ANTIBODIES WITH PROTHROMBINASE ACTIVITY IN LUPUS

凝血酶原是凝血酶的前体，凝血酶是血液凝固的中心酶。狼疮患者的凝血酶原结合自身抗体在临床上与血栓形成相关，但这种关联是矛盾的，因为传统的抗体作用机制预测血栓形成减少（例如，抗体介导的Xa因子对凝血酶原活化的抑制；加速凝血酶原清除）。在初步研究中，我们发现了这些抗体诱导血栓形成的一种新的有效机制，即催化凝血酶原的裂解。这一建议是基于这样的假设，即凝血酶原自身抗体通过从凝血酶原（凝血酶原的生理激活因子Xa）中产生类似凝血酶的活性来促进血栓形成。凝血酶原抗体的周转能力表明，它们可以发挥比可逆结合的化学计量抗体更有效的生物学效应。凝血酶原加工的产物，即凝血酶（或凝血酶样片段）也是催化剂，与可逆结合抗体相比，这将进一步放大凝血酶原的促凝作用。具体目的是：确定狼疮患者凝血酶原活性与血栓形成的统计相关性；明确凝血酶原自身抗体的生化特征及其潜在的临床效果；从狼疮患者中克隆催化高效的凝血酶原Fv构建物进行机制研究；使用体外模型系统确定抗体产生的凝血酶原片段的促凝作用；以及确定给予小鼠的Fv构建物是否诱导血栓形成。为此，我们将采用电泳、荧光和放射等方法比较狼疮患者和正常人多克隆IgG的凝血酶原裂解活性；对亲和纯化的抗凝血酶原抗体进行分析，以确定凝血酶原片段的动力学参数、特异性、裂解位点、辅因子需求和酶活性；重组凝血酶原Fv构建体将通过选择凝血酶原和与自身抗体中丝氨酸蛋白酶样催化位点反应的化学反应性抗原类似物从噬菌体展示文库中分离出来；抗体产生的凝血酶原片段模拟凝血酶对纤维蛋白原、凝血因子V、VIII和XI以及血小板的促凝作用的能力将在体外通过测定纤维蛋白的形成、各种凝血因子和蛋白酶激活受体1对血小板的裂解和活化来确定。通过测量循环凝血酶原的消耗、凝血因子的消耗和股静脉阻塞的增强来确定给药小鼠的凝血酶原Fv的血栓前作用。这些研究将允许评估凝血酶原自身抗体对狼疮高凝状态的贡献程度。如果我们的假设是有效的，我们的研究可以扩展到通过抑制自身抗体的凝血酶原活性来改善狼疮的血栓事件。

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.