Ghosh S, Sen P, Pendurthi U R, Rao L V M
Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, TX 75708, USA.
J Thromb Haemost. 2008 Sep;6(9):1525-33. doi: 10.1111/j.1538-7836.2008.03065.x. Epub 2008 Jul 4.
Recombinant coagulation factor VIIa (rFVIIa) has proven to be a safe and effective drug for treatment of bleeding episodes in hemophilic patients with inhibitors. However, rFVIIa is cleared from the circulation relatively quickly. Protein modification with poly(ethylene glycol) (PEG) can prolong the circulatory lifetime of proteins but it could also impair protein function by molecular shielding of the protein surface.
To characterize the interaction of glycoPEGylated rFVIIa - rFVIIa-10K PEG and rFVIIa-40K PEG - with tissue factor (TF), factor X (FX) and plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT).
The amidolytic and FX activation assays were employed to investigate the interaction of glycoPEGylated rFVIIa with its macromolecular substrate and inhibitors.
Both the glycoPEGylated rFVIIa analogs exhibited similar amidolytic activity as that of rFVIIa in the absence or the presence of relipidated TF. The analogs were as effective as rFVIIa in activating FX in the absence of TF. In the presence of TF, the glycoPEGylated rFVIIa variants, relative to rFVIIa, were slightly less effective at lower concentrations, but no significant differences were found among them in activating FX at saturating concentrations. Both AT/heparin and TFPI effectively inhibited the glycoPEGylated rFVIIa bound to relipidated TF or TF on stimulated endothelial cells. In contrast to their normal interaction with TF, the glycoPEGylated rFVIIa variants appeared to interact poorly with phospholipids.
The glycoPEGylated rFVIIa variants retained their catalytic activity and interacted efficiently with TF, FX and the plasma inhibitors. Further work with appropriate in vitro and in vivo model systems is needed to determine the feasibility of using glycoPEGylated rFVIIa to improve therapeutic options for bleeding disorders.
重组凝血因子VIIa（rFVIIa）已被证明是治疗有抑制物的血友病患者出血发作的一种安全有效的药物。然而，rFVIIa从循环中清除相对较快。用聚乙二醇（PEG）进行蛋白质修饰可以延长蛋白质的循环寿命，但也可能通过蛋白质表面的分子屏蔽损害蛋白质功能。
表征糖基化聚乙二醇化rFVIIa（rFVIIa-10K PEG和rFVIIa-40K PEG）与组织因子（TF）、因子X（FX）和血浆抑制剂、组织因子途径抑制剂（TFPI）和抗凝血酶（AT）之间的相互作用。
采用酰胺水解和FX激活试验研究糖基化聚乙二醇化rFVIIa与其大分子底物和抑制剂之间的相互作用。
在有无重新脂质化TF的情况下，两种糖基化聚乙二醇化rFVIIa类似物均表现出与rFVIIa相似的酰胺水解活性。在无TF的情况下，这些类似物在激活FX方面与rFVIIa一样有效。在有TF的情况下，相对于rFVIIa，糖基化聚乙二醇化rFVIIa变体在较低浓度下激活FX的效果略差，但在饱和浓度下激活FX时它们之间没有显著差异。AT/肝素和TFPI均能有效抑制与重新脂质化TF或刺激的内皮细胞上的TF结合的糖基化聚乙二醇化rFVIIa。与其与TF的正常相互作用相反，糖基化聚乙二醇化rFVIIa变体与磷脂的相互作用似乎较差。
糖基化聚乙二醇化rFVIIa变体保留了其催化活性，并与TF、FX和血浆抑制剂有效相互作用。需要使用合适的体外和体内模型系统进行进一步研究，以确定使用糖基化聚乙二醇化rFVIIa改善出血性疾病治疗选择的可行性。
