Luckow E A, Lyons D A, Ridgeway T M, Esmon C T, Laue T M
Department of Biochemistry, University of New Hampshire, Durham 03824.
Biochemistry. 1989 Mar 7;28(5):2348-54. doi: 10.1021/bi00431a055.
Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.
凝血因子Va重亚基（Vh）对凝血酶原亲和力的变化被认为在调节凝血酶生成速率中起重要作用。我们使用分析超速离心法，在23.3℃、pH 7.65、50 mM三（羟甲基）氨基甲烷、0.1 M氯化钠、0.1 mM苯甲脒以及2 mM钙离子或2 mM乙二胺四乙酸（EDTA）的条件下，测定了牛Vh对凝血酶原以及凝血酶原的凝血酶原1前体片段的亲和力。在这些条件下，Vh与凝血酶原形成1:1复合物，其解离常数（Kd）为10 μM，无论缓冲液中含有钙离子还是EDTA。当用凝血酶原1前体替代凝血酶原时，观察到相同的Kd。这表明凝血酶原的包含γ-羧基谷氨酸残基的片段1部分不影响结合。用人凝血酶原1前体替代牛分子也会形成1:1的Vh - 凝血酶原1前体复合物，其Kd稍弱（27 μM）。抗凝剂活化蛋白C对牛Vh进行离散蛋白水解会将Vh转化为对凝血酶原1前体几乎没有或没有亲和力的形式（Kd大于1 mM），而Vh的质量没有可检测到的变化。
