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Platelet-activating factor modulates endotoxin-induced macrophage procoagulant activity by a protein kinase
D S Kucey1, P Y Cheung, O D Rotstein
1Department of Surgery, Toronto General Hospital, Ontario, Canada.
Abstract:
Macrophage procoagulant activity is an important mediator of extravascular fibrin deposition at sites of infection and appears to contribute to the pathogenesis of several infectious disease processes. Previous studies have shown that the inflammatory mediator platelet-activating factor was able to prime macrophages for induction of procoagulant activity by bacterial lipopolysaccharide. The present studies were designed to examine the mechanism of this priming effect. Platelet-activating factor (100 nM) primed macrophages for procoagulant activity generation in response to endotoxin at concentrations as low as 100 ng/ml and also following exposure to Escherichia coli, Bacteroides fragilis, and Staphylococcus aureus. The priming effect occurred following a pretreatment with platelet-activating factor for as short as 1 min, suggesting a rapid activation event. Two different doses of the calcium ionophore ionomycin were used to mimic the peak and sustained effects of platelet-activating factor on cytoplasmic calcium levels (1 microM and 100 nM, respectively). Neither dose was able to mimic the priming effect. However, extracellular calcium was necessary for induction of procoagulant activity and the priming effect. By contrast, the protein kinase C agonist phorbol myristate acetate reproduced the priming phenomenon observed for platelet-activating factor. In further support of the concept that protein kinase C activation mediated the effect of platelet-activating factor, the specific protein kinase C inhibitor staurosporine reversed the ability of platelet-activating factor to augment induction of macrophage procoagulant activity by endotoxin. These data suggest mechanisms by which inflammatory mediators within the microenvironment of infection might modulate the host response to bacterial pathogens.
Insights
Platelet-activating factor primes macrophages for enhanced procoagulant activity during infection. Protein kinase C activation, not calcium levels, mediates this rapid inflammatory response, crucial for host defense against bacterial pathogens.
Area of Science:
- Immunology
- Cell Biology
- Pathogenesis
Background:
- Macrophage procoagulant activity (PA) drives fibrin deposition in infections.
- Platelet-activating factor (PAF) primes macrophages for PA induction by bacterial lipopolysaccharide (LPS).
Purpose of the Study:
- To investigate the mechanism behind PAF-mediated priming of macrophage PA.
- To elucidate the role of intracellular calcium and protein kinase C (PKC) in this priming effect.
Main Methods:
- Macrophages were pretreated with PAF and then exposed to bacterial endotoxin or whole bacteria.
- Calcium ionophore ionomycin and PKC activator phorbol myristate acetate (PMA) were used.
- PKC inhibitor staurosporine was employed to assess PKC involvement.
Main Results:
- PAF rapidly primed macrophages for PA induction by endotoxin and various bacteria.
- Neither calcium ionophore mimicked the PAF priming effect, but extracellular calcium was essential.
- PMA reproduced the priming effect, and staurosporine reversed PAF's augmentation of endotoxin-induced PA.
Conclusions:
- PKC activation is the primary mechanism for PAF-induced priming of macrophage PA.
- These findings reveal how inflammatory mediators modulate host responses to bacterial pathogens during infection.