Fay P J, Walker F J
University of Rochester School of Medicine, NY 14642.
Biochim Biophys Acta. 1989 Feb 2;994(2):142-8. doi: 10.1016/0167-4838(89)90153-2.
Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and component heavy and light chains isolated, the heavy chains were no longer a substrate for proteolysis by activated protein C. However, when factor VIII heavy chains were recombined with light chain, the reconstituted factor VIII activity was inactivated by activated protein C. The rate of factor VIII inactivation catalyzed by activated protein C was reduced by the presence of free light chain. The extent of this inhibition was dependent upon the concentration of light chain. Control experiments indicated that this protective effect of free light chain was not the result of inhibition of the activated protein C - lipid interaction. Fluorescence analysis demonstrated binding between the factor VIII light chain, chemically modified with eosin maleimide, and activated protein C, modified at its active site by dansyl-Glu-Gly-Arg chloromethyl ketone. Similar to proteolysis of factor VIII by activated protein C, this binding was dependent upon a lipid surface. Based upon the degree of fluorescence quenching, a spatial distance of 26 A was calculated separating the two fluorophores. These results demonstrate direct binding of activated protein C to the factor VIII light chain and suggest that this binding is an obligate step for activated protein C-catalyzed inactivation of factor VIII.
凝血因子VIII表现为一系列异二聚体，由一条83（81）kDa的轻链与一条可变大小（93至210 kDa）的重链非共价结合而成。活化蛋白C使凝血因子VIII失活，导致凝血因子VIII重链发生多处裂解。当凝血因子VIII亚基解离并分离出重链和轻链成分时，重链不再是活化蛋白C进行蛋白水解的底物。然而，当凝血因子VIII重链与轻链重新组合时，重组后的凝血因子VIII活性被活化蛋白C灭活。游离轻链的存在降低了活化蛋白C催化的凝血因子VIII失活速率。这种抑制程度取决于轻链的浓度。对照实验表明，游离轻链的这种保护作用不是抑制活化蛋白C与脂质相互作用的结果。荧光分析表明，用马来酰亚胺曙红化学修饰的凝血因子VIII轻链与在其活性位点用丹磺酰基 - 谷氨酸 - 甘氨酸 - 精氨酸氯甲基酮修饰的活化蛋白C之间存在结合。与活化蛋白C对凝血因子VIII的蛋白水解作用类似，这种结合依赖于脂质表面。根据荧光猝灭程度，计算出两个荧光团之间的空间距离为26埃。这些结果证明活化蛋白C与凝血因子VIII轻链直接结合，并表明这种结合是活化蛋白C催化凝血因子VIII失活的必要步骤。
