Stilbene衍生物之抗血小板作用及其機轉探討

The antiplatelet effect and mechanism of action of a stilbene derivative

江怡臻(Yi-Chun Chiang)

指導教授 ： 吳志中

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2016年)

摘要

血小板的活化在止血功能及動脈血栓形成扮演關鍵的角色。當內皮細胞受損露出內皮下基質，血小板會貼附其上進而活化並產生凝集反應，最終形成血栓達到止血的作用。但是在病理狀態下，過度的活化反而會造成血管栓塞。阻斷致效劑位於血小板膜上的受體而抑制血小板活化是抗血栓疾病的治療方法之一。
Thrombin為一強效血小板致效劑。Thrombin活化血小板透過作用在其受體：PAR1和PAR4。PAR1參與thrombin引起初始血小板凝集而達到止血作用，PAR4的活化則對於後期血栓穩固形成血栓較重要。選擇性抑制PAR4受體能減少因抑制PAR1活化訊號路徑而造成出血的副作用。因此本實驗的目的為自天然化合物或其化學衍生物中尋找新型PAR4拮抗劑。
在本實驗中發現一stilbene衍生物，compound 1，具有抑制PAR4-AP誘導引起的血小板凝集作用 ( IC50 = 2.5 ± 0.4 M)。對於其他血小板致效劑 (PAR1-AP，U46619，collagen) 則無顯著抑制作用。為了確認compound 1是否為選擇性PAR4拮抗劑，進一步探討其對於血小板顆粒釋放、細胞內鈣離子濃度增加以及下游訊號傳遞的影響。Compound 1能降低PAR4-AP誘導但不影響PAR1-AP誘導之血小板ATP釋放及細胞內鈣離子濃度上升。Compound 1抑制PAR4-AP引起但不影響AA引起TxB2和12-HETE合成。對於血小板下游訊號因子的活化，compound 1可選擇性抑制PAR4-AP刺激引起的p47、Akt、p38 MAPK及ERK磷酸化。在實驗中也發現compound 1合併PAR1或P2Y12受體拮抗劑具有加成抑制thrombin引起的血小板凝集作用。
綜合上述的實驗結果，compound 1可做為先導化合物進一步發展新型抗血小板藥物，可能對於預防及治療血栓疾病能有所助益。

關鍵字

血小板 ； PAR4 ； PAR4抑制劑 ； 凝血酶

並列摘要

Activation of platelets is crucial in haemostasis and arterial thrombosis. When endothelial injury occurs, platelets adhere to the subendothelial matrix and then aggregate with each other to form a platelet plug. However, platelets also cause thrombotic disorders under pathological conditions. Blockage of agonist receptors on platelet surface such as thrombin receptors, ADP receptors or GP IIb/IIIa will be new antithrombotic therapies.
Thrombin is a potent agonist that can induce human platelet activation via PAR1 and PAR4. PAR1 participates in the initial platelet aggregation in response to thrombin, while PAR4 is associated with the stability of platelet aggregation. Selective inhibition of PAR4 may not influence the PAR1 signaling and thereby decrease the unwanted bleeding or other complications. Therefore, our purpose is to find a novel PAR4 selective inhibitor.
In the present study, we found a stilbene derivative, compound 1, could inhibit platelet aggregation induced by PAR4-activating peptide (IC50 = 2.5 ± 0.4 M), but had no or less inhibitory effects on which induced by PAR1-activating peptide, U46619 or collagen. To further investigate if compound 1 is a selective PAR4 inhibitor, we examined the effect of compound 1 on granule secretion, intracellular Ca2+ mobilization and signaling transduction caused by PAR4 or other stimulators. Compound 1 inhibited PAR4-AP-, but not PAR1-AP-, induced ATP secretion and intracellular Ca2+ mobilization in human platelets. Compound 1 inhibited PAR4-AP-, but not AA-induced TXB2 and 12-HETE formation in human platelets. Compound 1 also selectively suppressed phosphorylation of p47, Akt, p38 MAPK, and ERK stimulated by PAR4-activating peptide in a dose-dependent manner. Compound 1 additionally inhibited thrombin-induced platelet aggregation with either a PAR1 antagonist or a P2Y12 receptor antagonist.
Taken together, our results suggest that compound 1 is a potential lead for the development of new antiplatelet drugs. These effects may be use to prevent and treat thrombotic disorders.

並列關鍵字

Platelets ； PAR4 ； PAR4 inhibitors ； Thrombin

參考文獻

Ablooglu AJ, Kang J, Petrich BG, Ginsberg MH, Shattil SJ (2009). Antithrombotic effects of targeting alphaIIbbeta3 signaling in platelets. Blood 113(15): 3585-3592.

Adams RJ, Albers G, Alberts MJ, Benavente O, Furie K, Goldstein LB, et al. (2008). Update to the AHA/ASA recommendations for the prevention of stroke in patients with stroke and transient ischemic attack. Stroke; a journal of cerebral circulation 39(5): 1647-1652.

Ahn KS, Kim JH, Oh SR, Ryu SY, Lee HK (2000). Inhibitory activity of stilbenes from medicinal plants on the expression of cell adhesion molecules on THP1 cells. Planta medica 66(7): 641-644.

Aloui C, Prigent A, Sut C, Tariket S, Hamzeh-Cognasse H, Pozzetto B, et al. (2014). The signaling role of CD40 ligand in platelet biology and in platelet component transfusion. International journal of molecular sciences 15(12): 22342-22364.

Ammar T, Scudder LE, Coller BS (1997). In vitro effects of the platelet glycoprotein IIb/IIIa receptor antagonist c7E3 Fab on the activated clotting time. Circulation 95(3): 614-617.