Duffy E J, Parker E T, Mutucumarana V P, Johnson A E, Lollar P
Department of Medicine, Emory University, Atlanta, Georgia 30322.
J Biol Chem. 1992 Aug 25;267(24):17006-11.
The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was modified at the active site with fluorescein-5-maleimide (Fl-M-FPR-fIXa). Titration of Fl-M-FPR-fIXa with fVIIIa at fixed PCPS resulted in a large, saturable increase in anisotropy (delta r = 0.09). The titration data were fit to a model assuming a reversible equilibrium between fVIIIa and fIXa, resulting in an apparent dissociation constant of 2 nM and a stoichiometry of 1 mol of fVIIIa/mol of Fl-M-FPR-fIXa. The initial velocity of factor X activation was measured under identical conditions except that active fIXa and factor X were included, which yielded binding parameters similar to those determined fluorometrically. Thus, the fluorescence method accurately reflects complex formation between fVIIIa and fIXa on the phospholipid surface, and the fVIIIa-fIXa interaction is not influenced by the presence of the substrate, factor X. Addition of fVIII to Fl-M-FPR-fIXa and PCPS produced a small, saturable increase in anisotropy (delta r = 0.03), followed by a larger increase (delta r = 0.07) upon addition of thrombin to activate fVIII. Thus, fVIII binds fIXa, but proteolytic modification of fVIII must occur before the complete fVIIIa-dependent structural change in the active site of fIXa, as reflected in the anisotropy change, occurs
在磷脂酰胆碱 - 磷脂酰丝氨酸（PCPS）囊泡存在的情况下，凝血酶激活的因子VIII（fVIIIa）可显著加速因子IXa（fIXa）对因子X的激活。在25℃下，通过使用在活性位点用荧光素 - 5 - 马来酰亚胺（Fl - M - FPR - fIXa）修饰的D - 苯丙氨酰 - 脯氨酰 - 精氨酰 - fIXa衍生物，以荧光法研究了该复合物中高度纯化的fVIIIa与fIXa之间的相互作用。在固定的PCPS条件下，用fVIIIa滴定Fl - M - FPR - fIXa导致各向异性大幅增加且呈饱和状态（Δr = 0.09）。滴定数据符合一个假设fVIIIa与fIXa之间存在可逆平衡的模型，得出表观解离常数为2 nM，化学计量比为1摩尔fVIIIa /摩尔Fl - M - FPR - fIXa。在相同条件下测量因子X激活的初始速度，但加入了活性fIXa和因子X，得到的结合参数与荧光法测定的参数相似。因此，荧光法准确反映了磷脂表面上fVIIIa与fIXa之间的复合物形成，并且fVIIIa - fIXa相互作用不受底物因子X存在的影响。向Fl - M - FPR - fIXa和PCPS中加入fVIII会使各向异性小幅增加且呈饱和状态（Δr = 0.03），随后加入凝血酶激活fVIII后各向异性会有更大增加（Δr = 0.07）。因此，fVIII与fIXa结合，但在fIXa活性位点发生完全依赖fVIIIa的结构变化（如各向异性变化所反映）之前，fVIII必须先进行蛋白水解修饰。
