Cleavage at both Arg306 and Arg506 is required and sufficient for timely and efficient inactivation of factor Va by activated protein C
Cleavage at both Arg306 and Arg506 is required and sufficient for timely and efficient inactivation of factor Va by activated protein C
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DOI：
10.1097/mbc.0b013e3283456c4e
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发表时间：
2011-06-01
影响因子：
1.1
通讯作者：
Kalafatis, Michael
中科院分区：
文献类型：
--
作者：
Barhoover, Melissa A.;Kalafatis, Michael
重链 Arg(306)、Arg(506) 和 Arg(679) 处裂解后，活化蛋白 C (APC) 使膜结合因子 Va 失活。本研究的目的是检查哪种切割对于失活最重要。重组因子 V 分子构建如下：因子 V-306Q（突变 R-306 -> Q）、因子 V-506Q（突变 R-506 -> Q）和因子 V-306Q/506Q（突变 R-306 -> Q 和 R-506 -> Q）。重组分子在哺乳动物细胞中表达、纯化，并在凝血测定中和使用纯化试剂和饱和浓度的因子 Va 的测定中与 APC 和脂质（因子 Vai）孵育 30 分钟之前和之后进行测定。凝血测定表明野生型因子 Vai (Vai(WT))、因子 Vai(306Q) 和因子 Vai(506Q) 缺乏活性，而因子 Vai 缺乏活性。与 APC 孵育 30 分钟后，Vai(306Q/506Q) 保持约 70% 的活性。用 APC 处理之前和之后与所有突变辅助因子分子组装的凝血酶原酶的动力学常数 (K-m) 值与与因子 Va(WT) 组装的凝血酶原酶发现的值相似。与因子 Vai(WT) 组装的凝血酶原显示 k(cat) 降低了 20 倍，而与因子 Vai(506Q) 组装的凝血酶原的 k(cat) 与与因子 Va(WT) 组装的凝血酶原酶相比，k(cat) 降低了两倍。相比之下，在类似的实验条件下，因子Vai(306Q)和因子Vai(306Q/506Q)没有表现出k(cat)的任何损失。总之，我们的数据表明，在 Arg(306) 或 Arg(506) 处带有单突变的 APC 处理的因子 Va 分子的活性取决于所使用的测定；并且无论采用何种测定法，在 Arg(306) 和 Arg(506) 处不存在 APC 切割位点的情况下，在膜表面存在的情况下，活性辅因子无法被 APC 显着灭活。血液凝固纤维蛋白溶解 22:317-324 (C) 2011 Wolters Kluwer Health |利平科特·威廉姆斯和威尔金斯。
Activated protein C (APC) inactivates membrane-bound factor Va following cleavages of the heavy chain at Arg(306), Arg(506), and Arg(679). The objective of this study is to examine which cleavage is most important for inactivation. The recombinant factor V molecules were constructed as follows: factor V-306Q (mutations R-306 -> Q), factor V-506Q (mutations R-506 -> Q), and factor V-306Q/506Q (mutations R-306 -> Q and R-506 -> Q). The recombinant molecules were expressed in mammalian cells, purified, and assayed prior and after incubation with APC and lipids for 30 min (factor Vai) in clotting assays and in an assay using purified reagents and saturating concentrations of factor Va. Clotting assays demonstrated that wild-type factor Vai (Vai(WT)), factor Vai(306Q), and factor Vai(506Q) were devoid of activity, whereas factor Vai(306Q/506Q) maintained approximately 70% activity following a 30 min incubation with APC. Prothrombinase assembled with all mutant cofactor molecules before and after treatment with APC had kinetic constant (K-m) values similar to values found with prothrombinase assembled with factor Va(WT). Prothrombinase assembled with factor Vai(WT) demonstrated a 20-fold reduction in k(cat), whereas prothrombinase assembled with factor Vai(506Q) had a two-fold reduction in k(cat) as compared with prothrombinase assembled with factor Va(WT). In contrast, factor Vai(306Q) and factor Vai(306Q/506Q) did not show any loss in k(cat) under similar experimental conditions. In conclusion, our data demonstrate that the activity of an APC-treated factor Va molecule bearing a single mutation at Arg(306) or Arg(506) depends on the assay used; and regardless of the assay employed, in the absence of the APC-cleavage sites at Arg(306) and Arg(506), the active cofactor is unable to be significantly inactivated by APC in the presence of a membrane surface. Blood Coagul Fibrinolysis 22:317-324 (C) 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.