Aristolochic Acid抑制血小板凝集作用之機轉探討

1. Aristolochic Acid抑制血小板凝集作用之機轉探討 馬兜鈴酸是一種來自馬兜鈴科(Aristolochiaceae)的生物鹼，已被證實有致癌性，且長期使用下將造成腎衰竭。最近的研究報告指出馬兜鈴酸可以抑制由蛇毒所引起的水腫現象、抗發炎，且有抑制血小板的活性的功效。然而，馬兜鈴酸在血小板上的藥理學功效尚未明確，因此我們有意探討馬兜鈴酸在血小板活化過程中，對於訊息傳遞方面的抑制機轉。由本研究結果顯示，馬兜鈴酸隨著濃度的增加(75-150 micromolar)，能有效地抑制collagen (1 microgram/ml)所引起的人類血小板凝集反應以及ATP釋放反應；且馬兜鈴酸(115和150 micromolar)可以抑制由collagen所刺激細胞內鈣離子的流動、phosphoinositide的增加和thromboxane A2的形成。此外，馬兜鈴酸(115和150 micromolar)可以增加細胞內nitrate的含量及vasodilator-stimulated phosphoprotein (VASP)的磷酸化。對於血小板內47 kDa蛋白質磷酸化，這是一個標記protein kinase C活性的方法。在本實驗中我們分別使用collagen (1 microgram/ml)和PDBu (150 nano molar)促進血小板47 kDa蛋白質磷酸化，發現馬兜鈴酸只能抑制由collagen所活化47 kDa蛋白質磷酸化。另外，馬兜鈴酸(115和150 micromolar)可以抑制由collagen (10 microgram/ml)所引起p38 MAPK的磷酸化反應但不能清除由collagen (1 microgram/ml)刺激血小板所導致的自由基。由結果證實，馬兜鈴酸抑制血小板活性的作用可能涉及下列路徑：(一)馬兜鈴酸可以抑制PLC的活性，接著進一步抑制phosphoinositide breakdown、鈣離子的流動、以及47 kDa 蛋白質的磷酸化(二)馬兜鈴酸可經由抑制p38 MAPK磷酸化來調控phospholipase A2的活性而使TXA2的含量減少而抑制血小板的活化。 (三)馬兜鈴酸會影響eNOS的活性，增加血小板細胞內NO的產生，可能進一步影響guanylate cyclase的活性，增加cyclic GMP的含量以誘發VASP磷酸化而抑制血小板的活化

2. Mechanisms Involved in the Antiplatelet Activity of Aristolochic Acid • Aristolochic acid (AsA) is an alkaloid from the plant Aristolochiaceae. The naturally occurring herbal toxin that can cause cancer and end-stage kidney failure. Recently, it had been reported that AsA could reduce edema induced by snake venom and possess anti-inflammation and anti-platelet activity of AsA. However, the mechanisms involved in anti-platelet activity of AsA is still unclear, and we are interested in investigating the effects on cellular signal transduction during the process of platelet activation. In this study, AsA concentration-dependently (75-150 micromolar) inhibited collagen (1 microgram/ml) induced human platelet aggregation and ATP release reaction. AsA (115 and 150 micromolar) inhibited intracellular Ca2+ mobilization, phosphoinositide breakdown, and thromboxane A2 formation stimulated by collagen (1 microgram/ml) in human platelets. In addition, AsA (115 and 150 micromolar) increased levels of nitrate and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Phosphorylation of 47 kDa proteins is a marker of protein kinase C activation, and can be triggered by collagen (1 microgram/ml) and PDBu (150 nano molar). In our experiments, we found AsA (115 and 150 micromolar) could inhibit phosphorylation of 47 kDa proteins by collagen (1 microgram/ml). Besides, AsA (115 and 150 micromolar) reduced p38 MAPK phosphorylation induced by collagen (10 microgram/ml), and had no effects on scavenging collagen (1 micro gram/ml)-induced hydroxyl radicals in platelets. In conclusion, our study suggested that the pathways of AsA (115 and 150 micromolar) about anti-platelet activity maybe involved the following: (1) AsA could regulate the activity of PLC and then inhibit phosphoinositide breakdown, intracellular Ca2+ mobilization and 47 kDa protein phosphorylation. (2) AsA significantly reduced thromboxane A2 formation maybe through inhibition of p38 MAPK phosphorylation and Ca2+ mobilization, which are responsibe for PLA2 activation. (3) AsA inhibted the activity of platelet by increasing the amount of NO and VASP phosphorylation .