Partial characterization and staphylocidal activity of thrombin-induced platelet microbicidal protein

Abstract

Thrombin-induced platelet microbicidal protein (PMP) is considered to play an important role in preventing an important role in preventing streptococcal endocarditis. However, the structural features and functions of PMPs have not been well characterized, and their antibacterial spectra against other common endocarditis pathogens, such as the staphylococci, are not known. Thrombin stimulation of washed rabbit platelets (10(8)/ml) yielded a PMP-rich preparation with a specific activity of approximately 25 U/mg of protein as determined by Bacillus subtilis bioassay. Twenty-eight clinical and laboratory Staphylococcus aureus isolates, exposed to a standardized PMP preparation (100 U/ml for 2 h at 37 degrees C), exhibited a Poisson-distributed heterogeneity to the bactericidal action of PMP, with approximately one-third designated as PMP resistant. Gel filtration chromatography (Sephadex G-50) identified the bioactive moiety within PMP preparations to be in the major protein elution peak; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) presumptively identified PMP as a low-molecular-weight (MW) (8,500) protein present only in such bioactive protein peaks. Both the bioactivity of PMP preparations and the low-MW protein band were removable by specific anionic membranes (e.g., cellulose-acetate/nitrate), as well as by a variety of anionic resins, further corroborating the suspected cationic charge of PMP. In addition, both PMP bioactivity and the low-MW protein band were recoverable by 1.5 M NaCl elution of the anionic membrane filters post-PMP adsorptive removal. Adsorption of bioactive PMP preparations by highly PMP-susceptible B. subtilis (10(8) CFU/ml, 30 min) resulted in a near-complete loss of residual bioactivity; in contrast, adsorption of bioactive PMP preparations with less PMP-susceptible S. aureus strains failed to reduce bioactivity. Significant lysozyme contamination of PMP-rich preparations was ruled out by determination of differences between bioactive PMP preparations and exogenous lysozyme as regards (i) relative heat stabilities; (ii) differential bactericidal activity versus B. subtilis and Micrococcus luteus; and (iii) SDS-PAGE protein profiles. These data show that the bioactive PMP protein moiety is of low MW, is heat stable, is probably cationic (similar to leukocyte-derived defensins), and possesses potent bactericidal activity against a significant percentage of S. aureus isolates.