化合物Than40抑制血小板凝集作用及機轉之探討

Abstract

異常的血小板活化對於血栓生成是相當關鍵的一個步驟。因此，許多研究及臨床試驗指出，抗血小板製劑有益於於預防和治療栓塞性的心血管疾病 (例如:心肌梗塞及中風)。血管受傷後導致內皮下基質曝露 (例如:collagen 及vWF)，循環中血小板因而有機會和它們接觸 (即adhesion)，初始不穩定的接合(tethering)使得血小板在內皮上基質滾動 (rolling)，導致血小板表面受體有更多機會和基質接觸，而產生比較穩固的結合，經由受體下方一連串的訊息傳遞終使血小板被活化(activation)、產生凝集(aggregation)最終是血栓塊(thrombus)生成。然而，目前使用的抗血小板藥物仍有許多限制(例如會增加出血風險、有抗藥性或無法口服等)，因此開發新的抗血小板藥物有其必要性。 經由一系列的化合物篩選，我們發現化合物Than40具抑制人類血小板凝集的活性，並且在活性測試所使用濃度下，不影響細胞膜完整性。本篇論文主要在探討其抗血小板之機轉。在人類血小板懸浮液中，Than40呈濃度相關性的抑制由Collagen 及AA引起的血小板凝集，其IC50分別為6.85 ± 0.31 nM和51.05 ± 2.51 nM。此外，Than40也呈濃度相關性抑制由collagen和AA引起的P-selectin 表現量及thromboxane B2生成。在酵素活性分析方面，Than40主要呈濃度相關性抑制COX-1酵素活性(IC50為50.4nM),於10 uM下亦會減少TXS活性，但對於COX-2及PLC影響較小。在訊息傳遞方面，Than40呈時間相關性地抑制collagen引起的Fyn、Syk、Akt、ERK1/2及p38的磷酸化；然而，對PI3K、cSrc、JNK1/2/3之影響則不明顯。從酵素活性分析及訊息傳遞的結果，我們推測Than40經由抑制GP (主要是GPVI)下游的訊息傳遞分子，而干擾collagen引起的血小板活化及凝集；然而，Than40也能直接抑制COX-1酵素活性，而減少AA引起的血小板活化反應。在動物實驗中，Than40呈濃度相關性抑制collagen引起ICR小鼠富含血小板血漿(PRP)之血小板凝集 (IC50為22.48 uM)。進一步將Than40於尾靜脈注射1 ug/g，亦能明顯降低ICR小鼠的血小板凝集能力及有效抑制螢光素前處理小鼠之腸繫膜小靜脈血栓生成而不會明顯影響尾巴出血程度及血小板數。另一方面，當口服投予3 ug/g，Than40能抑制血小板凝集而不會引起急性胃部之損傷，顯示其為口服有效且安全之抗血栓藥物。綜合上述結果，Than40於體內及體外實驗皆呈現強效抑制血小板之活性，此化合物可做為抗血栓藥物之優勢化設計和研發。

It is well-known that abnormal platelet activation is a key step in thrombogenesis. Therefore, antiplatelet agents have beneficial therapeutic effects in treating or preventing cardiovascular disease (such as myocardiac infarction and stroke). Upon blood vessel injury, platelets adhere to exposed extracellular matrix rapidly, such as collagen and von Willebrand factor, triggering sequential events, including tethering and rolling, platelet activation and platelet aggregation. However, novel antiplatelet agents with less side effects are still needed. We screened a series of synthetic compounds and found that Than40, a derivative of compound 5250, possesses concentration-dependent inhibition on platelet aggregation of washed human platelets induced by collagen (IC50=6.85 ± 0.31 nM) and arachidonic acid (IC50=51.05 ± 2.51 nM). We excluded its cytotoxicity by LDH release assay In addition, Than40 inhibited P-selectin expression and TXB2 formation induced by collagen or AA in a concentraion-dependent manner. In the enzyme activity assay, we found that Than40 primarily inhibited human platelet COX-1 in a concentration-dependent manner (IC50=50.4 nM) and secondarily inhibited TXS activity (at 10 uM), whereas it had little effect on enzyme activity of COX-2 and PLC. In the western blotting assay, Than40 inhibited collagen-induced phosphorylation of Fyn, Syk, Akt, ERK1/2 and p38 in a time-dependent manner, but phosphorylations of PI3K, cSrc and JNK1/2/3 were not affected. Therefore, we suppose that the suppression of GPVI downstream signaling transduction pathway may be responsible for the inhibitory effect of Than40 on collagen-induced platelet activation and aggregation, whereas the COX-1 inhibitory activity may contribute to the attenuated AA-induced ones. On the other hand, Than40 concentraion-dependently inhibited platelet aggregation of mice PRP induced by collagen (IC50=22.48 uM), which was about 100 folds higher than human PRP. When intravenously administered to mice (1ug/g), it impaired platelet aggregation of PRP caused by collagen. Furthermore, intravenously administered Than40 (1 ug/g) inhibited thrombus occlusion of the irradiated mesenteric vessels of fluorescein-pretreated mice with little impact on tail bleeding time and platelet counts. Upon oral administration of Than40 (3 ug/g), it exerted potent antiplatelet activity whereas it did not cause gastric lesions, implying that it is an orally active and safe antithrombotic agent. Taken together, these results indicate that Than40 has potent antiplatelet activity both in vivo and ex vivo, suggesting that it may be an attractive lead compound for developing antithrombotic agents.