Targeting thrombomodulin to circulating red blood cells augments its protective effects in models of endotoxemia and ischemia-reperfusion injury

Abstract

Endothelial thrombomodulin (TM) regulates coagulation and inflammation via several mechanisms, including production of activated protein C (APC). Recombinant APC and soluble fragments of TM (sTM) have been tested in settings associated with insufficiency of the endogenous TM/APC pathway, such as sepsis. We previously designed a fusion protein of TM [single-chain variable fragment antibody (scFv)/TM] targeted to red bloodcells (RBCs) to improvepharmacokinetics andantithrombotic effectswithout increasingbleeding.Here, scFv/TM was studied in mouse models of systemic inflammation and ischemia-reperfusion injury. Injected concomitantly with or before endotoxin, scFv/TM provided more potent protection against liver injury and release of pathological mediators than sTM, showing similar efficacy at up to 50-fold lower doses. scFv/TM provided protection when injected after endotoxin, whereas sTMdid not, and augmentedAPCproduction by thrombin~50-fold more than sTM. However, scFv/TM injected after endotoxin did not reduce thrombin/antithrombin complexes; nor did antibodies thatblockAPCanticoagulant activity suppress theprophylactic anti-inflammatory effect of scFv/TM. Therefore, similar to endogenous TM, RBC-anchored scFv/TM activates several protective pathways. Finally, scFv/TM wasmore effective at reducing cerebral infarct volume and alleviated neurological deficits than sTM after cerebral ischemia/reperfusion injury. These results indicate that RBC-targeted scFv/TM exerts multifaceted cytoprotective effects and may find utility in systemic and focal inflammatory and ischemic disorders.-Carnemolla, R., Villa, C. H., Greineder, C. F., Zaitseva, S., Patel, K. R., Kowalska, M. A., Atochin, D.N., Cines, D. B., Siegel, D. L., Esmon, C.T., Muzykantov, V.R.Targeting thrombomodulinto circulating red blood cells augments its protective effects inmodels of endotoxemia and ischemia-reperfusion injury.