• blood coagulation
• cardiovascular risk factors
• leptin
• microparticles
• obesity
• tissue factor

Background: Microparticles are phospholipid vesicles shed by cells upon activation or during apoptosis. Microparticles are involved in numerous physiological processes, including coagulation and inflammation. Leptin, synthesized by adipose tissue, has been implicated in the regulation of inflammation and the pathogenesis of thrombosis, and its concentration is linked to the risk of cardiovascular events.
Aim: The aim of our study is to test the hypothesis that one of the mechanisms whereby leptin increases the risk of cardiovascular events is linked to the induction of procoagulant microparticles by human peripheral blood mononuclear cells and to investigate the intracellular mechanisms leading to microparticles release upon incubation with leptin.
Methods: Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy donors. Cells were incubated with leptin. Leptin-stimulated cells were also pre-incubated (30 minutes) with a phospholipase C inhibitor, U73122; with verapamil, L-type calcium channel current blocker; with W-7, a calmodulin inhibitor or with three different inhibitors of mitogen activated protein kinases. Microparticle generation was assessed as phosphatidylserine concentration by a prothrombinase assay, after capturing the microparticles onto annexin V-coated wells and by cytofluorimentric analysis. Peripheral blood mononuclear cell-derived microparticles were discriminated first by size, as events conforming to a light scatter distribution within the 0.5-0.9μm bead range in a side scatter channel vs. forward scatter channel window and further identified as CD14 and annexin V positive events after incubation with fluoresceinisothyocianate-annexin V and allophycocyanin-anti CD14 antibody, in anallophycocyanin vs. fluoresceinisothyocianate window. Tissue factor expression on microparticles was measured with a one-stage clotting assay. Intracellular calcium concentration was assessed by a fluorescent probe.
Results: Leptin significantly stimulates the generation of tissue factor-bearing MP by peripheral blood PBMC, as assessed by phosphatidylserine quantification and clotting tests. These results are confirmed by cytofluorimetric analysis. U73122, PD98059 (an extracellular signal-regulated kinase1/2 inhibitor), and verapamil, significantly inhibit leptin-induced MP release. SP600125 (a p38 inhibitor), SB203580 (a c-JunN-terminal kinase inhibitor), and W-7, have no effect.
Conclusions: Leptin induces the release of tissue factor bearing microparticles with a procoagulant potential by peripheral blood mononuclear cells through a mechanism that involves phospholipase C,L-type calcium channels and extracellular signal-regulated kinase1/2 activation. These data are consistent with a role of leptin-induced procoagulant microparticles shed by peripheral blood mononuclear cells in vascular diseases linked to obesity.