Department of Pharmacology and Center for Targeted Therapeutics and Translational Nanomedicine of the Institute for Translational Medicine and Therapeutics (R.C., C.F.G., A.M.C., S.Z., and V.R.M.) and Department of Radiology, the Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (A.M.C.); Department of Molecular Medicine, Boston University School of Medicine, Boston, Massachusetts (K.R.P.); Department of Genetic Medicine, Weill Cornell Medical College, New York, New York (B.S.D.); Departments of Pathology and Biochemistry Molecular Biology, Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, and Howard Hughes Medical Institute, Oklahoma City, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma (C.T.E.).
J Pharmacol Exp Ther. 2013 Nov;347(2):339-45. doi: 10.1124/jpet.113.205104. Epub 2013 Aug 21.
Thrombomodulin (TM) is a glycoprotein normally present in the membrane of endothelial cells that binds thrombin and changes its substrate specificity to produce activated protein C (APC) that has antithrombotic and anti-inflammatory features. To compensate for loss of endogenous TM in pathology, we have fused recombinant TM with single chain variable fragment (scFv) of an antibody to mouse platelet endothelial cell adhesion molecule-1 (PECAM). This fusion, anti-PECAM scFv/TM, anchors on the endothelium, stimulates APC production, and provides therapeutic benefits superior to sTM in animal models of acute thrombosis and inflammation. However, in conditions of oxidative stress typical of vascular inflammation, TM is inactivated via oxidation of the methionine 388 (M388) residue. Capitalizing on the reports that M388L mutation renders TM resistant to oxidative inactivation, in this study we designed a mutant anti-PECAM scFv/TM M388L. This mutant has the same APC-producing capacity and binding to target cells, yet, in contrast to wild-type fusion, it retains APC-producing activity in an oxidizing environment in vitro and in vivo. Therefore, oxidant resistant mutant anti-PECAM scFv/TM M388L is a preferable targeted biotherapeutic to compensate for loss of antithrombotic and anti-inflammatory TM functions in the context of vascular oxidative stress.
血栓调节蛋白（TM）是一种糖蛋白，正常存在于内皮细胞的膜上，它能与凝血酶结合并改变其底物特异性，生成具有抗血栓和抗炎特性的活化蛋白 C（APC）。为了弥补病理状态下内源性 TM 的缺失，我们将重组 TM 与抗鼠血小板内皮细胞黏附分子-1（PECAM）单链可变片段（scFv）融合。这种融合蛋白，抗 PECAM scFv/TM，锚定于内皮细胞，刺激 APC 的生成，并在急性血栓形成和炎症的动物模型中提供优于 sTM 的治疗益处。然而，在血管炎症中典型的氧化应激条件下，TM 会因甲硫氨酸 388（M388）残基的氧化而失活。利用 TM 的 M388L 突变使其对氧化失活具有抗性的报道，在本研究中，我们设计了一种突变型抗 PECAM scFv/TM M388L。该突变体具有相同的 APC 生成能力和与靶细胞的结合能力，但与野生型融合蛋白不同的是，它在体外和体内氧化环境中保留了 APC 生成活性。因此，抗氧化突变型抗 PECAM scFv/TM M388L 是一种更理想的靶向生物治疗药物，可补偿血管氧化应激情况下抗血栓和抗炎 TM 功能的缺失。
