ANTIBODIES WITH PROTHROMBINASE ACTIVITY IN LUPUS

凝血酶原是凝血酶的前体，凝血酶是血液凝固中的中心酶。 来自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，但这种相关性是矛盾的，因为抗体作用的常规机制预测血栓形成减少（例如，抗体介导的因子Xa对凝血酶原活化的抑制;加速凝血酶原清除）。 在初步研究中，我们确定了这些抗体可诱导血栓形成的新的有效机制，即凝血酶原的催化裂解。 该提议基于凝血酶原酶自身抗体通过从凝血酶原产生凝血酶样活性（如凝血因子Xa，凝血酶原的生理激活剂）来促进血栓形成的假设。 凝血酶原酶抗体的周转能力表明，它们可以发挥比可逆结合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，其将进一步放大凝血酶原酶的促凝血作用。具体目标是：确定狼疮患者中凝血酶原酶活性与血栓形成的统计学相关性;定义凝血酶原酶自身抗体与其潜在临床作用相关的生化特征;从狼疮患者克隆催化有效的凝血酶原酶Fv构建体用于机理研究;使用体外模型系统确定抗体产生的凝血酶原片段的促凝血作用;并确定给予小鼠的Fv构建体是否诱导血栓形成。 为了这些目的，将通过电泳、荧光和放射性方法比较来自狼疮和正常受试者的多克隆IgG的凝血酶原切割活性;将分析亲和纯化的抗凝血酶原抗体以确定动力学参数、特异性、切割位点、辅因子要求和凝血酶原片段的酶活性;通过使用凝血酶原和与自身抗体中发现的丝氨酸蛋白酶样催化位点反应的化学反应性抗原类似物进行选择，从噬菌体展示文库中分离重组凝血酶原酶Fv构建体;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以及血小板的促凝血作用的能力将在体外通过测量纤维蛋白形成、各种凝血因子和血小板上蛋白酶激活受体1的切割和活化来确定。 将通过测量循环凝血酶原的消耗、凝血因子的消耗和股静脉的增强闭塞来确定给予小鼠的凝血酶原酶Fv的促血栓形成作用。这些研究将允许评估凝血酶原酶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酶活性来改善狼疮中的血栓形成事件。

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.