Abstract 972: Anchoring Of Thrombomodulin On Endothelium Protects Against Mouse Lung Ischemia-reperfusion Injury
The endothelial cell (EC) transmembrane protein thrombomodulin (TM) plays a key role in controlling the intertwined pathological processes of thrombosis and inflammation both through and independent of protein C and several EC luminal molecular partners. Cardiovascular, pulmonary and infectious diseases are often associated with a loss of endothelial TM, aggravating thrombosis and inflammation, leading to tissue ischemia and injury. We postulated that anchoring of TM to the endothelial luminal determinant Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) would alleviate the loss of endogenous TM in these settings. To do so, we synthesized a fusion protein consisting of an anti-PECAM-1 single-chain antibody variable fragment (scFv) fused with the extracellular domain of mouse TM (Leu17-Ser517). This fusion product (scFv/TM):
activates protein C in the presence of thrombin as efficiently as soluble sTM;
binds to mouse PECAM-1;
binds the pro-inflammatory cytokine HMGB-1; and
after IV injection accumulates in mouse lungs anchored to pulmonary endothelial PECAM.
We tested the protective effect of targeting scFv/TM to the pulmonary endothelium in an ischemia-reperfusion (I/R) model that simulates lung transplantation and cardio-pulmonary bypass, which often leads to pulmonary thrombosis, inflammation and parenchymal injury. Unilateral in situ lung I/R (120 min/150 min) in mice caused a ∼45 % loss of endogenous TM and enhanced fibrin deposition and inflammation (elevated leukocyte myeloperoxidase activity) in the affected compared with the contralateral control lung. Injection of scFv/TM 30 min prior to I/R attenuated pulmonary fibrin deposition and activation of the pro-inflammatory early growth response gene-1 pathway, to a significantly greater extent than the same amount of sTM (P < 0.005 and P < 0.01, respectively). Moreover, I/R induced lung leukocyte infiltration was greatly diminished by scFv/TM (p < 0.05, compared to PBS treated group), while no significant attenuation was induced by the same dose of sTM. In conclusion, the modular structure of by anti-PECAM scFv/TM permits flexible targeting and replenishment of the anti-thrombotic and anti-inflammatory properties of TM at diverse sites of vascular injury.