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 blood cells (RBCs) to improve pharmacokinetics and antithrombotic effects without increasing bleeding. 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 sTM did not, and augmented APC production by thrombin ∼50-fold more than sTM. However, scFv/TM injected after endotoxin did not reduce thrombin/antithrombin complexes; nor did antibodies that block APC anticoagulant activity suppress the prophylactic anti-inflammatory effect of scFv/TM. Therefore, similar to endogenous TM, RBC-anchored scFv/TM activates several protective pathways. Finally, scFv/TM was more 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 thrombomodulin to circulating red blood cells augments its protective effects in models of endotoxemia and ischemia-reperfusion injury.

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes

“…TM shows promise in the treatment of sepsis, 53 and RBC-coupled TM has demonstrated superiority to soluble TM in mouse models. 20,21 Coupling TM to either epitope resulted in efficacious RBC drug carriers as measured by enzymatic activity and in a humanized microfluidic model of inflammatory thrombosis. However, RhCEcoupled TM showed higher specific activity in vitro and improved efficacy in our microfluidic model.…”

“…10,[17][18][19][20] For example, coupling of thrombomodulin (TM) to murine RBCs improves its efficacy in thrombotic, 20 inflammatory, and ischemia-reperfusion injuries. 21 Previous reports have generally used fusion proteins, antibodies, and peptides to couple therapeutics to the surface of murine and porcine, but not human, RBCs. Coupling to murine RBCs is typically accomplished by derivatives of Ter119, an antibody to an epitope associated with glycophorin A (GPA), 22 or with ERY1 peptide, whose putative target is GPA.…”

ICAM-1–targeted thrombomodulin mitigates tissue factor–driven inflammatory thrombosis in a human endothelialized microfluidic model

“…[48][49][50][51][52] Our priority was to establish a comparatively straightforward model, suitable for testing and comparing targeted scFv/TM with other antithrombotic drugs. 18,[53][54][55] Although the model lacks subendothelial components of a true vessel, including epithelial tissues, it allows for control over endothelial and leukocyte activation, manipulation and treatment of human WB, and quantification of therapeutic effects with high spatial and temporal resolution. While the present focus was on abrogation of fibrin deposition, additional readouts of inflammatory biology such as leukocyte adhesion and generation of neutrophil extracellular traps were also shown to be possible using this model system.…”

Vascular Drug Delivery Using Carrier Red Blood Cells: Focus on RBC Surface Loading and Pharmacokinetics

“…Drug delivery systems based on surface loading can find utility for the prolongation and redistribution of agents that are supposed to work in the bloodstream and other sites accessible for RBCs ( Figure 1), including vascular endothelium, hepatic sinuses, pathological sites of RBC diapedesis and hemorrhages, and sites of surveillance and phagocytosis of senescent RBC. These drug cargoes include drugs regulating blood fluidity, inflammation, decoy and capture systems for pathological agents, immunological reactions, and some of the applications described above for RBCencapsulated agents [19][20][21][22]. In addition, RBC drug delivery systems can be surface-modified to confer specific affinity to therapeutic sites of interest, including those of vascular injury [13,14,23,24].…”