The PROWESS phase III clinical trial showed that activated protein C (APC) reduced mortality where neither purely anticoagulant nor purely anti-inflammatory agents were effective. Thus, APC infused into humans, as in animals, exerts both anticoagulant and anti-inflammatory actions. Recent in vitro and in vivo studies support a new paradigm in which APC exerts protective anti-inflammatory and anti-apoptotic direct effects on cells via mechanisms involving activation of Protease Activated Receptor 1 (PAR1) by APC bound to Endothelial Protein C Receptor (EPCR). Studies suggest that APC's anti-inflammatory and anti-apoptotic in vivo activities may result from its ability to alter gene expression profiles. Thus, we suggest that recombinat APC is very promising for treatment of thrombotic disorders. We propose to generate and characterize recombinant human and murine APC mutants as potential therapeutic agents and to use murine model systems to characterize their in vivo antithrombotic and anti-inflammatory activities. For in vitro characterizations, APC's anticoagulant activity and APC's direct effects on cells will be determined in studies of the EPCR-dependent, PAR1 -dependent abilities of APC to inhibit staurosporine-induced apoptosis and to alter endothelial cell gene expression. To assess APC's in vivo activities, we propose to use murine models and murine APC mutants and to determine for wt-APC and novel murine APC mutants the following: 1) antithrombotic activity; 2) anti-inflammatory activity; and 3) alteration of gene expression in the absence of injury. In preliminary work, we identified two APC mutants deficient in anticoagulant activity with normal anti apoptotic activity. Such APC variants may provide APC's beneficial EPCR-dependent, PAR1 -dependent direct effects on cells with reduced risk of serious bleeding. Because APC protectively targets both clotting factors and cell surface receptors, the results of this project may lead directly to development of novel, multifunctional agents for treatment of a variety of thrombotic disorders.
