Identifying pathways involved in leptin-dependent aggregation of human platelets

OBJECTIVE: To investigate the role of phospholipase C ( PLC), phospholipase A(2) (PLA(2)), calcium, and protein kinase C (PKC) in mediating leptin-enhanced aggregation of human platelets. DESIGN: In vitro, ex vivo study. SETTING: Outpatient's Service for Prevention and Treatment of Obesity at the University Hospital of Messina, Italy. SUBJECTS: In total, 14 healthy normal-weight male ( age 31.4 +/- 1.9 y; body mass index 22.7 +/- 0.6 kg/m(2)) subjects. MEASUREMENTS: Adenosine diphosphate-(ADP-) induced platelet aggregation and platelet free calcium were measured after incubation of platelets with leptin alone ( 5 - 500 ng/ml), or leptin ( 50 and 100 ng/ml) in combination with anti-human leptin receptor long form antibody (anti-ObRb-Ab, 1: 800 - 1: 100 dilutions), PLC inhibitor U73122 (3.125 - 25 muM), PLA(2) inhibitor AACOCF3 (1.25 - 10 muM), or PKC inhibitor Ro31- 8220 (1.25 - 10 muM). RESULTS: Platelet stimulation with leptin leads to a significant and dose-dependent increase in ADP- induced platelet aggregation and platelet free calcium concentrations. Leptin effects on both platelet aggregation and calcium mobilization were completely abated by the co-incubation with leptin and anti-ObRb-Ab. Leptin-induced platelet aggregation was dose-dependently inhibited by U73122, AACOCF3, or Ro31-8220. The effect of leptin on intracellular calcium was inhibited in a dose-dependent manner by incubation with U73122 and AACOCF3, but not with Ro31-8220. CONCLUSIONS: Our study confirms that leptin is able to enhance ADP- induced aggregation of human platelets, and raise the possibility that PLC, PKC, PLA(2), and calcium could play a relevant role in mediating the proaggregating action of leptin.