Involvement of p38 MAPK Phosphorylation and Nitrate Formation in Aristolochic Acid-Mediated Antiplatelet Activity

Abstract

Aristolochic acid (AsA) is produced from Aristolochia fangchi, and has been used as a Chinese herbal medicine. AsA possesses various biological activities including antiplatelet, antifungal, and anti-inflammatory properties. The aim of this study was to examine the mechanisms of AsA in inhibiting platelet aggregation. AsA (75 – 150 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen (1 μg/mL) than other agonists. AsA (115 and 150 μM)  inhibited collagen-induced platelet activation accompanied by [Ca^+ 2]i mobilization, thromboxane A₂ (TxA₂) formation and phosphoinositide breakdown. On the other hand, AsA also markedly increased levels of NO/cyclic GMP, and cyclic GMP-induced vasodilator-stimulated phosphoprotein phosphorylation. AsA inhibited p38 MAPK but not ERK1/2 phosphorylation in washed platelets. In conclusion, the most important findings of this study suggest that the inhibitory effects of AsA possibly involve the (1) inhibition of the p38 MAPK-cytosolic phospholipase A₂-arachidonic acid-TxA₂-[Ca^+ 2]i cascade, and (2) activation of NO/cyclic GMP, resulting in inhibition of phospholipase C. These results imply that Aristolochia fangchi treatment alone or in combination with other antiplatelet drugs, may result in alteration of hemostasis in vivo.

Key words

Aristolochia fangchi - Aristolochiaceae - aristolochic acid - nitrate - p38 MAPK - phospholipase C - platelet aggregation

References

• 1 Vanherweghem J L, Depierreux M, Tielemans C, Abramowicz D, Dratwa M, Jadoul M. et al . Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs.  Lancet. 1993;  341 387-91
• 2 Rietjens I M, Martena M J, Boersma M G, Spiegelenberg W, Alink G M. Molecular mechanisms of toxicity of important food-borne phytotoxins.  Mol Nutr Food Res. 2005;  49 131-58
• 3 Schmeiser H H, Bieler C A, Wiessler M, van Ypersele de Strihou C, Cosyns J P. Detection of DNA adducts formed by aristolochic acid in renal tissue from patients with Chinese herbs nephropathy.  Cancer Res. 1996;  56 2025-8
• 4 Sheu J R, Lee C R, Lin C H, Hsiao G, Ko W C, Chen Y C. et al . Mechanisms involved in the antiplatelet activity of Staphylococcus aureus lipoteichoic acid in human platelets.  Thromb Haemost. 2000;  72 617-21
• 5 Vishwanath B S, Gowda T V. Interaction of aristolochic acid with Vipera russelli phospholipase A₂: its effect on enzymatic and pathological activities.  Toxicon. 1987;  25 929-37
• 6 Wu T S, Ou L F, Teng C M. Aristolochic acids, aristolactam alkaloids and amides from Aristolochia kankauensis.  Phytochemistry. 1994;  36 1063-8
• 7 Sheu J R, Lin C H, Peng C H, Huang T F. Triflavin, an Arg-Gly-Asp-containing peptide, inhibits the adhesion of tumor cells to matrix protein via binding to multiple integrin receptors expressed on human hepatoma cells.  Proc Soc Exp Biol Med. 1996;  213 71-9
• 8 Grynkiewicz G, Poenie M, Tsien R Y. A new generation of Ca⁺² indicator with greatly improved fluorescence properties.  J Biol Chem. 1985;  260 3440-50
• 9 Li Z, Ajdic J, Eigenthaler M, Du X. A predominant role for cAMP-dependent protein kinase in the cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein and platelet inhibition in humans.  Blood. 2003;  101 4423-9
• 10 Zhao Y, Brandish P E, DiValentin M, Schelvis J P, Babcock G T, Marletta M A. Inhibition of soluble guanylate cyclase by ODQ.  Biochemistry. 2000;  39 10 848-54
• 11 Mangin P, Yuan Y, Goncalves I, Eckly A, Freund M, Cazenave J P. et al . Signaling 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 32 880-91
• 12 McDonald L J, Murad F. Nitric oxide and cyclic GMP signaling.  Proc Soc Exp Biol Med. 1996;  211 1-6
• 13 Bugaud F, Nadal-Wollbold F, Levy-Toledano S, Rosa J P, Bryckaert M. Regulation of c-jun-NH2 terminal kinase and extracellular-signal regulated kinase in human platelets.  Blood. 1999;  94 3800-5
• 14 Coulon L, Calzada C, Moulin P, Vericel E, Lagarde M. Activation of p38 mitogen-activated protein kinase/cytosolic phospholipase A₂ cascade in hydroperoxide-stressed platelets.  Free Radic Biol Med. 2003;  35 616-25