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Purification and in vitro activities of rabbit platelet microbicidal proteins
M R Yeaman1, Y Q Tang, A J Shen
1Department of Medicine, Los Angeles County-Harbor UCLA Medical Center, Torrance, California 90509, USA. yeaman@afp76.humc.edu
Abstract:
Recent in vitro studies have demonstrated that rabbit platelets release a small, cationic antimicrobial protein in response to thrombin stimulation under physiological conditions (M. R. Yeaman, S. M. Puentes, D. C. Norman, and A. S. Bayer, Infect. Immun. 60:1202-1209, 1992). This observation prompted our present investigation, focused on determining the array of antimicrobial proteins contained within rabbit platelets and their in vitro activity against common bloodstream pathogens. A group of small (6.0- to 9.0-kDa), cationic proteins with in vitro antimicrobial activity was purified from whole and thrombin-stimulated rabbit platelets by gel filtration and reversed-phase high-performance liquid chromatography. Purified proteins in micromolar concentrations (10 to 40 microg/ml) exerted in vitro microbiostatic and/or microbicidal activities against Staphylococcus aureus, Escherichia coli, and Candida albicans in a dose-dependent manner. The antimicrobial activities of proteins purified from rabbit platelet acid extracts were generally inversely related to pH, with maximal activity observed at pH 5.5. In contrast, the predominant protein isolated from thrombin-stimulated rabbit platelets, though biochemically and microbiologically similar to proteins extracted by acid, exhibited antimicrobial activities which were modestly enhanced at pH 7.2 compared with pH 5.5. Amino acid compositional analyses in combination with molecular mass determinations suggest that the majority of these proteins are distinct molecules not derived from a single common precursor. Collectively, these data indicate that rabbit platelets contain proteins which exert potent in vitro antimicrobial activity against bacterial and fungal pathogens which commonly invade the bloodstream. Moreover, several of these proteins were released from platelets stimulated with thrombin under physiological conditions and exerted potent antimicrobial activities in physiological pH ranges. These observations support the hypothesis that platelets serve an important role in host defense against infection, via localized release of antimicrobial proteins in response to stimuli associated with tissue injury or microbial colonization.
Insights
Rabbit platelets contain antimicrobial proteins that fight common bloodstream pathogens like Staphylococcus aureus and E. coli. These platelet antimicrobial proteins are released upon stimulation, suggesting a role in host defense against infection.
Area of Science:
- Biochemistry
- Microbiology
- Immunology
Background:
- Platelets are known for their role in hemostasis.
- Previous studies suggested platelets release antimicrobial proteins.
Purpose of the Study:
- To identify antimicrobial proteins in rabbit platelets.
- To determine their activity against bloodstream pathogens.
Main Methods:
- Purification of proteins using gel filtration and HPLC.
- In vitro antimicrobial activity assays against Staphylococcus aureus, Escherichia coli, and Candida albicans.
- Amino acid analysis and molecular mass determination.
Main Results:
- Purified cationic proteins (6.0-9.0 kDa) showed dose-dependent antimicrobial activity.
- Maximal activity for acid-extracted proteins was at pH 5.5.
- A protein from thrombin-stimulated platelets showed enhanced activity at pH 7.2.
- Proteins appear to be distinct molecules.
Conclusions:
- Rabbit platelets contain potent antimicrobial proteins active against bacterial and fungal pathogens.
- These proteins are released under physiological conditions and active at physiological pH.
- Platelets play a role in host defense through localized release of antimicrobial proteins.