No abstract

The possible involvement of platelet-activating factor (PAF) in the pathogenesis of endotoxemia, was investigated by using a binding assay to patients' platelets, complemented with the extraction and chemical characterization of PAF obtained from patients' platelets. Platelets from 12 human volunteers had 281 +/- 63 freely accessible high affinity binding sites (PAF-receptors) per platelet; whereas this number was of 49 +/- 37 PAF-receptors per platelet, n = 14 samples, P less than 0.01, in a group of 13 patients with positive blood culture. A group of patients with respiratory or cardiovascular disturbances and negative blood culture had 253 +/- 74, accessible receptors per platelet (n = 19 samples from 16 patients, P less than 0.01 as compared to septic patients, which was not significantly different when compared to control individuals). Patients with sepsis possessed significant amounts of PAF associated to their platelets, whereas this mediator could not be isolated from platelets of patients with respiratory or cardiovascular disturbances and negative blood culture, nor from platelets of control individuals. PAF was also assayed in whole blood samples and found at high concentrations in sepsis patients. These data indicate that occupancy of PAF receptors in combination with high amounts of platelet-associated PAF, is a common finding in patients with sepsis.

In A-431 cells, platelet-activating factor (PAF) induces the expression of c-fos and TIS-1 genes in both the absence and the presence of cycloheximide in a structurally specific and receptor-coupled manner. We have now investigated the molecular mechanisms of this response, particularly in relation to the role of protein kinases. Pretreatment of cells with genistein or methyl-2,5-dihydroxycinnamate (tyrosine kinase inhibitors) or staurosporine (a protein kinase C inhibitor) for 20 min abolished the c-fos expression induced by PAF. Interestingly, when genistein was added 90 s after addition of PAF, no inhibition was observed. Similarly, staurosporine did not inhibit c-fos expression when added 8 min after PAF addition to the cells. These inhibitions were dose-dependent (IC50 for staurosporine was 180 nM, and for genistein 50 microM). Simultaneous addition of PAF and phorbol 12-myristate 13-acetate (PMA) did not give a synergistic effect on c-fos expression. Pretreatment of cells with PMA had no effect on [3H]PAF binding, but abolished the PAF-induced gene expression. PAF-stimulated gene expression was desensitized if cells were pretreated with PAF. Interestingly, epidermal growth factor was able to stimulate c-fos expression in PAF-desensitized cells, and thus indicated involvement of distinct mechanisms for the two stimuli. Forskolin, an activator of adenylate cyclase, did not induce c-fos expression and had no effect on the PAF response. Exposure of cells to PAF for as little as 1 min, followed by its removal, was sufficient to activate the gene expression and demonstrated the rapidity and the exquisite nature of the signalling involved in this process. It is concluded that activation of PAF receptor (a proposed G-protein-coupled receptor) causes rapid production of signals which induce the expression of c-fos gene and that this is mediated via tyrosine kinase and protein kinase C.

No abstract