1-(5-Methyl-2-furyl)-3-phenyl-2-propen-1-one 抑制血小板凝集作用機轉之探討

Abstract

本篇論文主要探討化合物p95wu33a 抑制人類血小板凝集的作用 機轉。在人類血小板懸浮液中，p95wu33a 呈濃度相關性的抑制由 collagen ( 10μg/ml ), AA ( 200μM ), U46619 ( 1μM ) 和thrombin(0.1U/ml) 引起的血小板凝集，其IC50 分別為0.23±0.02, 0.41±0.05,13.38±0.28 and 285.12±3.39 μM。p95wu33a 也會抑制由collagen 引起的P-selectin 表現量及thomboxane B2 生成，對於蛋白質tyrosine phosphorylation 訊息傳遞方面，在PLCγ2、PI3K、Syk、Src、LAT 這些位置都有抑制磷酸化的情形。Fibrinogen 結合到活化的醣蛋白受體GPIIb-IIIa 是血小板凝集的最終反應路徑。然而p95wu33a 並不影響fibrinogen 結合到活化態的GPIIb-IIIa。除此之外，p95wu33a 能夠顯著的加強sodium nitroprusside ( SNP )的抑制血小板凝集能力。同時也證 實了p95wu33a 能獨自顯著提升血小板內的cGMP 含量，同時加強SNP 提升胞內cGMP 的能力。在ODQ 的存在下，p95wu33a 對collagen 引 起的血小板凝集抑制作用有加強的現象。p95wu33a 能抑制由AA 和 collagen 引起的thromboxan B2 生成，對cPLA2 活性沒有直接的影響。顯示其作用非來自對內生性c P L A 2 的活性抑制， 但有可能對arachidonate-thromboxane A2 生合成路徑上的enzyme 有抑制的能力。在血小板內鈣離子濃度變化的實驗中，我們發現p95wu33a 呈濃度相關性抑制collagen 引起的Ca2+ mobilization。除此之外，在血小板附著實驗中，p95wu33a 也能以濃度相關性的抑制血小板附著到collagen 上。在動物實驗中，p95wu33a 在以10μg/g 尾靜脈注射時,能顯著的降低ICR小鼠的血小板凝集能力。此外在相同劑量下， 對於尾巴出血程度只有些微增加。综合上述結果，p95wu33a 這種具有PDE5 抑制劑和sGC 活化劑之化合物可做為抗血栓藥物之優勢化設計和研發。

In the present study, the mechanism of antiplatelet activity of p95wu33a, a synthetic 1-( 5-methyl-2furyl )-3-phenyl-2-propen-1-one, was investigated. p95wu33a concentration-dependently inhibited platelet aggregation stimulated by collagen ( 10μg/ml ), AA ( 200μM ), U46619( 1μM ) and thrombin (0.1U/ml) with IC50 values of 0.23±0.02, 0.41±0.05,13.38±0.28 and 285.12±3.39 μM in washed human platelets, respectively. p95wu33a also inhibited collagen-induced P-selectin expression and thromboxane B2 formation. In addition, p95wu33a attenuated tyrosine phosphorylation of a number of signal proteins including PLCγ2、PI3K、Syk、Src、and LAT. Fibrinogen binding to activated glycoprotein GPIIb-IIIa is the final common pathway of platelet aggregation. However, p95wu33a did not affect fibrinogen binding to activated GPIIb-IIIa. This suggests that the inhibitory effect of p95wu33a is not through acting as a direct antagonist of GPIIb-IIIa. Sodium nitroprusside (SNP) markedly potentiated the platelet-inhibitory effect of p95wu33a. It was demonstrated that p95wu33a itself markedly increased guanosine 3’,5’-cyclic monophosphate (cGMP) level and Sodium nitroprusside potentiated the elevated cGMP by p95wu33a. The inhibitory effect of p95wu33a on collagen-induced platelet aggregation and cGMP content was markly enhanced in the presence of ODQ, an inhibitor of sGC. p95wu33a inhibited the biosynthesis of thromboxan B2 induced by arachidonic acid and collagen. However, it had no inhibitory on cytosolic phospholipase A2, suggesting that it dose not affect the endogenous cPLA2 activity, but impairs arachidonate - thromboxane A2 biosynthesis pathway. In Fura-2 loaded platelets, p95wu33a inhibited intracellular calcium mobilization triggered by collagen. Similar to other nucleotide-elevating agent, p95wu33a dose-dependently inhibited platelet adhesion to collagen. p95wu33a significantly reduced the ex vivo platelet aggregation of PRP in ICR mice, as it was intravenously administered at a dose of 10μg/g, whereas p95wu33a at the same dose cause a slight prolongation of tailing bleeding time. In conclusion, the promising antithrombotic profile of p95wu33a, PDE5 inhibitor and sGC activator, suggests that it is an attractive lead compound for developing antiplatelet agents.