Leptin signalling and leptin-mediated activation of human platelets: Importance of JAK2 and the phospholipases Cγ2 and A₂

Summary

Leptin enhances agonist-induced platelet aggregation, and human platelets have been reported to express the leptin receptor. However, the pathways and mediators lying downstream of leptin binding to platelets remain, with few exceptions, unknown. In the present study, we sought to gain further insight into the possible role of leptin as a platelet agonist. Stimulation of platelets with leptin promoted thromboxane generation and activation of α_IIbβ₃, as demonstrated by PAC-1 binding. Furthermore, it increased the adhesion to immobilised fibrinogen (p<0.001) and induced cytoskeletal rearrangement of both platelets and Meg01 cells. Leptin time- and dose-dependently phosphorylated the intracellular signalling molecules JAK2 and STAT3, although the importance of STAT3 for leptin-induced platelet activation remains to be determined. Important intracellular mediators and pathways activated by leptin downstream of JAK2 were found to include phosphatidylinositol-3 kinase, phospholipase Cγ2 and protein kinase C, as well as the p38 MAP kinase-phospholipase A2 axis. Accordingly, incubation with the specific inhibitors AG490, Ly294002, U73122, and SB203580 prevented leptin-mediated platelet activation. These results help delineate biologically relevant leptin signalling pathways in platelets and may improve our understanding of the mechanisms linking hyperleptinaemia to the increased thrombosis risk in human obesity.

Keywords

Leptin - janus kinase - phospholipase - platelets - signal transduction

^* current address: Torrey Pines Institute for Molecular Studies, San Diego, California, USA

• References

• 1 Loskutoff DJ, Samad F. The adipocyte and hemostatic balance in obesity: studies of PAI-1. Arterioscler Thromb Vasc Biol 1998; 18: 1-6.
• 2 Matsuzawa Y, Funahashi T, Nakamura T. Molecular mechanism of metabolic syndrome X: contribution of adipocytokines adipocyte-derived bioactive substances. Ann NY Acad Sci 1999; 892: 146-154.
• 3 Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998; 395: 763-770.
• 4 Gomez-Ambrosi J, Salvador J, Silva C. et al. Increased cardiovascular risk markers in obesity are associated with body adiposity: role of leptin. Thromb Haemost 2006; 95: 991-996.
• 5 Konstantinides S, Schäfer K, Koschnick S. et al. Leptin-dependent platelet aggregation and arterial thrombosis suggests a mechanism for atherothrombotic disease in obesity. J Clin Invest 2001; 108: 1533-1540.
• 6 Bodary PF, Westrick RJ, Wickenheiser KJ. et al. Effect of leptin on arterial thrombosis following vascular injury in mice. J Am Med Assoc 2002; 287: 1706-1709.
• 7 Corsonello A, Perticone F, Malara A. et al. Leptindependent platelet aggregation in healthy, overweight and obese subjects. Int J Obes Relat Metab Disord 2003; 27: 566-573.
• 8 Nakata M, Yada T, Soejima N. et al. Leptin promotes aggregation of human platelets via the long form of its receptor. Diabetes 1999; 48: 426-429.
• 9 Bjorbaek C, Kahn BB. Leptin signalling in the central nervous system and the periphery. Recent Prog Horm Res 2004; 59: 305-331.
• 10 Elbatarny HS, Maurice DH. Leptin-mediated activation of human platelets: involvement of a leptin receptor and phosphodiesterase 3A-containing cellular signalling complex. Am J Physiol Endocrinol Metab 2005; 289: E695-E702.
• 11 Corsonello A, Malara A, De Domenico D. et al. Identifying pathways involved in leptin-dependent aggregation of human platelets. Int J Obes Relat Metab Disord 2004; 28: 979-984.
• 12 Giandomenico G, Dellas C, Czekay RP. et al. The leptin receptor system of human platelets. J Thromb Haemost 2005; 3: 1042-1049.
• 13 Kasirer-Friede A, Cozzi MR, Mazzucato M. et al. signalling through GP Ib-IX-V activates alpha IIb beta 3 independently of other receptors. Blood 2004; 103: 3403-3411.
• 14 Kramer RM, Roberts EF, Um SL. et al. p38 mitogen- activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2. J Biol Chem 1996; 271: 27723-27729.
• 15 Wolk R, Berger P, Lennon RJ. et al. Plasma leptin and prognosis in patients with established coronary atherosclerosis. J Am Coll Cardiol 2004; 44: 1819-1824.
• 16 Konstantinides S, Schäfer K, Neels JG. et al. Inhibition of endogenous leptin protects mice from arterial and venous thrombosis. Arterioscler Thromb Vasc Biol 2004; 24: 2196-2201.
• 17 Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelet paradigm. Blood 1998; 91: 2645-2657.
• 18 Leng L, Kashiwagi H, Ren XD. et al. RhoA and the function of platelet integrin alphaIIbbeta3. Blood 1998; 91: 4206-4215.
• 19 Davi G, Guagnano MT, Ciabattoni G. et al. Platelet activation in obese women: role of inflammation and oxidant stress. J Am Med Assoc 2002; 288: 2008-2014.
• 20 Bates SH, Stearns WH, Dundon TA. et al. STAT3 signalling is required for leptin regulation of energy balance but not reproduction. Nature 2003; 421: 856-859.
• 21 Ruggeri ZM. Platelets in atherothrombosis. Nat Med 2002; 8: 1227-1234.
• 22 Mangin P, Yuan Y, Goncalves I. et al. Signalling role for phospholipase C gamma 2 in platelet glycoprotein Ib alpha calcium flux and cytoskeletal reorganization. Involvement of a pathway distinct from FcR gamma chain and Fc gamma RIIA. J Biol Chem 2003; 278: 32880-32891.
• 23 Gratacap MP, Payrastre B, Viala C. et al. Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C-gamma2 is an early key event in FcgammaRIIA-mediated activation of human platelets. J Biol Chem 1998; 273: 24314-24321.
• 24 Falasca M, Logan SK, Lehto VP. et al. Activation of phospholipase C gamma by PI 3-kinase-induced PH domain-mediated membrane targeting. EMBO J 1998; 17: 414-422.
• 25 Chang YJ, Holtzman MJ, Chen CC. Differential role of Janus family kinases (JAKs) in interferongamma- induced lung epithelial ICAM-1 expression: involving protein interactions between JAKs, phospholipase Cgamma, c-Src, and STAT1. Mol Pharmacol 2004; 65: 589-598.
• 26 Saklatvala J, Rawlinson L, Waller RJ. et al. Role for p38 mitogen-activated protein kinase in platelet aggregation caused by collagen or a thromboxane analogue. J Biol Chem 1996; 271: 6586-6589.
• 27 Cao Q, Mak KM, Ren C. et al. Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAK-mediated H2O2-dependant MAPK pathways. J Biol Chem 2004; 279: 4292-4304.
• 28 Ogunwobi O, Mutungi G, Beales IL. Leptin stimulates proliferation and inhibits apoptosis in Barrett’s esophageal adenocarcinoma cells by cyclooxygenase- 2-dependent, prostaglandin-E2-mediated transactivation of the epidermal growth factor receptor and c-Jun NH2-terminal kinase activation. Endocrinology 2006; 147: 4505-4516.
• 29 Sakurai K, Matsuo Y, Sudo T. et al. Role of p38 mitogen- activated protein kinase in thrombus formation. J Recept Signal Transduct Res 2004; 24: 283-296.