Mechanism of Staphylocoagulase-activated Blood Clotting

描述(申请人提供)：拟议研究的主要目标是确定金黄色葡萄球菌蛋白(SC)激活人类凝血的分子机制，以及该机制在心内膜炎发病机制中的作用。SC与人凝血酶原(Pro)紧密结合，并在酶原中诱导形成一个功能性催化部位，而不需要通常严格要求的肽键断裂。这种独特的构象激活机制被认为涉及SC和Pro的初始相遇，随后在两个或两个以上离散的构象变化中激活催化位点和占据调节原外显子I。其机制可能涉及原外显子I占据和催化位点激活之间的构象连接，通过SC与活性构象的高亲和力结合来稳定，而不需要将SC氨基末端插入到Pro催化域的结合口袋中。SC-Pro将纤维蛋白原(FBG)转化为纤维蛋白(FBN)的独特专一性机制被认为是为了绕过和抑制Pro激活的正常反应。这一机制是心内膜炎心脏瓣膜上血小板-FBN-细菌繁殖体繁殖的中心。SC-Pro/T络合物的FBG闭合活性被认为涉及到通过在SC-Pro/T络合物上表达FBG结合外位而特异性识别FBG作为底物，此外还涉及催化位点特异性的变化。提出了利用新型活性部位标记的Pro荧光衍生物的生化、生物物理和结构方法来检验SC激活Pro构象的假说及其将FBG转化为FBN的特异性基础。具体目标是：(1)确定SC激活Pro构象的热力学机制；(2)确定构象激活过程中单个分子事件的动力学途径；(3)阐明FBG作为SC-Pro/T复合物底物的特异性识别机制；(4)确定与Pro/T物种结合的SCI-327的三维结构。所提出的研究对于理解SC如何在丝氨酸蛋白酶酶原激活中绕过对肽键断裂的严格要求具有基础性意义。这些研究将为SC激活Pro在心内膜炎发病机制中的作用提供新的见解，并最终可能开发出基于抑制SC激活的凝血功能的抗生素辅助治疗。

DESCRIPTION (provided by applicant): The broad goal of the proposed studies is to define the molecular mechanism of the activation of human blood coagulation by the S. aureus protein, staphylocoagulase (SC), and the role of the mechanism in the pathogenesis of endocarditis. SC binds tightly to human prothrombin (Pro) and induces formation of a functional catalytic site in the zymogen without the usual strictly required peptide bond cleavages. This unique conformational activation mechanism is hypothesized to involve initial encounter of SC and Pro, followed by activation of the catalytic site and occupation of regulatory proexosite I in two or more discrete conformational changes. The mechanism may involve conformational linkage between proexosite I occupation and catalytic site activation, stabilization by high affinity binding of SC to the active conformation, and is unlikely to require insertion of the SC amino-terminus into a binding pocket in the Pro catalytic domain. The mechanism underlying the unique specificity of SC-Pro to convert fibrinogen (Fbg) to fibrin (Fbn) is hypothesized to bypass and inhibit the normal reactions of Pro activation. This mechanism is central to the propagation of platelet- Fbn-bacteria vegetations on heart valves in endocarditis. Fbg clofting activity of the SC-Pro/T complexes is hypothesized to involve specific recognition of Fbg as a substrate through expression of a Fbg-binding exosite on the SC-Pro/T complexes, in addition to changes in catalytic site specificity. Biochemical, biophysical, and structural approaches employing novel active site-labeled fluorescent derivatives of Pro are proposed to test hypotheses for conformational activation of Pro by SC and the basis of its specificity for conversion of Fbg to Fbn. Specific Aims are: (1) To determine the thermodynamic mechanism of conformational activation of Pro by SC; (2) To define the kinetic pathway of individual molecular events in conformational activation; (3) To elucidate the mechanism of specific recognition of Fbg as a substrate of SC-Pro/T complexes; and (4) To determine the three dimensional structures of SCI-327 bound to Pro/T species. The proposed studies are of fundamental significance in understanding how SC can circumvent the otherwise strict requirement for peptide bond cleavage in serine proteinase zymogen activation. The studies will provide new insight into the role of activation of Pro by SC in the pathogenesis of endocarditis and may ultimately allow therapy adjunctive to antibiotics to be developed based on inhibition of SC-activated blood coagulation.