ANTIBODIES WITH PROTHROMBINASE ACTIVITY IN LUPUS

狼疮中具有凝血酶原活性的抗体

基本信息

批准号：
6650264
负责人：
Perumal Thiagarajan
金额：
$ 22.69万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-30 至 2005-08-31
项目状态：
已结题

项目摘要

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.

凝血酶原是凝血酶的前体，凝血酶是血液凝固的核心酶。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 因子对凝血酶原激活的抑制；加速凝血酶原清除）。在初步研究中，我们发现了这些抗体可以诱导血栓形成的一种新的有效机制，即凝血酶原的催化裂解。该提议基于这样的假设：凝血酶原酶自身抗体通过凝血酶原（如凝血酶原的生理激活剂 Xa 因子）产生类似凝血酶的活性来促进血栓形成。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，与可逆结合的抗体相比，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；定义与其潜在临床效果相关的凝血酶原酶自身抗体的生化特征；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血作用；并确定给予小鼠的 Fv 构建体是否诱导血栓形成。为此，将通过电泳、荧光和放射测量方法比较来自狼疮和正常受试者的多克隆 IgG 的凝血酶原裂解活性；将分析亲和纯化的抗凝血酶原抗体，以确定凝血酶原片段的动力学参数、特异性、切割位点、辅因子要求和酶活性；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，可以从噬菌体展示文库中分离重组凝血酶原酶Fv构建体；抗体生成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通过测量纤维蛋白形成、各种凝血因子和蛋白酶激活受体的裂解和激活在体外测定1 血小板上。给予小鼠的凝血酶原酶Fv的促血栓作用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闭塞的增强来确定。这些研究将有助于评估凝血酶原酶自身抗体对狼疮高凝状态的影响程度。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活性来改善狼疮的血栓形成事件。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin.

DOI：
10.1021/bi030167f
发表时间：
2004-04
期刊：
Biochemistry
影响因子：
2.9
作者：
A. Le;S. Dasgupta;S. Planque;S. Paul;P. Thiagarajan
通讯作者：
A. Le;S. Dasgupta;S. Planque;S. Paul;P. Thiagarajan

Polymorphisms beta2-glycoprotein I: phospholipid binding and multimeric structure.

β2-糖蛋白 I 多态性：磷脂结合和多聚体结构。

DOI：
10.1016/s0049-3848(02)00392-4
发表时间：
2002
期刊：
Thrombosis research
影响因子：
7.5
作者：
Gushiken,FranciscaC;Le,Anhqueyn;Arnett,FrankC;Thiagarajan,Perumal
通讯作者：
Thiagarajan,Perumal

Characterization of autoantibodies against sulfatide from a V-gene phage-display library derived from patients with systemic lupus erythematosus.

来自系统性红斑狼疮患者的 V 基因噬菌体展示文库的硫苷脂自身抗体的表征。

DOI：
10.1016/j.jim.2004.10.001
发表时间：
2004
期刊：
Journal of immunological methods.
影响因子：
0
作者：
Guchhait,Prasenjit;Lopez,JoseA;Thiagarajan,Perumal
通讯作者：
Thiagarajan,Perumal

Sulfatides: targets for anti-phospholipid antibodies.

脑硫脂：抗磷脂抗体的靶标。

DOI：
10.1161/01.cir.0000095030.44185.6a
发表时间：
2003
期刊：
Circulation.
影响因子：
0
作者：
Merten,M;Motamedy,S;Ramamurthy,S;Arnett,FC;Thiagarajan,P
通讯作者：
Thiagarajan,P

New targets for antithrombotic drugs.

抗血栓药物的新靶点。

DOI：
10.2165/00129784-200202040-00002
发表时间：
2002
期刊：
American journal of cardiovascular drugs : drugs, devices, and other interventions
影响因子：
0
作者：
Thiagarajan,Perumal
通讯作者：
Thiagarajan,Perumal