| [Objective] To explore the bactericidal activity of recombinant human platelet phospholipase A2 (PLA2) in vitro and its mechanism, and to provide important information for developing new antibacterial drugs.[Methods] We studied the bactericidal activity of recombinant human platelet PLA2 in four aspects: in vitro bactericidal activity, factors of affected bactericidal activity, the interactions of the PLA2 and antibacterial drugs and mechanisms. 1 .Assay for bactericidal activity : Incubated bacteria solution with different concentration of the PLA2at 37# for 2 hours in a water bath. Then diluted the reactive solution and poured agar plates, and grew the bacteria in 37 癈 for 18-20 hours. Counted the colony formed unit (CFU) and calculated the bactericidal rate of PLA2. The drug resistance of Staphylococcus aureus (S.aureus) induced by the PLA2 was observed in drug resistant inducing test. 2.The affecting factors (pH, serum albumin,. concentration of bacteria, Ca2+ , Mg2+, heparin) were determined. 3. S.aureus was treated by the PLA2 or antibacterial drug alone or by the combination of both. Antibacterial activity was determined by counting CFU of bacteria. 4. By means of electron microscope, the morphologic changes of S.aureus were observed after the incubated with PLA2. Bacterial populations with different growth phases wastested for the PLA2 sensitivity. The postantibiotic effect (PAE) of the PLA2 against S.aureus was performed by routine colonial counting method.[Results] 1. The recombinant human platelet possessed potent bactericidal activity on the gram-positive(G+) bacteria, such as Staphylococcus aureus , Staphylococcus epidermidis, Enterococcus facecalis, Bacillus subtilis, Bacillus anthrax. The minimal bactericidal concentration (MBC) of the PLA2 were 6~200ng/ml when incubated for 2h . The PLA2 required for highly concentration to against gram-negative (G-) bacteria, its MBC were 20-55u g/ml when incubated for 2h. The PLA2 also had good bactericidal activity against drug-resistant G+ bacteria, such as meticillin-resistant Staphylococcus aureus (MRSA), meticillin-resistant Staphylococcus epidermidis (MRSE) and drug-resistant Enterococcus facecalis , its MBC were 100~300 ng/ml. 2. The bactericidal activity of platelet PLA2 was influenced by pH in media, and the bactericidal rates of PLA2 were 44.4%, 99.8% and 99.5% respectively in the pH of 5.0,7.4 and 9.0. It was easy for the PLA2 to be combined with serum albumin and its bactericidal activity is affected by serum albumin in media. The MBC of it against S.aureus increased with concentration of bacteria incubated in media. 1-5 mM Ca2+ were suitable for bactericidal activity of the PLA2 EGTA, heparin and MgCl2 significantly inhibitted bactericidal activity of recombinant human platelet PLA2, and the inhibition rate could reach 100% when the concentration of EGTA was 5mM, MgCl2 was 10mM, heparin was 0.03U/ml. 3.Benzylpenicillin, cefradine, fosfomycin and vancomycin significantly increased bactericidal activity of the PLA2. Erythromycin and clindamycin inhibited the bactericidal activity of the PLA2. ofloxacin and Amikacin had no effect on the PLA2 bactericidal activity. 4. Electron microscope examination found that the cell wall and membrane of S.aureus appeared cleft and segregation after the PLA2 incubated for 30~60min. We can see some S.aureus broken down completely at 2h. Mid-logarith phases S. aureus were more susceptible to the PLA2 than stationary ones. The PAE of PLA2 (160ng/ml) against S.aureu was 6h when incubated for Ih and the PAE>24h of 320ng/ml PLA2 [Conclusions] 1.The recombinant human platelet PLA2 possesses potent bactericidal activity against G+ bacteria as well as their drug-resistant strains. 2. The bactericidal activity of the PLA2 is affected by pH, serum albumin and concentration of bacteria in media. 3. The effects of the PLA2 bactericidal activity were synergistic with cell-wall active antibacterial drugs, antagonistic with bacteriostatic antibacterial drugs. 4. Ca2+, cationic property, cell-wall structure of bact...
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