Prothrombin is the precursor of thrombin, the central enzyme in blood coagulation. Prothrombin binding autoantibodies from lupus patients are clinically associated with thrombosis, but the association is paradoxical, because conventional mechanisms of antibody action predict decreased thrombus formation (e.g., antibody mediated inhibition of prothrombin activation by factor Xa; accelerated prothrombin clearance). In Preliminary Studies, we identified a new and potent mechanism by which these antibodies can induce thrombosis, i.e. the catalytic cleavage of prothrombin. This proposal is based on the hypothesis that prothrombinase autoantibodies promote thrombus formation by generating thrombin-like activities from prothrombin, like factor Xa, the physiological activator of prothrombin. The turnover capability of prothrombinase antibodies suggests that they can exert substantially more potent biological effects than reversibly binding stoichiometric antibodies. The products of prothrombin processing, i.e., thrombin (or thrombin-like fragments) are also catalysts, which will further amplify the procoagulant effect of the prothrombinases compared to reversibly binding antibodies.
The specific aims are: to determine the statistical correlation of prothrombinase activity to thrombosis in lupus patients; define the biochemical characteristics of the prothrombinase autoantibodies relevant to their potential clinical effects; clone catalytically efficient prothrombinase Fv constructs from lupus patients for mechanistic studies; determine the procoagulant effects of the antibody-generated prothrombin fragments using in vitro model systems; and, determine whether the Fv constructs administered to mice induce thrombosis. To these ends, the prothrombin cleaving activity of polyclonal IgG from lupus and normal subjects will be compared by electrophoretic, fluorimetric and radiometric methods; affinity purified anti-prothrombin antibodies will be analyzed to determine kinetic parameters, specificity, cleavage sites, cofactor requirements, and enzymatic activity of prothrombin fragments; recombinant prothrombinase Fv constructs will be isolated from phage display libraries by selection using prothrombin and chemically reactive antigen analogs reactive with serine protease-like catalytic sites found in autoantibodies; the ability of antibody-generated prothrombin fragments to mimic the procoagulant effects of thrombin on fibrinogen, coagulation factors V, VIII and XI, and platelets will be determined in vitro by measuring fibrin formation, cleavage and activation of the various coagulation factors and protease activated receptor 1 on platelet. The prothrombotic effects of prothrombinase Fv administered to mice will be determined by measuring depletion of circulating prothrombin, consumption of coagulation factors and enhanced occlusion of the femoral vein. These studies will permit assessment of the extent to which the prothrombinase autoantibodies contribute toward the hypercoagulable state in lupus. If our hypotheses are valid, our studies can be extended to ameliorating the thrombotic events in lupus via inhibition of the prothrombinase activity of the autoantibodies.
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