Sørensen B B, Rao L V
Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Gentofte, Denmark.
Blood Coagul Fibrinolysis. 1998 Mar;9 Suppl 1:S67-71.
The coagulation cascade is initiated by binding of plasma activated or non-activated factor VII [FVII(a)] to cell surface tissue factor (TF). TF-induced coagulation plays a primary role not only in haemostasis but also in the pathogenesis of various thrombotic disorders. Recent studies with animal model systems showed that the administration of active site-inhibited FVIIa (FVIIai) blocked TF-FVIIa-induced fibrin and thrombus formation. These data suggest that FVIIai competes with plasma FVII(a) for a limited number of TF sites expressed on cells either constitutively or induced after the perturbation. To obtain insights into the mechanism(s) by which FVIIai is effective in inhibiting TF-FVIIa induced coagulation in vivo, we compared the interaction of FVIIai and FVIIa with TF using a variety of competition assays and direct binding assays. The TF-FVIIa amidolytic activity competition assay showed that FVIIai bound with a threefold higher affinity than that of FVIIa to TF relipidated in phosphatidylcholine (PC) vesicles, whereas no significant differences were found between FVIIa and FVIIai binding to TF if it had been relipidated in mixed phospholipid vesicles containing PC and phosphatidylserine (PS). When FVIIa and FVIIai binding to TF was analysed in a FXa generation assay, we found that FVIIai bound to TF in PCPS vesicles with two- to fivefold higher affinity than that of FVIIa, whereas the affinity of FVIIai for TF in PC vesicles was seven- to 10-fold higher than that of FVIIa. Direct binding analysis to TF, immobilized on a sensor chip or on a cell surface, showed a faster association and a slower dissociation of FVIIai to TF compared with that of FVIIa. Equilibrium binding to cell surface TF showed that the affinity of FVIIai was fivefold higher than that of FVIIa to non-functional TF, whereas both FVIIa and FVIIai bound functional TF with the same high affinity. The enhanced affinity of FVIIai to TF, particularly to non-functional TF, would make FVIIai a valuable reagent to block TF-induced coagulation before it is triggered by cell injury or a pathological stimuli.
凝血级联反应由血浆活化或未活化的因子VII [FVII(a)] 与细胞表面组织因子 (TF) 结合启动。TF诱导的凝血不仅在止血过程中起主要作用，在各种血栓形成性疾病的发病机制中也起主要作用。最近在动物模型系统中的研究表明，给予活性位点抑制的FVIIa (FVIIai) 可阻断TF - FVIIa诱导的纤维蛋白和血栓形成。这些数据表明，FVIIai与血浆FVII(a) 竞争细胞上组成性表达或在扰动后诱导表达的有限数量的TF位点。为了深入了解FVIIai在体内有效抑制TF - FVIIa诱导的凝血的机制，我们使用各种竞争试验和直接结合试验比较了FVIIai和FVIIa与TF的相互作用。TF - FVIIa酰胺水解活性竞争试验表明，FVIIai与重新脂质化于磷脂酰胆碱 (PC) 囊泡中的TF结合的亲和力比FVIIa高3倍，而如果TF重新脂质化于含有PC和磷脂酰丝氨酸 (PS) 的混合磷脂囊泡中，FVIIa和FVIIai与TF的结合未发现显著差异。当在FXa生成试验中分析FVIIa和FVIIai与TF的结合时，我们发现FVIIai与PCPS囊泡中的TF结合的亲和力比FVIIa高2至5倍，而FVIIai与PC囊泡中TF的亲和力比FVIIa高7至10倍。对固定在传感器芯片或细胞表面的TF进行直接结合分析表明，与FVIIa相比，FVIIai与TF的结合和解离更快。与细胞表面TF的平衡结合表明，FVIIai与无功能TF的亲和力比FVIIa高5倍，而FVIIa和FVIIai与有功能TF的结合亲和力相同。FVIIai与TF，特别是与无功能TF的亲和力增强，将使FVIIai成为在细胞损伤或病理刺激触发TF诱导的凝血之前阻断该过程的有价值试剂。
