The major scientific goal of this Pathway to Independence (K99/ROO) Career Development Award application is to understand the molecular pathophysiology of thrombotic and inflammatory disorders by studying novel mechanisms for cytoprotective actions of vitamin K-dependent coagulation proteases. This application focuses initially on the role of membrane receptors in the regulation of the cellular protein C pathway and later on the exploration of novel mechanisms for cytoprotective activities of coagulation proteases. The major career development goal of the applicant is to expand his technical and academic experience required for a successful transition into an independent investigator. These studies will provide the opportunity and solid basis to apply successfully for future independent NIH R01 funding focused on the molecular mechanistic studies centered on the crossroads of coagulation and inflammation. Novel hypotheses on the functional proteomics of cytoprotective actions by blood coagulation proteases will be tested using biochemical and cellular biology methods. The clinical and therapeutic implications of the proposed studies are clear from the large clinical trials, where activated protein C (ARC), but not other anticoagulants reduced mortality in severe sepsis patients and implied that the unique combination of APC's anticoagulant activity and direct activity on cells is the basis for APC's success. My published work and unpublished preliminary data lead directly to the proposed studies and provide strong support for my hypotheses. In testing these hypotheses, I propose: 1) To characterize the formation of endothelial cell membrane receptor complexes between thrombomodulin, endothelial protein C receptor and protease activated receptor-1 required for APC generation and APC's direct effects on cells; 2) To clarify the potential beneficial and detrimental functional properties of platelet factor 4 for APC generation and APC's direct effects on cells; 3) To identify novel themes and mechanisms for APC and fVlla cytoprotective actions on cells by exploration of the similarities and differences between APC and fVlla anti-apoptotic activities; and 4) To establish whether meizothrombin has anti-apoptotic activity, as predicted, and if this activity requires cofactor-dependent and PAR-dependent mechanisms. If the proposed studies are successful, they will increase our knowledge and may lead to improved treatment of a variety of disorders in which thrombosis, apoptosis and inflammation contribute to pathogenesis.
