Platelet aggregation and activation are important components of hemostatic and inflammatory responses. Activated platelets may play an important role in the modulation of the host response during periodontitis. Microorganisms commonly found in dental plaque have been shown to aggregate and activate platelets. Certain strains of Streptococcus sanguis, the predominant organism in dentaTplaque, expresses a surface protein, platelet aggregation-associated protein (PAAP) which activates platelets in a complement-independent manner. An oral microorganism which is a periodontal pathogen, Porphyromonasgmgivalis, has been shown in vitro to aggregate and activate human platelets. P. gingivalis expresses a 100 kDa cell surface protein which is cross reactive with lgG antibodies to the S. sanguis PAAP. The identify of the 100-kDa P. gingivalis PAAP is not clear. Several P. gingivalis hemagglutinins and adhesion proteins have been identified, however their relationship to P. gingivalis PAAP is uncertain. There is also evidence that a secreted P. gingivalis protein. Protease I, can caus e in vitro platelet aggregation and activation. The work supported by this grant will be to evaluate the surface proteins of P. gingivalis for binding to cell surface proteins expressed by human platelets. Specifically, protein extracts or P. gingivalis, outer membrane vesicles, and a firnbria negative mutant, DPG3, will be separated by SDS-PAGE and transferred to nitrocellulose membranes. Cell surface proteins and glycoproteins of non-activated human platelets will be biotinylated and extracted from whole cell lysates. The platelet proteins will be incubated with the nitrocellulose membranes retaining the P. gingivalis proteins. Protein interactions will be identified by incubations with streptavidin conjugated to horseradish peroxidase and an appropriate substrate. Reactive bands will be removed from duplicate gels and their N-termini sequenced to identify P. gingivalis proteins which interact with cell surface proteins of human platelets. The sequences will be used in a reverse genetics approach to identify and isolate the genes encoding the proteins. Gene disruption will allow for identification of the proteins which play important roles in platelet aggregation. Key words: Porphyromonas gingivalis, human platelets, cell surface proteins
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