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

凝血酶原和纤溶酶原是两种中枢止血酶原，它们是通过切割An而被蛋白分解激活的。 激活循环。新形成的N-末端插入到结合口袋中并触发官能团的形成 活动站点。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)，通过插入自己的基因来劫持这一机制 N-末端进入宿主酶原口袋，构象激活催化位点。SK-纤溶酶原 复合体可将纤溶酶原激活为纤溶酶。SC和SK与酵母原的络合物 成熟的蛋白酶可裂解纤维蛋白(原)，但不能被宿主的抗凝血酶和抗纤溶酶所渗透。 需要其他方法来控制他们不想要的活动。我们的单抗(MAbs) SC和SK N-末端阻断复合体的形成和活性，对抗感染相关的血栓形成和 细菌在体内传播。这说明了基于机制的单抗在日益增长的环境中的可行性 抗生素耐药性。SC和SK具有不依赖于纤维蛋白(原)的额外的、未完全定义的结合部位 在底物识别中发挥作用的底物识别。我们的建议旨在确定唯一的SC和SK 序列及其与酵素的复合体中的构象表位，促进结合 纤维蛋白(原)，在底物和锚定模式下。我们的集团在SC和SK方面拥有长期的专业知识- 介导的酶原激活，我们最近在鉴定纤维蛋白(原)片段D结合方面取得了良好的进展 到SC的C末端重复序列。然而，SK-纤溶酶(原)复合体与宿主纤维蛋白(原)的相互作用 仍然没有被很好地理解。我们的短期目标是定义纤维蛋白(原)结合，增强辅因子- 通过纤维蛋白(原)的酶原反应，鉴定结合表位，并在体内开发有效的单抗 添加到我们现有的抗体库中。我们将我们的结构功能和机械专业知识与此相结合 单抗开发专家(比尔·丘奇博士)以及SC和SK作用的小鼠模型的应用专家(Dr。 彼得·帕尼齐)。目标1将定义SC-凝血酶原复合体与纤维蛋白(原)的双重相互作用机制， 目的是确定用于阻断纤维蛋白(原)结合的合适的线性和构象表位。目标2 将描述化脓性链球菌SK变种的纤维蛋白(原)依赖的纤溶酶原激活机制 日期没有很好地定义，相同的目标是确定SK变异体上的纤维蛋白(原)结合表位。目标 3将在体内测试我们针对SC和SK N端的人源化mAbs，并选择紧密结合的抗 用于体内研究的纤维蛋白(原)结合部位单抗。未来资金周期的长期目标是发展 与多种血清型和等位基因变异发生交叉反应，并可能符合临床前条件的单抗 和临床测试。这些单抗的鸡尾酒将支持患者的止血系统， 纤溶酶介导的细菌传播和不需要的凝血酶原激活，而不会导致细菌耐药性。

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.