Tetramethylpyrazine 抗血小板凝集作用之機轉探討

Mechanisms involved in the antiplatelet activity of tetramethylpyrazine

甘雅珍(KAN Ya-Chen)

指導教授 ： 許準榕

臺北醫學大學/醫學院/醫學科學研究所/碩士(1998年)

摘要

Tetramethylpyrazine (TMP) 是由天然物 "川芎" 中所萃取純化之生物鹼，本篇論 文主要的研究目的，在於探討TMP抑制血小板凝集反應之作用機轉。 在人類血小板懸浮液中，TMP (0.5-1.5 mM) 隨著濃度的增加而有效地抑制由多種 活化劑 (如ADP, collagen及U46619) 所引起之血小板凝集及ATP的釋放反應。TMP 並不 會影響血小板細胞膜的流動性，即使將濃度提高到1.5 mM，也沒有明顯的差異。 此外，TMP (0.5-1.5 mM) 亦隨著劑量的增加而抑制由collagen (5 ug/ml) 所引起 [3H]inositol monophosphate的生成，而且TMP (0.5-1.5 mM) 同樣也明顯地抑制 collagen所引起細胞內鈣離子濃度的增加。再者TMP (0.5-1.5 mM) 會抑制 collagen所 引起細胞內thromboxane B2之形成。 在高濃度 (1.0 mM) 時，TMP明顯地影響FITC-triflavin與血小板表面之 glycoprotein IIb/IIIa complex的結合。Triflavin是由Trimeresurus flavoviridis 蛇毒中所純化的單鏈peptide對血小板細胞膜上的GP IIb/IIIa受體具有專一性的拮抗作 用。 在人類血小板懸浮液中，較低濃度的TMP (50, 100和200 uM) 會隨著劑量的增加而 明顯的抑制由collagen (5 ug/ml) 所引起的血小板內鈣離子的釋放，其抑制的程度分 別為45.4 %, 75.5 % 及88.3 % 。再者，TMP (50, 100和200 uM) 並不會有意義的增加 血小板細胞內cyclic AMP的濃度，卻能明顯的提高血小板細胞內cyclic GMP的濃度，這 也暗示著較低劑量的TMP是否會藉由促進血小板內nitric oxide (NO)的合成，而增加 cyclic GMP的含量；因此，我們利用chemiluminescence的方法來進一步偵測TMP對血小 板細胞內nitric oxide (NO) 的影響，實驗結果亦發現TMP (50, 100和200 uM) 明顯的 增加了血小板細胞內nitric oxide (NO) 的生成濃度。 為進一步探討TMP增加NO的生成是否藉由影響血小板細胞內之nitric oxide synthetase (NOS) 而來，利用Western blot的方法來觀察NOS蛋白的表現情形，實驗的 結果也顯示TMP (50和200 uM) 可藉由活化血小板細胞內之cNOS而促進NO的生成。 綜合以上的結果我們判定，低劑量的TMP (50, 100和200 uM) 對血小板凝集反應之 抑制的作用機轉可能經由活化血小板內的constitutive nitric oxide synthetase (cNOS)，進而促進NO的生成，接著活化guanylate cyclase，使得細胞內cyclic GMP的 濃度增加，再使得細胞內鈣離子釋放濃度降低；而在較高劑量下 (0.5-1.5 mM) 抑制血 小板凝集的作用可能經由抑制phosphoinositide breakdown和thromboxane A2之形成， 以及干擾纖維蛋白原與GP IIb/IIIa complex之結合而抑制血小板的凝集反應。

關鍵字

血小板 ； 凝集作用 ； 川芎

並列摘要

Tetramethylpyrazine (TMP) is one of the active ingredient of a Chinese herbal medicine. In this study, TMP was tested for its antiplatelet activities in human platelet suspensions. In human platelets, TMP (0.5-1.5 mM) dose-dependently inhibited both platelet aggregation and ATP-release reaction induced by a variety of agonists (i.e., ADP, collagen and U46619). TMP (0.5 mM) did not significantly change the fluorescence of platelet membranes labeled with dipheylhexatriene (DPH), even at the a higher concentration (1.5 mM). Furthermore, TMP (0.5-1.5 mM) dose-dependently inhibited [3H] inositol monophosphate formation stimulated by collagen (5 ug/ml) in [3H] myoinositol loaded platelets. In addition, TMP (0.5-1.5 mM) also dose-dependently inhibited the intracellular free Ca+2 rise of Fura 2-AM loaded platelets stimulated by collagen (5 ug/ml). Moreover, TMP (0.5-1.5 mM) inhibited thromboxane B2 formation stimulated by collagen. At a higher concentration (1.0 mM), TMP has also been shown to influence the binding of FITC-triflavin to platelet glycoprotein IIb/IIIa complex. Triflavin, is a specific glycoprotein IIb/IIIa complex antagonist, purified from Trimeresurus flavoviridis venom. Furthermore, in human platelet suspension, tetramethylpyrazine (50-200 uM) also dose-dependently inhibited the intracellular free Ca+2 rise of Fura 2-AM loaded platelets stimulated by collagen (5 ug/ml). Tetramethylpyrazine (50-200 uM) did not significantly affect the cyclic AMP levels, whereas it significantly increased the cyclic GMP levels in human platelets. Additionally, we also found that tetramethylpyrazine (50-200 uM) markedly increased nitric oxide formation by the chemiluminescence technique. By means of Western blot analysis, it was demonstrated that tetramethylpyrazine (50-200 uM) significantly affected the protein expression of NO synthase in human platelets both in dose-dependent and time-dependent manners. Based on the above observations, we suggest that (1) at a lower concentration (50-200 uM), the antiplatelet activity of tetramethylpyrazine may be directly involved in the activation of constitutive NO synthase of platelets and subsequent to induce the formation of NO, resulting in increasing the cGMP levels in human platelets. (2) At a higher concentrations (0.5-1.5 mM), the antiplatelet activity of TMP may possibly be involved in the inhibition of phosphoinositide breakdown and thromboxane A2 formation, and interuption of the glycoprotein IIb/IIIa complex in platelet surface membrane.

並列關鍵字

TMP ； platelet aggregation ； glycoprotein IIb/IIIa complex ； thromboxane A2 ； intracellular calcium