Carboxypeptidases in Thrombosis and Inflammation

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DESCRIPTION (provided by applicant): Inflammatory and procoagulant responses are often protective and yet may contribute to maladies such as sepsis and heart attacks. This study focuses on elucidating the role(s) of two carboxypeptidases (CP) in regulating the fibrinolytic and inflammatory responses. The two CP, activated TAFI (TAFla) and carboxypeptidase N (CPN) both remove C-terminal lysine and arginine residues from protein substrates. However, while TAFla prefers Arg, CPN prefers Lys substrates. Also, TAFI requires activation and may play a role in "on demand" situations whereas CPN is constitutively active and may play a "house-keeping" role. TAFla inhibits fibrinolysis and could exacerbate thrombosis whereas CPN may not. In contrast CPN is considered to play a predominant role in regulating inflammation by inactivating anaphylatoxins generated by complement activation whereas the role of TAFI in this process is unknown. How are these two enzymes independently regulated and how does the presence of one affect the total or overall activity? We believe that regulation of fibrinolysis and inflammation are mediated to different extents by TAFla and CPN but are not mutually exclusive. They act synergistically in order to maintain exquisite control of both hemostatic and inflammatory responses. Our specific aims are to; 1) determine the correlation between functional and antigenic concentrations of TAFI in human plasma, 2) to assess the antifibrinolytic effects of TAFla and CPN in a purified system, 3) to determine the role of glycosaminoglycans on cell surfaces to participate in plasmin-mediated TAFI activation, 4) to determine the effect of CPN and TAFla on inhibition of C3a-mediated calcium ion efflux. Activity assays will be performed in a 2 x 2 matrix varying both concentrations of TAFla and CPN in order to assess synergistic effects. In the long-term this understanding this will lead to new therapeutic interventions which may specifically and significantly affect thrombosis without negatively affecting inflammatory responses or vice versa.