Department of Laboratory Medicine, Division of Clinical Chemistry, The Wallenberg Laboratory, Lund University, MAS, Malmo, Sweden.
J Thromb Haemost. 2010 Jan;8(1):129-36. doi: 10.1111/j.1538-7836.2009.03657.x. Epub 2009 Oct 23.
Activated protein C (APC) inhibits factor Va (FVa) by cleaving at Arg306, Arg506 and Arg679. Protein S serves as cofactor, in particular for the Arg306 site, and a protein S-mediated relocation of the active site of APC closer to the membrane has been proposed as a mechanism. Recently, it was demonstrated that FVa, which was mutated at all three APC-cleavage sites (FVa-306Q/506Q/679Q), could still be cleaved by APC. These sites were close to Arg306 and Arg506 but not further defined.
To identify and characterize the additional APC-cleavage sites in FVa.
The cDNA for FV-306Q/506Q/679Q was used as a template to create FV variants with one or more possible cleavage sites being mutated. The FV variants were expressed and their sensitivity for APC characterized functionally and with Western blotting.
The additional APC-cleavage sites were located at Lys309, Arg313, Arg316, Arg317 and Arg505. FVa-306Q/309Q/313Q/316Q/317Q/505Q/506Q/679Q (denoted 8M-FVa) was APC resistant. To investigate individual sites, they were mutated back using 8M-FV as a template. The kinetics of APC-degradation of these variants demonstrated that protein S was equally efficient in enhancing the APC effect for all the novel sites.
Multiple APC-cleavage sites close to Arg306 and a single site close to Arg506 were identified. Protein S was equally efficient as APC cofactor for all novel sites. The stimulation by protein S of the Arg505 cleavage argues against a specific protein S-mediated stimulation of cleavage at Arg306 due to relocation of the APC active site closer to the membrane.
活化蛋白 C（APC）通过裂解 Arg306、Arg506 和 Arg679 抑制因子 Va（FVa）。蛋白 S 作为辅因子，特别是对 Arg306 位点，并且据提议 APC 的活性位点向膜的蛋白 S 介导的重新定位是一种机制。最近，证明了在所有三个 APC 切割位点（FVa-306Q/506Q/679Q）处突变的 FVa 仍可被 APC 切割。这些位点靠近 Arg306 和 Arg506，但没有进一步定义。
鉴定和表征 FVa 中的其他 APC 切割位点。
使用 FV-306Q/506Q/679Q 的 cDNA 作为模板，创建一个或多个可能的切割位点发生突变的 FV 变体。表达 FV 变体并通过功能和 Western blot 分析表征其对 APC 的敏感性。
其他 APC 切割位点位于 Lys309、Arg313、Arg316、Arg317 和 Arg505。FV-306Q/309Q/313Q/316Q/317Q/505Q/506Q/679Q（表示为 8M-FVa）对 APC 具有抗性。为了研究单个位点，它们使用 8M-FV 作为模板进行了回突。这些变体的 APC 降解动力学表明，所有新位点的蛋白 S 在增强 APC 效应方面同样有效。
鉴定了靠近 Arg306 的多个 APC 切割位点和靠近 Arg506 的单个位点。蛋白 S 作为所有新位点的 APC 辅因子同样有效。蛋白 S 对 Arg505 切割的刺激反对由于 APC 活性位点向膜的更接近而导致蛋白 S 介导的 Arg306 切割的特异性刺激。
