MECHANISMS OF PLATELET BINDING BY VIRIDANS STREPTOCOCCI

血小板和细菌的结合是血小板聚集的一个假定的中心事件。 心内膜炎的发病机制然而，无论是机制， 细菌-血小板结合，也不是它的贡献的过程中， 感染，已被定义。这项建议的目的是隔离和 表征草绿色组链球菌的表面组分， 介导与人血小板的结合。为了提高 成功的配体纯化，概述了两种不同的策略， 其可以单独或组合使用，以回收 配体。作为第一种方法，我们将采用转座子诱变， 产生血链球菌菌株M99的非结合变体。的 然后通过免疫亲和性回收链球菌配体 色谱法;如果需要，将进行额外的纯化， 使用FPLC和HPLC。作为配体纯化的替代方法， 描述了多步色谱法（离子交换，凝胶 渗透和反相色谱法），其中配体是 从全菌的变溶素提取物中回收。 一旦分离，结合特性（动力学，饱和度， 可逆性）的配体与其血小板受体将是 评估。血小板受体的数量及其结合亲和力 将通过划痕分析来确定配体的含量。以确认 完整链球菌与血小板的粘附是由配体介导的， 我们将检测纯化的配体是否抑制血小板-链球菌 结合，如通过流式细胞术测定所测量。抑制研究将 也可以进行，其中链球菌与IgG预孵育 F（ab '）2对配体特异。为了确定配体的结合 对于血小板聚集是重要的，我们还检查了 纯化的配体和上述F（ab ′）2片段对血小板聚集的影响 被链球菌感染 此外，血小板结合在心内膜炎中的作用将被 在动物模型中进行评估。转座子的相对毒力- 将检查诱导的非结合突变体，如通过其能力测量的 引发并延续内分泌感染通过评估血小板- 细菌结合在细胞和分子水平，这项研究 应该有助于确定结合在发病机制中的重要性， 心内膜炎此外，这项工作可以为确定 血小板的免疫功能，并检查相互作用 链球菌与其他细胞系，如内皮细胞， 单核细胞

The binding of platelets and bacteria is a postulated central event in the pathogenesis of endocarditis. However, neither the mechanism for bacterial-platelet binding, nor its contribution to the course of infection, have been defined. The aim of this proposal is to isolate and characterize the surface component of viridans group streptococci that mediates binding to human platelets. To enhance the likelihood of successful ligand purification, two distinct strategies are outlined, which can be employed independently or in combination, to recover the ligand. As a first approach, we will employ transposon mutagenesis to produce a nonbinding variant of Streptococcus sanguis strain M99. The streptococcal ligand will then be recovered via immuno-affinity chromatography; if needed, additional purification will be performed, using FPLC and HPLC. As an alternative method for ligand purification, a multi-step chromatographic procedure is described (ion exchange, gel permeation, and reverse phase chromatography), in which the ligand is recovered from mutanolysin extracts of whole bacteria. Once isolated, the binding properties (kinetics, saturability, reversibility) of the ligand with its platelet receptor will be assessed. The number of platelet receptors, and their binding affinity for the ligand will be determined by Scratched analysis. To confirm that adherence of intact streptococci to platelets is mediated by the ligand, we will examined if the purified ligand inhibits platelet-streptococcal binding, as measured by a flow cytometric assay. Inhibition studies will also be performed, in which streptococci are preincubated with IgG F(ab')2 specific for the ligand. To determine if binding by the ligand is important for platelet aggregation, we also examine the effect of the purified ligand and the above F(ab')2 fragments on platelets aggregation by streptococci. In addition, the role of platelet binding in endocarditis will be evaluated in an animal model. The relative virulence of the transposon- induced nonbinding mutant will be examined, as measured by its ability to initiate and perpetuate endocardial infection. By assessing platelet- bacterial binding at the cellular and molecular level, this research should help define the significance of binding in the pathogenesis of endocarditis. Moreover, this work may provide a basis for defining the immunologic functions of platelets, and for examining the interaction of streptococci with other cell lines, such as endothelial cells and monocytes.