Influence of platelets and platelet microbicidal protein susceptibility on the fate of Staphylococcus aureus in an in vitro model of infective endocarditis.

The pathogenesis of endovascular infections, such as infective endocarditis (IE), is multifaceted, involving interactions among microorganisms (such as Staphylococcus aureus), host plasma proteins, endothelial cells, and platelets (23). The outcome of these interactions represents a pivotal step in either the limitation or progression of IE (3, 5, 9, 12, 23). However, the exact role of platelets in this regard has been enigmatic (3, 5, 9, 10, 12, 23–25, 28, 32). Recent evidence from several laboratories suggests that platelets serve an important host defense function against the induction and evolution of endovascular infection (7, 24–37; R. C. Mercier, M. R. Yeaman, A. S. Bayer, and M. J. Rybak, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother. [ICAAC], abstr. B-61, 1997). The antimicrobial functions of mammalian platelets have been recently linked to their release of a family of small, cationic peptides termed platelet microbicidal proteins (PMPs) (32, 37; Y. Q. Tang, M. R. Yeaman, and M. E. Selsted, Blood 86:910, abstr. 3626, and Blood 86:556, abstr. 2212, 1995; J. Krijgsveld, S. A. Zaat, A. J. Kuijpers, G. H. Engbers, J. Feijen, and J. Dankert, Abstr. 38th ICAAC, abstr. F-179, 1998). For example, Yeaman et al. isolated and characterized seven antimicrobial peptides from quiescent and thrombin-stimulated rabbit platelets (37). Furthermore, Tang et al. discovered that human platelets likewise contain a group of peptides that exert potent and synergistic antimicrobial activities, including platelet factor-4, platelet basic peptide (PBP), connective tissue activating peptide-III (CTAP-III), and neutrophil activating peptide-2 (NAP-2; both CTAP-III and NAP-2 are derivatives of PBP) (Tang et al., abstr. 3626). Subsequently, Krijgsveld et al. confirmed that C-terminal modifications of human CTAP-III and NAP-2 also have direct antimicrobial properties (so-called “thrombocidins”) (Krijgsveld et al., 38th ICAAC). Moreover, both thrombin (generated at sites of damaged or infected endothelium) and infecting pathogens themselves (N. Azizi, C. Li, A. J. Shen, A. S. Bayer, and M. R. Yeaman, Abstr. 36th ICAAC, abstr. G-54, 1996) are potent agonists which stimulate platelets to release PMPs (2, 32, 37). Both in vitro and in vivo evidence now exists substantiating the role of platelets and PMPs in antimicrobial host defense. In vitro, PMPs exert rapid and potent (at nanomolar to micromolar concentrations) microbicidal activities against pathogens that commonly gain access to the bloodstream, such as S. aureus, coagulase-negative staphylococci, viridans group streptococci, and Candida albicans (26, 29, 37). PMPs achieve these effects in microenvironmental conditions relevant to the vascular compartment, such as physiologic ionicity and pH, as well as in the presence of serum (19). PMPs have also been shown to interfere with platelet adherence and aggregation due to S. aureus and C. albicans in vitro and potentiate the microbicidal and postexposure growth-inhibitory effects of conventional antibiotics in vitro (3, 30, 34–36). In vivo data also support the role of platelets in host defense. For example, Sullam et al. showed that cardiac vegetations from thrombocytopenic rabbits infected with PMP-susceptible viridans group streptococci had significantly higher bacterial densities compared to controls with normal platelet counts (25). Related studies have shown that in vitro PMP susceptibility correlates with a decreased propensity of clinical bloodstream isolates to cause IE (1, 26). Furthermore, in experimental IE, PMP susceptibility in S. aureus or C. albicans correlates with reduced microbial proliferation of these pathogens in vegetations and the spleen compared to isogenic PMP-resistant counterpart strains (8, 9, 33). Taken together, these in vitro and in vivo observations provide evidence that platelets play important roles in antimicrobial host defense, probably through localized elaboration of PMPs. Recently, an in vitro pharmacokinetic and pharmacodynamic model has been developed to simulate IE (4, 6, 15, 17, 20, 22); R. C. Mercier, and M. J. Rybak, Abstr. 8th Eur. Congr. Clin. Microbiol. Infect. Dis. abstr. P395, 1997; Mercier et al., 37th ICAAC). This model has been shown to closely parallel key events in IE pathogenesis that are observed in the well-characterized rabbit model (e.g., colonization via initial adherence and proliferation of organisms to high densities within simulated IE vegetations) (16, 20, 21; G. W. Kaatz, S. M. Seo, J. R. Aeschlimann, H. H. Houlihan, R. C. Mercier, and M. J. Rybak, Abstr. 37th ICAAC, abstr. F-11, 1997; G. W. Kaatz, S. M. Seo, J. R. Aeschlimann, H. H. Houlihan, R. C. Mercier, and M. J. Rybak, Abstr. 37th ICAAC, abstr. B-25, 1997). Like the in vivo model, the in vitro model poses a rigorous challenge to antimicrobial agent efficacy, due to the presence of high organism densities in the setting of a complex biomatrix comprising the simulated vegetation. Additionally, this model minimizes the use of experimental animals, is cost- and time-effective, and allows precise control of key experimental variables (such as bacterial densities, platelet counts, and specific platelet agonists). With the above considerations in mind, the objective of the present study was to examine the influence of platelets on the survival of isogenic S. aureus strains differing in PMP susceptibility profiles within simulated vegetations in an in vitro model of IE.