Laboratoire d'hémobiologie EA4174, Faculté RTH Laennec, Université de Lyon, Université Lyon 1, Lyon, France.
J Thromb Haemost. 2010 Feb;8(2):286-93. doi: 10.1111/j.1538-7836.2009.03675.x. Epub 2009 Oct 30.
Factor VIII (FVIII) and its activated form (FVIIIa) are subject to proteolysis that dampens their cofactor function. Among the proteases that attack FVIII (activated factor X (FXa), activated protein C (APC) and plasmin), only APC cleaves within the FVIII A2 domain at R562 to fully abolish FVIII activity.
We investigated the possible involvement of the FXa cleavage at R562 within the A2 domain in the process of FVIII inactivation.
An antibody (GMA012/R8B12) that recognizes the carboxy-terminus extremity of the A2 domain (A2C) was used to evaluate FXa action. A molecule mutated at R562 was also generated to assess the functional role of this particular residue.
The appearance of the A2C domain as a function of time evidenced the identical cleavage within the A2 domain of FVIII and FVIIIa by FXa. This cleavage required phospholipids and occurred within minutes. In contrast, the isolated A2 domain was not cleaved by FXa. Von Willebrand factor and activated FIX inhibited the cleavage in a dose-dependent manner. Mutation R562K increased both the FVIII specific activity and the generation of FXa due to an increase in FVIII catalytic efficiency. Moreover, A2C fragment could not be identified from FVIII-R562K cleavage. In summary, this study defines a new mechanism for A2 domain-mediated FVIII degradation by FXa and implicates the bisecting of the A2 domain at R562.
因子 VIII（FVIII）及其激活形式（FVIIIa）易受蛋白酶水解，从而减弱其辅助因子功能。在攻击 FVIII 的蛋白酶中（激活的因子 X（FXa）、激活的蛋白 C（APC）和纤溶酶），只有 APC 在 FVIII A2 结构域的 R562 处切割，从而完全消除 FVIII 活性。
我们研究了 FXa 在 A2 结构域内 R562 处的切割是否可能参与 FVIII 失活过程。
使用一种识别 A2 结构域羧基末端（A2C）的抗体（GMA012/R8B12）来评估 FXa 的作用。还生成了一个突变 R562 的分子，以评估该特定残基的功能作用。
随着时间的推移，A2C 结构域的出现证明了 FXa 对 FVIII 和 FVIIIa 的 A2 结构域进行了相同的切割。这种切割需要磷脂，并在数分钟内发生。相比之下，分离的 A2 结构域不会被 FXa 切割。血管性血友病因子和激活的 FIX 以剂量依赖性方式抑制切割。突变 R562K 由于 FVIII 催化效率的提高，增加了 FVIII 的比活性和 FXa 的产生。此外，无法从 FVIII-R562K 切割中鉴定出 A2C 片段。总之，本研究定义了 FXa 介导的 A2 结构域 FVIII 降解的新机制，并暗示 R562 处 A2 结构域的二分。
