Einthoven Laboratory for Experimental Vascular Medicine, Dept of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands.
Thromb Res. 2010 May;125(5):444-50. doi: 10.1016/j.thromres.2009.12.005. Epub 2010 Jan 3.
The procoagulant function of activated factor V (FVa) is inhibited by activated Protein C (APC) through proteolytic cleavages at R306, R506 and R679. Recombinant FVa mutated at all three APC-cleavage sites, FVa-GQA, was still inactivated by APC through at least two cleavages in the heavy chain of FVa; relatively rapid cleavage at R(x1) close to residue 506 and slower cleavage at R(x2) nearby residue 306. We investigated the exact location of these two cleavages, by substitution of arginines by glutamine within the R(x1)-region (R501, R505 or R510) and the R(x2)-region (R313, R316, R317 or R321). Immunoblot and kinetic analyses of the inactivation of activated R(x1)-mutants by APC revealed that using mutant FVa-GQA-505Q no R(x2)-R(x1) fragment was formed and that the inactivation reaction was first order with a rate constant of 1.0 x 10(4) M(-1) s(-1), similar to the rate constant of R(x2) cleavage (k(2)=1.3 x 10(4) M(-1) s(-1)). No single arginine could be pinpointed identified as R(x2). Individual replacement of arginine by glutamine at positions 313, 316, 317 or 321 in FV-GQA-505Q did not result in the disappearance of R(x2) as judged from kinetic and immunoblot analyses. However, replacement of all four arginines by glutamine completely prevented formation of the R(x2)-R(709) fragment. We conclude that substitution of arginine 506 by glutamine as in FV-Leiden, leads to the detection of a novel cleavage site at arginine 505 (R(x1)). Substitution of arginine 306 by glycine, like in FV-Cambridge, reveals several alternative cleavage sites near arginine 306, which together constitute a secondary cleavage site.
激活的因子 V（FVa）的促凝功能通过蛋白 C（APC）在 R306、R506 和 R679 处的蛋白水解裂解而受到抑制。在所有三个 APC 裂解位点处发生突变的重组 FVa（FVa-GQA）仍然通过 FVa 重链中的至少两个裂解被 APC 失活；在靠近残基 506 的 R(x1)处快速裂解，在靠近残基 306 的 R(x2)处缓慢裂解。我们通过在 R(x1)-区（R501、R505 或 R510）和 R(x2)-区（R313、R316、R317 或 R321）内用谷氨酰胺取代精氨酸，研究了这两个裂解的确切位置。APC 失活激活的 R(x1)-突变体的免疫印迹和动力学分析表明，使用突变体 FVa-GQA-505Q 不形成 R(x2)-R(x1)片段，失活反应为一级反应，速率常数为 1.0 x 10(4) M(-1) s(-1)，与 R(x2)裂解的速率常数（k(2)=1.3 x 10(4) M(-1) s(-1)）相似。不能确定单个精氨酸为 R(x2)。在 FV-GQA-505Q 中，单独用谷氨酰胺取代位置 313、316、317 或 321 的精氨酸，从动力学和免疫印迹分析来看，并不导致 R(x2)的消失。然而，用谷氨酰胺完全取代所有四个精氨酸完全阻止了 R(x2)-R(709)片段的形成。我们得出结论，如 FV-Leiden 中精氨酸 506 被谷氨酰胺取代，导致在精氨酸 505（R(x1)）处检测到一个新的裂解位点。如 FV-Cambridge 中精氨酸 306 被甘氨酸取代，揭示了精氨酸 306 附近的几个替代裂解位点，这些位点共同构成了一个次要裂解位点。
