Roles of fibrin(ogen) in conformational activation of hemostatic proteinase precursors

纤维蛋白（原）在止血蛋白酶前体构象激活中的作用

基本信息

批准号：
10453034
负责人：
INGRID M VERHAMME
金额：
$ 49.57万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-10 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10453034
关键词：
Active Sites Antibiotic Resistance Antibodies Antiplasmin Antithrombins Bacterial Endocarditis Bacterial Proteins Binding Binding Sites Blocking Antibodies Blood Coagulation Disorders C-terminal Catalytic Domain Church Coagulation Process Collaborations Combined Modality Therapy Complex Cryoelectron Microscopy Data Development Environment Enzyme Precursors Epitopes Equilibrium Evaluation Extracellular Matrix Proteins Fibrin Fibrinogen Fluorescence Funding Future Goals Hemostatic Agents Hemostatic function Host Defense Mechanism Human In Vitro Infection Innate Immune System Kinetics Knowledge Mediating Methods Modeling Molecular Conformation Monoclonal Antibodies N-terminal Patients Peptide Hydrolases Physiological Plasmin Plasminogen Play Preclinical Testing Proteins Prothrombin Regulation Resources Risk Role Scanning Serotyping Staphylocoagulase Staphylococcus aureus Streptococcus pyogenes Streptokinase Structure Surface System Terminal Repeat Sequences Testing Therapeutic Embolization Therapeutic antibodies Thrombin Thrombosis Tissues Trypsin Vaccines Variant Virulence Factors Work activation product bacterial resistance base cofactor design expression vector genetic variant humanized monoclonal antibodies in vivo in vivo evaluation in vivo imaging inhibitor mouse model murine monoclonal antibody novel novel therapeutics prevent protein complex research clinical testing side effect

项目摘要

Prothrombin and plasminogen, two central hemostatic zymogens, are activated proteolytically by cleavage of an activation loop. The newly formed N-terminus inserts into a binding pocket and triggers formation of a functional active site. The activation products thrombin and plasmin respectively form and degrade fibrin, but physiological regulation prevents uncontrolled clotting and promiscuous plasmin-mediated tissue degradation. The bacterial virulence factors, staphylocoagulase (SC) and streptokinase (SK), hijack this mechanism by inserting their own N-termini into the host zymogen pockets, and conformationally activating the catalytic site. The SK-plasminogen complex proteolytically activates plasminogen to plasmin. Both the SC and SK complexes with the zymogens and the mature proteases cleave fibrin(ogen) but are impervious to host antithrombin and antiplasmin, and alternative methods are needed to zcontrol their unwanted activity. Our monoclonal antibodies (mAbs) against the SC and SK N-termini block complex formation and activity, counteracting infection-related thrombosis and bacterial spreading in vivo. This illustrates mechanism-based mAb feasibility in an environment of increasing antibiotic resistance. SC and SK have additional, incompletely defined binding sites for fibrin(ogen) independent of substrate recognition, that play a role in localization. Our proposal aims to identify unique SC and SK sequences, and conformational epitopes in their complexes with the zymogens, that promote binding of fibrin(ogen), both in substrate and anchoring modes. Our group has long-standing expertise with SC and SK- mediated zymogen activation, and we recently made good progress identifying fibrin(ogen) fragment D binding to the C-terminal repeats of SC. However, interactions of the SK-plasmin(ogen) complexes with host fibrin(ogen) are still not well understood. Our short-term goals are to define fibrin(ogen) binding, enhancement of cofactor- zymogen reactivity by fibrin(ogen), identify binding epitopes, and develop in vivo effective mAbs that will be added to our existing antibody arsenal. We combine our structure-function and mechanism expertise with that of experts in mAb development (Dr. Bill Church), and in application of mouse models of SC and SK action (Dr. Peter Panizzi). Aim 1 will define dual interaction mechanisms of the SC-prothrombin complex with fibrin(ogen), with the goal of identifying suitable linear and conformational epitopes for blocking fibrin(ogen) binding. Aim 2 will delineate fibrin(ogen)-dependent plasminogen activation mechanisms of S. pyogenes SK variants that to date are not well defined, with the same goal of identifying fibrin(ogen)-binding epitopes on the SK variants. Aim 3 will test our humanized mAbs targeting the N-termini of SC and SK in vivo, and select tight-binding anti- fibrin(ogen) binding site mAbs for in vivo studies. Long-term goals for future funding cycles are the development of mAbs that that cross-react with a wide range of serotypes and allelic variants, and may qualify for pre-clinical and clinical testing. Cocktails of these mAbs would support the patient's hemostatic system by minimizing plasmin-mediated bacterial spreading and unwanted prothrombin activation without causing bacterial resistance.

