ANTIBODIES WITH PROTHROMBINASE ACTIVITY IN LUPUS

凝血酶原是凝血酶的前体，凝血酶是凝血的中心酶。狼疮患者的凝血酶原结合自身抗体在临床上与血栓形成有关，但这种联系是矛盾的，因为抗体作用的传统机制预测血栓形成减少(例如，抗体介导的Xa因子抑制凝血酶原激活；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、有效的机制，即催化裂解凝血酶原。这一建议基于如下假设，即凝血酶原酶自身抗体通过凝血酶原产生凝血酶样活性来促进血栓形成，如凝血酶原的生理激活剂Xa因子。凝血酶原酶抗体的周转能力表明，与可逆结合的化学计量抗体相比，它们可以发挥更强大的生物学效应。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，与可逆结合的抗体相比，这将进一步放大凝血酶原酶的促凝血作用。具体目的是：确定凝血酶原酶活性与狼疮患者血栓形成的统计相关性；确定凝血酶原酶自身抗体与其潜在临床效果相关的生化特征；从狼疮患者克隆催化高效的凝血酶原酶FV结构用于机制研究；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FV构建体注射到小鼠身上是否会导致血栓形成。为此，将通过电泳法、荧光法和放射学方法比较狼疮患者和正常人多克隆抗体的凝血酶原裂解活性；亲和纯化的抗凝血酶原抗体将被分析以确定凝血酶原片段的动力学参数、特异性、裂解部位、辅因子要求和酶活性；重组凝血酶原酶Fv结构将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抗原类似物进行筛选而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各种凝血因子和蛋白水解酶激活受体1的纤维蛋白形成、裂解和激活来确定。凝血酶原酶FV对小鼠的血栓前作用将通过测量循环中凝血酶原的消耗、凝血因子的消耗和股静脉的强化闭塞来确定。这些研究将评估凝血酶原酶自身抗体在多大程度上有助于狼疮的高凝状态。如果我们的假设成立，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.