MECHANISMS OF PLATELET BINDING BY VIRIDANS STREPTOCOCCI

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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.