Targeting thrombomodulin to circulating red blood cells augments its protective effects in models of endotoxemia and ischemia-reperfusion injury.
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.
Department of Pharmacology, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.|Center for Targeted Therapeutics and Translational Nanomedicine, Institute for Translational Medicine and Therapeutics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.
Ronald
R
Carnemolla
Department of Pharmacology, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.|Center for Targeted Therapeutics and Translational Nanomedicine, Institute for Translational Medicine and Therapeutics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.|Department of Pathology and Laboratory Medicine, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.
Carlos H
CH
Villa
Department of Pharmacology, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.|Center for Targeted Therapeutics and Translational Nanomedicine, Institute for Translational Medicine and Therapeutics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.
Colin F
CF
Greineder
Department of Pharmacology, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.|Center for Targeted Therapeutics and Translational Nanomedicine, Institute for Translational Medicine and Therapeutics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.|Department of Pathology and Laboratory Medicine, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.
Sergei
S
Zaitsev
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Kruti R
KR
Patel
Division of Hematology, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.|Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.
M Anna
MA
Kowalska
Division of Cardiology, Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Dmitriy N
DN
Atochin
Department of Pathology and Laboratory Medicine, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.
Douglas B
DB
Cines
Department of Pathology and Laboratory Medicine, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.
Don L
DL
Siegel
Department of Pathology, Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA; and.|Department of Biochemistry and Molecular Biology, Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA.
Charles T
CT
Esmon
Department of Pharmacology, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA; muzykant@mail.med.upenn.edu.|Center for Targeted Therapeutics and Translational Nanomedicine, Institute for Translational Medicine and Therapeutics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; USA.
Vladimir R
VR
Muzykantov
D000818:Animals; D019446:Endotoxemia / Q000517:prevention & control*; D004912:Erythrocytes / Q000378:metabolism*; D007249:Inflammation / Q000188:drug therapy; D008297:Male; D050576:Membrane Fusion Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D015427:Reperfusion Injury / Q000517:prevention & control*; D018180:Thrombomodulin / Q000008:administration & dosage* / Q000737:chemistry / Q000627:therapeutic use*
D050576:Membrane Fusion Proteins; D018180:Thrombomodulin
coagulation; drug delivery; inflammation; pharmacokinetics; sepsis