Tetramethylpyrazine 抗血小板凝集作用之機轉 探討
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。
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.
