ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?
Objective: Although recent advances in the treatment of congestive heart disease, mortality among patientsâ€™ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.
Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(Ï‰)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30ÂµM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.
Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50ÂµM (64.35Â±4.641) and 100ÂµM (52.72Â±4.192) concentration of capsaicin as compared to vehicle control (85.82Â±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53Â±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38Â±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.
Conclusion: In our study, capsaicin (50 ÂµM, 100ÂµM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.
2. National Institutes of Health: National Heart, Lung and Blood Institute. NHLBI Morbidity and Mortality Chart Book. National Heart, Lung and Blood Institute; 2009.
3. Libby P, Ridker PM, Hansson GK. Leducq transatlantic network on atherothrombosis. Inflammation in atherosclerosis: from pathophysiology to practice. J Am Coll Cardiol 2009;54:2129-38.
4. Davi G, Patrono C. Platelet activation and atherothrombosis. N Engl J Med 2007;357:2482-94.
5. Furie B, Furie BC. Mechanisms of thrombus formation. N Engl J Med 2008;359:938-49.
6. Cosemans JM, Munnix IC, Wetzker R, Heller R, Jackson SP, Heemskerk JW. Continuous signalling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization. Blood 2006;108:3045-52.
7. Jackson SP, Nesbitt WS, Kulkarni S. Signaling events underlying thrombus formation. J Thromb Haemost 2003;1:1602-12.
8. Gibbins JM. Platelet adhesion signalling and the regulation of thrombus formation. J Cell Sci 2004;117:3415-25.
9. Heusch G, Post H, Michel MC, Kelm M, Schulz R. Endogenous nitric oxide and myocardial adaptation to ischemia. Circ Res 2000;87:146-52.
10. Gewaltig MT, Kojda G. Vasoprotection by nitric oxide: mechanisms and therapeutic potential. Cardiovasc Res 2002; 55:250-60.
11. Radomski MW, Palmer RM, Moncada S. The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmacol 1987;92:639â€“46.
12. Trepakova ES, Cohen RA, Bolotina VM. Nitric oxide inhibits capacitative action influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca2+-ATPase-dependent refilling of Ca2+stores. Circ Res 1999;84:201â€“9.
13. Wang GR, Zhu Y, Halushka PV, Lincoln TM, Mendelsohn ME. Mechanism of platelet inhibition by nitric oxide: in vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase. Proc Natl Acad Sci USA 1998;95:4888-93.
14. Schlossmann J, Ammendola A, Ashman K, Zong X, Huber A, Neubauer G, et al. Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta. Nature 2000;404:197â€“201.
15. Maurice DH, Haslam RJ. Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP. Mol Pharmacol 1990;37:671-81.
16. Radomski MW, Palmer RM, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987;2:1057-8.
17. Kosuge S, Inagaki Y, Okumura H. Studies on the pungent principles of red pepper. Part 11. Determination and contents of the two pungent principles. J Agric Chem Soc 1962;36:251.
18. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;389:816-24.
19. Meller ST, Dykstra C, Gebhart GF. Production of endogenous nitric oxide and activation of soluble guanylate cyclase are required for N-methyl-D-aspartate-produced facilitation of the nociceptive tail-flick reflex. Eur J Pharmacol 1992;214:93â€“6.
20. Wang JP, Hsu MF, Teng CM. Antiplatelet effect of capsaicin. Thromb Res 1984;36:497-507.
21. Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 1999;398:436-41.
22. Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem 2007;76:387â€“17.
23. Yang D, Luo Z, Ma S, Wong WT, Ma L, Zhong J, et al. Activation of TRPV1 by dietary capsaicin improves endothelium-dependent vasorelaxation and prevents hypertension. Cell Metab 2010;12:130-41.
24. Ching LC, Kou YR, Shyue SK, Su KH, Wei J, Cheng LC, et al. Molecular mechanisms of activation of endothelial nitric oxide synthase mediated by transient receptor potential vanilloid type 1. Cardiovasc Res 2011;91:492-501.
25. Shand RA, Butler KD, Davies JA, Menys VC, Wallis RB. The kinetics of platelet and fibrin deposition on to damaged rabbit carotid arteries in vivo: involvement of platelets in the initial deposition of fibrin. Thromb Res 1987;45:505â€“15.
26. Wang JP, Hsu MF, Hsu TP, Teng CM. Antihemostaticand antithrombotic effects of capsaicin in comparison with aspirin and indomethacin. Thromb Res 1985;37:669â€“79.
27. Eguchi Y, Takahari Y, Higashijima N, Ishizuka N, Tamura N, Kawamura Y, et al. Nicorandil attenuates FeCl-induced thrombus formation through the inhibition of reactive oxygen species production. Circ J 2009;73:554-61.
28. Mittelstadt SW, Nelson RA, Daanen JF, King AJ, Kort ME, Kym PR, et al. Capsaicin-induced inhibition of platelet aggregation is not mediated by transient receptor potential vanilloid type 1. Blood Coagul Fibrinolysis 2012;23:94-7.