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Anchoring Fusion Thrombomodulin to the Endothelial Lumen Protects against Injury-induced Lung Thrombosis and Inflammation

¹ Department of Pharmacology, ² Institute for Environmental Medicine, ³ Division of Pulmonary Medicine, and ⁵ Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and ⁴ California NanoSystems Institute, University of California Santa Barbara, Santa Barbara, California

Correspondence and requests for reprints should be addressed to Vladimir R. Muzykantov, M.D., Ph.D, 1 John Morgan Building, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, PA 19104. E-mail: muzykant{at}mail.med.upenn.edu

Rationale: Endothelial thrombomodulin (TM) regulates thrombosis and inflammation. Diverse forms of pulmonary and vascular injury are accompanied by down-regulation of TM, which aggravates tissue injury. We postulated that anchoring TM to the endothelial surface would restore its protective functions.

Objectives: To design an effective and safe strategy to treat pulmonary thrombotic and inflammatory injury.

Methods: We synthesized a fusion protein, designated scFv/TM, by linking the extracellular domain of mouse TM to a single-chain variable fragment of an antibody to platelet endothelial cell adhesion molecule-1 (PECAM-1). The targeting and protective functions of scFv/TM were tested in mouse models of lung ischemia-reperfusion and acute lung injury (ALI) caused by intratracheal endotoxin and hyperoxia, both of which caused approximately 50% reduction in the endogenous expression of TM.

Measurements and Main Results: Biochemical assays showed that scFv/TM accelerated protein C activation by thrombin and bound mouse PECAM-1 and cytokine high mobility group-B1. After intravenous injection, scFv/TM preferentially accumulated in the mouse pulmonary vasculature. In a lung model of ischemia-reperfusion injury, scFv/TM attenuated elevation of early growth response-1, inhibited pulmonary deposition of fibrin and leukocyte infiltration, and preserved blood oxygenation more effectively than soluble TM. In an ALI model, scFv/TM, but not soluble TM, suppressed activation of nuclear factor-B, inflammation and edema in the lung and reduced mortality without causing hemorrhage.

Conclusions: Targeting TM to the endothelium using an scFv anchor enhances its antithrombotic and antiinflammatory effectiveness in models of ALI.

Key Words: vascular targeting • acute lung injury • PECAM-1 • protein C • pulmonary endothelium

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject Thrombomodulin (TM) is a pivotal endothelial molecule that helps maintain tissue homeostasis and prevents tissue injury. However, TM expression and activity are suppressed in disorders such as acute lung injury (ALI), aggravating thrombosis, inflammation, and tissue injury. What This Study Adds to the Field Endothelial anchoring of a recombinant TM fusion construct targeted to platelet-endothelial cell adhesion molecule-1 attenuated tissue damage in mouse models of lung ischemia-reperfusion and ALI to a greater extent than nontargeted soluble TM, without causing bleeding. Vascular targeting of TM may provide a novel approach to prevent or treat various forms of ALI.