Rosing J, Tans G, Govers-Riemslag J W, Zwaal R F, Hemker H C
J Biol Chem. 1980 Jan 10;255(1):274-83.
The kinetic parameters of the conversion of bovine prothrombin into thrombin by activated bovine blood clotting factor X (Xa) have been determined in the absence and presence of Ca2+, activated bovine factor V (Va) and phospholipid (dioleoylphosphatidylcholine/dioleoylphosphatidylserine, 1:1; mol/mol). In the absence of accessory components, the Km for prothrombin is 131 microM, which is well above its concentration in bovine plasma of about 1.5 microM. The Vmax of thrombin formation is 0.61 mol min-1 mol of Xa-1 under these conditions. In the presence of 7.5 microM phospholipid, the Km drops to 0.058 microM and the Vmax slightly increases to 2.25 mol min-1 mol of Xa. For the complete prothrombinase complex (Xa, Va, Ca2+, and 7.5 microM phospholipid), a Km for prothrombin of 0.21 microM and a Vmax of 1919 mol min-1 mol of Xa-1 is found. The Vmax of thrombin formation slightly increases when more phospholipid is present in our experiments and there is a considerable increase of the Km for prothrombin at higher phospholipid concentrations. Preliminary calculations show that the prothrombin density at the phospholipid surface at the Km is independent of the phospholipid concentration. This indicates that the Km measured in the presence of phospholipid has to be regarded as an apparent Km and the local prothrombin concentration determines the kinetics of activation. Prothrombin activation by prothrombinase complexes of different compositions was followed by gel electrophoresis in the presence of sodium dodecyl sulfate. Both in the absence and presence of phospholipid but without factor Va, prethrombin 2 is the main product formed during the initial stages of steady state prothrombin activation. In the presence of factor Va, thrombin is the main end product and minute amounts of prethrombin 2 are formed. This shift in the reaction pathway of prothrombin activation caused by factor Va will contribute to the observed increase of the Vmax measured in the presence of factor Va.
在不存在和存在Ca2+、活化牛因子V（Va）和磷脂（二油酰磷脂酰胆碱/二油酰磷脂酰丝氨酸，1:1；摩尔/摩尔）的情况下，已测定了活化牛凝血因子X（Xa）将牛凝血酶原转化为凝血酶的动力学参数。在不存在辅助成分的情况下，凝血酶原的Km为131μM，远高于其在牛血浆中的浓度（约1.5μM）。在这些条件下，凝血酶形成的Vmax为0.61摩尔·分钟-1·摩尔Xa-1。在存在7.5μM磷脂的情况下，Km降至0.058μM，Vmax略有增加至2.25摩尔·分钟-1·摩尔Xa。对于完整的凝血酶原酶复合物（Xa、Va、Ca2+和7.5μM磷脂），发现凝血酶原的Km为0.21μM，Vmax为1919摩尔·分钟-1·摩尔Xa-1。在我们的实验中，当存在更多磷脂时，凝血酶形成的Vmax略有增加，并且在较高磷脂浓度下凝血酶原的Km有相当大的增加。初步计算表明，在Km时磷脂表面的凝血酶原密度与磷脂浓度无关。这表明在存在磷脂的情况下测得的Km必须被视为表观Km，局部凝血酶原浓度决定了活化动力学。在十二烷基硫酸钠存在下，通过凝胶电泳跟踪不同组成的凝血酶原酶复合物对凝血酶原的活化。在不存在和存在磷脂但没有因子Va的情况下，凝血酶原2是稳态凝血酶原活化初始阶段形成的主要产物。在存在因子Va的情况下，凝血酶是主要终产物，仅形成微量的凝血酶原2。因子Va引起的凝血酶原活化反应途径的这种转变将导致在存在因子Va时测得的Vmax增加。
