Enzymatic complexes bound to monocytes or neutrophils exhibit kcat values similar to the platelet-bound complex. Complexes bound to lymphocytes are only 25% as active.
辅因子因子Va和酶因子Xa的膜结合钙依赖性复合物构成凝血酶原酶凝血复合物，该复合物催化凝血酶原向凝血酶的蛋白水解转化。凝血酶原激活动力学分析允许计算因子Va和因子Xa与分离的凝血酶激活的人血小板和分离的白细胞亚群功能相互作用的化学计量和结合参数。我们的动力学方法表明，因子Xa以表观解离常数（Kd）等于1.18±0.53×10^(-10) M和kcat等于19±7摩尔凝血酶/秒/摩尔结合的因子Xa的量，结合到凝血酶激活的血小板表面上约2700±1000（n = 8）个功能位点。正常血小板中因子V的储存阻止了对功能性因子Va血小板结合位点的类似测定。使用来自因子V抗原缺陷个体的血小板进行的因子Va和因子Xa滴定表明，因子Va和因子Xa在凝血酶激活的血小板表面形成1:1化学计量复合物。两种结合等温线均由相同的表观Kd（约等于10^(-10) M）控制，并表达相同的kcat/位点（14 - 17 s^(-1)）。因子Xa - 血小板结合参数不会因使用不同的血小板激动剂、抗凝剂的选择或血小板洗涤程序而改变。凝血酶原激活动力学还表明，单核细胞、淋巴细胞和嗜中性粒细胞分别具有16,000、45,000和8,000个因子Va - 因子Xa受体位点/细胞，它们均由约等于10^(-10) M的表观Kd控制。与单核细胞或嗜中性粒细胞结合的酶复合物表现出与血小板结合复合物相似的kcat值。与淋巴细胞结合的复合物活性仅为25%。
