Involvement of a serine protease in the synthesis of platelet-activating factor by endothelial cells stimulated by tumor necrosis factor-α or interleukin-1α

It has been shown that production of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells (EC) stimulated with tumor necrosis factor (TNF)-α and interleukin (IL)-1α requires the synthesis of new proteins and is regulated by anti-proteinases. Here, we demonstrate that TNF-α and IL-1α induce the expression by EC of a 34-kDa diisopropyl fluorophosphate-binding protein immunoprecipitated by an antihuman elastase antibody. This protein is released in the medium and cleaves the chromogenic substrate N-methoxysuccinyl- Ala-Ala-Pro-Val p-anilide, which is specific for elastase. The generation of this elastase-like protein seems to be important for the synthesis of PAF induced by TNF-α and IL-1α, as suggested by the following observations: (a) it precedes the synthesis of PAF; (b) the inhibitors of serine protease and anti-human elastase antibody prevent the synthesis of PAF and the activation of 1-O-alkyl-2-lyso-glycerophosphocholine acetyl-CoA: acetyltransferase, which is a key enzyme of the PAF remodelling pathway; (c) elastase, at concentrations similar to that detectable in the medium of cytokine-activated EC, elicits a rapid synthesis of PAF by EC. High-performance liquid chromatography-tandem mass spectrometric analysis of bioactive PAF demonstrates that the molecular species produced after stimulation of EC with TNF-α, IL-1α or elastase are similar, with a predominant synthesis of the alkyl species. These results indicate that TNF-α and IL-1α stimulate the production of a serine protease which is critical in the activation of enzymes involved in PAF synthesis, suggesting the potential involvement of this mechanism in the regulation of EC functions.