Factor VIIIa regulates substrate delivery to the intrinsic factor X-activating complex.
Factor VIIIa regulates substrate delivery to the intrinsic factor X-activating complex.
复制标题
因子 VIIIa 调节底物向内因子 X 激活复合物的传递。
DOI：
10.1111/j.1742-4658.2005.05070.x
复制
发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Saenko,EvgueniL
中科院分区：
文献类型：
--
作者：
Panteleev,MikhailA;Ananyeva,NatalyaM;Greco,NicholasJ;Ataullakhanov,FazoilI;Saenko,EvgueniL
凝血因子X（fX）被激活的因子IX（fIXa）和VIII（fVIIIa）激活需要在带负电荷的磷脂膜上组装酶-辅因子-底物fIXa-fVIIIa-fX复合物。 使用流式细胞术，我们探索了fIXa、fVIIIa和fX的中间膜结合二元复合物的形成。凝血因子与0.8 µm磷脂囊泡（25/75磷脂酰丝氨酸/磷脂酰胆碱）的配位结合研究表明，fVIII（fVIIIa）、fIXa和fX分别与每个囊泡的32700 ± 5000（33 200 ± 14 100）、20 000 ± 4500和30 500 ±1300个结合位点结合，表观Kd值分别为76± 23（71 ± 5）、1510 ± 430和223 ± 79 nm。 FVIII在10 nm下可诱导fIXa（1810 ± 370，20 ± 5 nm）和fX（12 630 ± 690，14 ± 4 nm）的额外高亲和力位点出现，而fX在100 nm下可诱导fIXa（541 ± 67，23 ± 5 nm）的高亲和力位点出现。 fVIII和fVIIIa对fIXa或fX结合的影响相似。fIXa激活fX的表观米氏常数是fVIIIa浓度的线性函数，斜率为1.00 ± 0.12，固有Km值为8.0 ± 1.5 nm，与反应速率受fVIIIa-fX复合物形成限制的假设一致。 此外，观察到fX激活率与fVIIIa-fX复合物的形成之间存在直接相关性。fX、fVIIIa、磷脂浓度和磷脂酰丝氨酸含量的滴定表明，在高fVIIIa浓度下，反应速率受游离fX而不是膜结合fX浓度的调节。所获得的结果揭示了在磷脂膜上形成高亲和力fVIIIa-fX复合物，并表明它们通过将fX锚定和递送至酶复合物来调节fX活化的作用。
Activation of coagulation factor X (fX) by activated factors IX (fIXa) and VIII (fVIIIa) requires the assembly of the enzyme–cofactor–substrate fIXa–fVIIIa–fX complex on negatively charged phospholipid membranes. Using flow cytometry, we explored formation of the intermediate membrane‐bound binary complexes of fIXa, fVIIIa, and fX. Studies of the coordinate binding of coagulation factors to 0.8‐µm phospholipid vesicles (25/75 phosphatidylserine/phosphatidylcholine) showed that fVIII (fVIIIa), fIXa, and fX bind to 32 700 ± 5000 (33 200 ± 14 100), 20 000 ± 4500, and 30 500 ± 1300 binding sites per vesicle with apparentKdvalues of 76 ± 23 (71 ± 5), 1510 ± 430, and 223 ± 79 nm, respectively. FVIII at 10 nminduced the appearance of additional high‐affinity sites for fIXa (1810 ± 370, 20 ± 5 nm) and fX (12 630 ± 690, 14 ± 4 nm), whereas fX at 100 nminduced high‐affinity sites for fIXa (541 ± 67, 23 ± 5 nm). The effects of fVIII and fVIIIa on the binding of fIXa or fX were similar. The apparent Michaelis constant of the fX activation by fIXa was a linear function of the fVIIIa concentration with a slope of 1.00 ± 0.12 and an intrinsicKmvalue of 8.0 ± 1.5 nm, in agreement with the hypothesis that the reaction rate is limited by the fVIIIa–fX complex formation. In addition, direct correlation was observed between the fX activation rate and formation of the fVIIIa–fX complex. Titration of fX, fVIIIa, phospholipid concentration and phosphatidylserine content suggested that at high fVIIIa concentration the reaction rate is regulated by the concentration of free fX rather than of membrane‐bound fX. The obtained results reveal formation of high‐affinity fVIIIa–fX complexes on phospholipid membranes and suggest their role in regulating fX activation by anchoring and delivering fX to the enzymatic complex.