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

凝血酶原和纤溶酶原是两种主要的止血酶原，它们通过蛋白水解被激活， 激活回路新形成的N-末端插入结合口袋，并触发功能性蛋白的形成。 活性部位活化产物凝血酶和纤溶酶分别形成和降解纤维蛋白，但生理上 调节防止不受控制的凝血和混杂的纤溶酶介导的组织降解。细菌 毒力因子葡萄球菌凝固酶（SC）和链激酶（SK）通过插入它们自己的 N-末端插入宿主酶原口袋，并构象激活催化位点。SK-纤溶酶原 复合物蛋白水解激活纤溶酶原为纤溶酶。SC和SK均与酶原复合 成熟蛋白酶切割纤维蛋白（原），但不受宿主抗凝血酶和抗纤溶酶的影响， 需要替代的方法来控制它们不需要的活性。我们的单克隆抗体（mAb）针对 SC和SK N-末端阻断复合物形成和活性，抵抗感染相关血栓形成， 细菌在体内传播。这说明了基于机制的mAb在日益增加的环境中的可行性。 抗生素耐药性SC和SK具有额外的、不完全确定的纤维蛋白（原）非依赖性结合位点 在定位中起作用的底物识别。我们的建议旨在确定独特的SC和SK 序列，以及它们与酶原的复合物中的构象表位，其促进 纤维蛋白（原），无论是在基板和锚定模式。我们的团队在SC和SK方面拥有长期的专业知识- 介导的酶原激活，我们最近取得了很好的进展，确定纤维蛋白（原）片段D结合 然而，SK-纤溶酶（原）复合物与宿主纤维蛋白（原） 仍然没有被很好地理解。我们的短期目标是确定纤维蛋白（原）结合，增强辅因子- 通过纤维蛋白（原）的酶原反应性，鉴定结合表位，并开发体内有效的mAb， 添加到我们现有的抗体库中。我们将联合收割机的结构功能和机械专业知识与此相结合， mAb开发专家（Bill Church博士）和SC和SK作用小鼠模型应用专家（Dr. Peter Panizzi）。目的1将确定SC-凝血酶原复合物与纤维蛋白（原）的双重相互作用机制， 目的是鉴定用于阻断纤维蛋白（原）结合的合适的线性和构象表位。目的2 将描述S.化脓性链球菌SK变体， 日期没有很好地定义，具有鉴定SK变体上的纤维蛋白（原）结合表位的相同目标。目的 3将在体内测试我们的靶向SC和SK的N-末端的人源化mAb，并选择紧密结合的抗- 用于体内研究的纤维蛋白（原）结合位点mAb。未来供资周期的长期目标是 与广泛的血清型和等位基因变体交叉反应的mAb，并可能符合临床前 和临床试验。这些mAb的混合物将通过最小化患者的血液中的凝血因子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激活，而不引起细菌耐药性。

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.