Submitted on November 12, 2008
Revised on April 3, 2009
Accepted on April 6, 2009
Activated Protein C Protects against Ventilator-induced Pulmonary Capillary Leak
James H Finigan1, Adel Boueiz2, Emily Wilkinson1, Rachel Damico2, Jarrett Skirball2, Hyun Hae Pae2, Mahendra Damarla3, Emile Hasan2, David B. Pearse2, Sekhar P Reddy4, Dmitry N Grigoryev2, Christopher Cheadle2, Charles T. Esmon5, Joe G.N. Garcia6, and Paul M Hassoun7*
1 Case Western Reserve University School of Medicine Department of Medicine, Cleveland, OH
2 Johns Hopkins University School of Medicine
3 ohns Hopkins University School of Medicine
4 The Johns Hopkins University Bloomberg School of Public Health
5 Oklahoma Medical Research Foundation
6 University of Chicago
7 Johns Hopkins University
* To whom correspondence should be addressed. E-mail: phassou1{at}jhmi.edu.
The coagulation system is central to the pathophysiology of acute lung injury. We have previously demonstrated that the anticoagulant activated protein C (APC) prevents increased endothelial permeability in response to edemagenic agonists in endothelial cells, and that this protection is dependent on the endothelial protein C receptor (EPCR). We currently investigate the effect of APC in a mouse model of ventilator-induced lung injury (VILI). C57BL/6J mice received spontaneous ventilation (control) or mechanical ventilation (MV) with high (HVT, 20 ml/kg) or low (LVT, 7ml/kg) tidal volumes for 2 h, and were pretreated with APC or vehicle via jugular vein 1 h prior to MV. In separate experiments, mice were ventilated for 4 h and received APC 30 and 150 min after starting MV. Indices of capillary leakage included broncho-alveolar lavage (BAL) total protein and Evans blue dye (EBD) assay. Changes in pulmonary EPCR protein and Rho-associated kinase (ROCK) were assessed using SDS-PAGE. Thrombin generation was measured via plasma thrombin-antithrombin complexes. HVT induced pulmonary capillary leakage as evidenced by significant increases in BAL protein and EBD extravasation, without significantly increasing thrombin production. HVT also caused significant decreases in pulmonary, membrane-bound EPCR protein levels and increases in pulmonary ROCK-1. APC treatment significantly decreased pulmonary leakage induced by MV when given either before or after initiation of MV. Protection from capillary leakage was associated with restoration of EPCR protein expression and attenuation of ROCK-1 expression. In addition, mice over-expressing EPCR on the pulmonary endothelium were protected from HVT-mediated injury. Finally, gene microarray analysis demonstrated that APC significantly altered the expression of genes relevant to vascular permeability at the ontology (e.g., blood vessel development) and specific gene (e.g., MAPK-associated kinase 2 and integrin 
6) levels. These findings indicate that APC is barrier protective in VILI and that EPCR is a critical participant in APC-mediated protection.
