Equine platelets inhibit E. coli growth and can be activated by bacterial lipopolysaccharide and lipoteichoic acid although superoxide anion production does not occur and platelet activation is not associated with enhanced production by neutrophils

Abstract

Activated platelets can contribute to host defense through release of products with bactericidal actions such as antimicrobial peptides and reactive oxygen species (ROS), as well as by forming heterotypic aggregates with neutrophils and enhancing their antimicrobial properties. Whilst release of vasoactive mediators from equine platelets in response to stimuli including bacterial lipopolysaccharide (LPS) has been documented, neither ROS production, nor the effects of activated platelets on equine neutrophil ROS production, have been reported. This study first sought evidence that activated equine platelets inhibit bacterial growth. Platelet superoxide production in response to stimuli including Escherichia coli-derived LPS and lipoteichoic acid (LTA) from Staphylococcus aureus was then determined. The ability of LPS and LTA to up-regulate platelet P-selectin expression and induce platelet–neutrophil aggregate formation was investigated and the effect of co-incubating activated platelets with neutrophils on superoxide production measured.Growth of E. coli was inhibited in a time-dependent manner, and to a similar extent, by addition of platelet rich plasma (PRP) or platelet poor plasma (PPP) obtained by centrifugation of PRP. Activation of platelets in PRP by addition of thrombin led to a significant increase in the inhibitory action between 0.5 and 2h.Although phorbol myristate acetate (PMA) caused superoxide production by equine platelets in a protein kinase C-dependent manner, thrombin, platelet activating factor (PAF), LPS, LTA and formyl-methionyl-leucyl phenylalanine (FMLP) were without effect. LPS and LTA did induce platelet activation, measured as an increase in P-selectin expression (% positive cells: 17±3 (un-stimulated); 63±6 (1μg/ml LPS); 64±6 (1μg/ml LTA); n=5) but not platelet superoxide production or heterotypic aggregate formation. Co-incubation of activated platelets with neutrophils did not increase neutrophil superoxide production.This study has demonstrated for the first time that when activated, equine platelets, like those of other species, are capable of releasing ROS that could assist in bacterial killing. However, the findings suggest that neither superoxide production by platelets nor enhancement of production by neutrophils is likely to play a significant role. Nevertheless, as has been reported in man, equine PPP and PRP did inhibit E. coli growth in vitro, and addition of thrombin significantly increased the inhibitory effect of PRP. This suggests that products released from activated platelets could contribute to antimicrobial activity in the horse. The factors in equine plasma and released by activated platelets that are responsible for inhibiting bacterial growth have yet to be determined.