凝血酶原和纤溶酶原（两个中央止血酶原）通过切割的蛋白水解激活激活环。新形成的N末端插入到结合口袋中，并触发功能的形成活性站点。活化产物凝血酶和纤溶酶分别形成并降解纤维蛋白，但生理调节可防止不受控制的凝结和渗透纤溶酶介导的组织降解。细菌毒力因子，葡萄球菌酶（SC）和链球菌酶（SK），通过插入自己的机制来劫持这种机制 N-末端进入宿主的Zymogen口袋，并在构象上激活催化位点。 Sk- ---纤溶酶原复杂的蛋白水解将纤溶酶原激活为纤溶酶。 Zymogens的SC和SK络合物都成熟的蛋白酶裂解纤维蛋白（OGEN），但不渗透宿主抗凝血酶和抗蛋白蛋白，并且需要替代方法来Zcontrol的不良活动。我们的单克隆抗体（mAb） SC和SK N-Termini阻滞复合物的形成和活性，抵消与感染相关的血栓形成和细菌在体内扩散。这说明了基于机制的mAb可行性在增加的环境中抗生素抗性。 SC和SK具有纤维蛋白（OGEN）独立的其他未完全定义的结合位点底物识别，在本地化中起作用。我们的建议旨在确定独特的SC和SK 序列和与Zymogen的复合物中的构象表位，促进结合的结合纤维蛋白（OGEN），无论是底物还是锚定模式。我们的小组在SC和SK-拥有长期的专业知识介导的酶原激活，我们最近取得了良好的进步，以识别纤维蛋白（OGEN）片段D结合 SC的C末端重复序列。但是，SK-质蛋白（OGEN）复合物与宿主纤维蛋白（OGEN）的相互作用仍然不太了解。我们的短期目标是定义纤维蛋白（OGEN）结合，增强辅因子的结合 - 纤维蛋白（OGen）的酶原反应性，鉴定结合表位，并在体内发展有效mAb，这将是添加到我们现有的抗体武器库中。我们将我们的结构功能和机制专业知识结合起来 MAB开发专家（Bill Church博士）以及SC和SK Action的鼠标模型的应用（博士 Peter Panizzi）。 AIM 1将定义与纤维蛋白（OGEN）的SC-凝结蛋白复合物的双重相互作用机制，目的是识别用于阻断纤维蛋白（OGEN）结合的合适的线性和构象表位。目标2 将描述纤维蛋白（OGEN）依赖性纤溶酶原激活机制，用于链球菌SK变体日期不是很好的定义，其目标是识别SK变体上的纤维蛋白（OGEN）结合表位。目的 3将测试针对体内SC和SK的N末端的人源化mAB，并选择紧密结合的抗 - 纤维蛋白（OGEN）结合位点mAb用于体内研究。未来资金周期的长期目标是发展与广泛的血清型和等位基因变体交叉反应的mAb，并且可能有资格参加临床前和临床测试。这些mAb的鸡尾酒可以通过最小化来支持患者的止血系统纤溶酶介导的细菌扩散和不需要的凝血酶原活化，而不会引起细菌耐药性。