MOLECULAR BIOLOGY, PLATELET AGGREGTION, S. SANGUIS

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Certain strains of viridans streptococci (Agg+), including the predominant member of the oral commensal flora, Streptococcus sanguis, induce human platelets to aggregate in vitro. When viridans streptococci entered the body, they may become significant pathogens or even etiological agents of certain severe human diseases by interacting with platelets, such as bacterial endocarditis, Behcet's syndrome, etc Three different surface antigens of S. sanguis (Adh+, Agg+) have been proposed to be involved in the interaction with platelets, which are class I antigen (adhesin), class II antigen, which is also called platelet-aggregation associated protein (PAAP), and class III antigen, which has ecto-ATPase activity. S. sanguis cells bind to platelets by class I antigen, and then class II antigen triggers and activates the release of the dense granules and the aggregation of platelets, the class III antigen amplifies the reaction by hydrolyzing ATP released from dense granules into ADP. Although various cell-free forms of PAAP have been identified, these molecules do not directly induce platelet aggregation. Direct evidence needs to be provided to prove that PAAP on the cell surface functions as an unique factor to activate and aggregate platelets. The specific aims of this project which so far have been accomplished includes (1) developed procedures for isolating an isogenic PAAP mutant of S. sanguis using transposon Tn916 and isogenic revertants, (2) cloned the PAAP gene (paap) from S. sanguis, (3) isolated and purified the portion of the recombinant PAAP (rPAAP) expressed by the cloned gene in E. coli and compared it functionally with the cell-free forms of PAAP isolated from S. sanguis, (4) partially sequenced the gene, and (5) characterized the gene by analyzing the sequencing data using specific sequencing software and comparing the sequence with genebanks. Further studies need to be done on sequencing and making a gene specific insertional mutant of S. sanguis.