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

Abstract

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-kB, 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.

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

“…Second, the benefit of ICAM-targeted TM over its soluble counterpart supports the hypothesis that localization of the protein to the cellular membranes enhances its functional activity. 19,60,61 In fact, the difference in potency between these 2 agents may be more significant than their relative concentrations would suggest, as only a fraction of hTM/R6.5 molecules would have bound to the cell membrane. While ICAM-1 targeting of hTM/R6.5 was limited to hematopoietic and ECs in these studies, other cell types, such as epithelium, express ICAM-1, and may be both an important source of TF 24 and a valuable target of this approach.…”

Sirt1 protects against thrombomodulin down-regulation and lung coagulation after particulate matter exposure

“…17 TM also has anti-inflammatory activity. [18][19][20] TM is highly expressed in lung endothelial cells. 20 TF/TFPI, t-PA/PAI-1, and TM/protein C systems are known to play distinct regulatory roles in coagulation and fibrinolysis pathways.…”

“…[18][19][20] TM is highly expressed in lung endothelial cells. 20 TF/TFPI, t-PA/PAI-1, and TM/protein C systems are known to play distinct regulatory roles in coagulation and fibrinolysis pathways. The molecular mechanisms underlying their regulation after PM exposure, nevertheless, remain elusive.…”

Update on Acute Lung Injury and Critical Care Medicine 2009

“…Investigators addressed the role of coagulation factor components on lung injury from sepsis and other clinically relevant injuries (27). In a novel study, others found that increased local expression of coagulation factor Xa contributes to the fibrotic response in human and mouse lung injury, a potentially important pathway after ALI (28).…”