Abstract 6: Co-targeting of Thrombomodulin and the Endothelial Protein C Receptor Protects the Pulmonary Vasculature in a Mouse Model of Acute Endothelial Injury

Abstract

Background Recombinant activated protein C is protective in animal models of sepsis, stroke, and focal thrombosis, but has a poor side effect profile. Thrombomodulin (TM) and the Endothelial Protein C Receptor (EPCR) are endothelial membrane proteins, which partner to bind thrombin and activate protein C at sites of inflammation, ischemia, and thrombosis. Both proteins can be lost from the endothelial surface in a variety of disease states. We have shown previously that targeted delivery of TM to the Platelet Endothelial Cell Adhesion Molecule (PECAM-1) on the endothelial surface is protective in mouse models of lung inflammation and ischemia. Unlike endogenous TM, however, recombinant TM targeted to PECAM-1 does not effectively partner with endogenous EPCR. Objective We hypothesized that co-delivery of TM and EPCR would enhance in vitro activity and therapeutic efficacy in vivo. Methods TM and EPCR were fused to single chain fragments of two anti-PECAM antibodies, which are known to demonstrate collaborative binding enhancement. TM and EPCR fusion proteins were tested on PECAM-expressing cells and mouse endothelial cells. Fusion proteins were injected intravenously in C57BL/6 mice prior to intratracheal injection of endotoxin. Expression of VCAM-1 was measured using qPCR and MIP-2 was quantified in bronchoalveolar lavage (BAL) fluid. Endothelial barrier dysfunction was quantified by measuring transendothelial leak of 125I-labeled albumin. Results TM and EPCR fusion proteins target PECAM-1 on endothelial cells and demonstrate collaborative binding. Independent of binding effects, the EPCR fusion protein results in a 50% increase in the activation of protein C by cell-bound TM, an effect blocked by anti-EPCR antibody. Consistent with in vitro results, the combination of TM and EPCR fusion proteins enhances protection in endotoxin-induced lung injury, reducing VCAM expression, BAL MIP-2, and transendothelial leak of albumin (p < 0.001), as compared to TM fusion protein alone. Conclusion Co-targeting of TM and EPCR to PECAM-1 enhances activation of protein C and represents a novel and promising therapeutic strategy for the treatment of diseases involving acute endothelial injury.