C1 inhibitor (C1INH) is a serine proteinase inhibitor (serpin) that regulates activation of the classical complement pathway and the contact (kinin-generating) systems by inactivation of C1r, C1s, plasma kallikrein and factor XII. Heterozygosity for C1INH deficiency or for expression of a dysfunctional C1INH protein results in hereditary angioedema (HAE). In the first Aim, an animal model for HAE will be developed using gene targeting technology. We hypothesize that C1INH-/-mice will not survive and that C1NH +/- mice will develop angioedema. To test the hypothesis that angioedema is mediated via contact system activation, deficient mice will be reconstituted with recombinant C1INH mutants with altered target proteinase specificities, and will be mated with bradykinin receptor 2 (Bk2R) and with C2 deficient mice. To test the hypothesis that inhibition of bradykinin will interfere with symptoms, the HAE mice will the treated with Bk2R antagonists. The mechanism of action of androgens in angioedema also will be analyzed. To test the hypothesis that the contact system plays a role in the pathogenesis of septic shock, the susceptibility of C1INH +/- mice to endotoxin shock will be determined. Infusions of C1INH proteins with selective inhibitory activities will define the roles of the complement and contact systems.
The second Aim i s directed toward structure-function analyses. To test the hypothesis that stable complex formation requires extensive insertion of the reactive center loop (RCL) into beta sheet A, RCL mutants and mutants that alter the stability of sheet A will be examined. To test the hypothesis that sites within and outside the RCL determine target protease specificity, P2 mutants, distal loop mutants, and non-RCL mutants in alpha1-antitrypsin:C1INH chimeras will be tested. To test the hypothesis that amino terminal-truncated C1INH more efficiently inhibits surface associated proteases, its ability to inhibit immune complex-mediated complement activation, and inactivate high molecular weight kininogen (HK)-bound kallikrein and surface-bound factor XIIa will be examined. To compare and contrast the structure of C1INH with other serpins, recombinant truncated C1INH will be crystallized. In addition to definition of the biologic roles of C1INH and characterization of its function, these studies also may lead to improved therapy of HAE and of other diseases in which activation of the complement or contact systems plays a role.
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