化合物PPA45之拮抗血小板凝集作用及機轉之探討

Abstract

動脈粥狀硬化栓塞(Atherothrombosis)是一種血管系統慢性發展之疾病，在臨床上 其表現不明顯，也不容易被發現，然而當其發展到一定的複雜程度時，會變成有 危害生命危險的疾病如急性冠狀動脈疾病，中風和暫時性腦缺血發作。其中不正 常的血小板活化是形成栓塞的重要原因之一。在經過一系列化合物NP-313 衍生 物的篩選，在人類血小板懸浮液( washed human platelet suspension )，我們發現化 合物PPA45 呈現濃度相關性抑制由U46619；collagen；arachidonic acid；thrombin 和thapsigargin 所引發之血小板凝集，其IC50 分別為1.37 ± 0.13，1.72 ± 0.11， 1.59 ± 0.16，3.21 ± 0.37 和21.41 ± 1.93 μM，PPA45 也呈濃度相關性抑制由上述 活化劑所誘導人類血小板之P-selectin 表現。雖然PPA45 會抑制collagen 刺激人 類血小板TXA2 的產生，但卻不會影響由thrombin 和arachidonic acid 刺激所產生 之TXA2。在鈣離子的訊息傳遞方面，PPA45 可以抑制collagen 和thrombin 所刺 激人類血小板而引發的鈣離子的濃度上升的作用，但卻不影響thapsigargin 所引起的游離鈣離子之濃度。在EGTA 存在下，我們也發現PPA45 不會影響collagen和thrombin 刺激而導致血小板內儲存鈣離子之釋放，然而PPA45 可以抑制由PMA 和OAG 所導致的人類血小板凝集，和抑制OAG 刺激所增加的人類血小板鈣離子濃度作用。這些結果顯現PPA45 可能透過DAG 路徑的訊息分子如PKC來抑制血小板的凝集，而藉由和SOCC 不相關的鈣離子通道如TRPC6 來抑制鈣離子的入流。至於PPA45 在訊息傳遞方面的作用，PPA45 會抑制collagen 所導 致的FAK，Akt，PKC，Fyn，Syk，PLCγ2，p38 和ERK 訊息分子活化，但是卻不影響c-Src 和JNK 活化情形。此外，PPA45 也會抑制thrombin 刺激的FAK, Fyn, p38 and ERK 的磷酸化表現情況，但一樣不影響c-Src 和JNK 的磷酸化表現。然而PPA45 在動物體內試驗卻沒有表現明顯的抗血栓活性，這可能與PPA45 在體內的藥效學和藥物動力學有關。綜之，我們證明PPA45 可透過抑制PKC 和多種kinases 所引起的訊息傳遞作用，而抑制血小板凝集的作用，若經過適當的結 構修飾將可成為潛力抗血小板的化合物。

Atherothrombosis is a progressive chronic disease in vascular system, and is always clinically silent, whereas it becomes complicated by thrombous formation causing life-threatening events including acute coronary syndrome, stroke, and transient ischaemic attack. The abnormality of platelet activation has a crucial role in thrombosis. After screening several derivatives of NP-313, PPA45 displays the inhibitory effect on U46619-, collagen-, arachidonic acid-, thrombin- and thapsigargin-induced platelet aggregation in a concentraction-dependent manner, with the IC50 values : 1.37 ± 0.13, 1.72 ± 0.11, 1.59 ± 0.16, 3.21 ± 0.37, and 21.41 ± 1.93 μM, respectively. PP45 concentration-dependently inhibited P-selectin expression of platelet activated by these agonists. PPA45 inhibited the formation of TXA2 induced by collagen, but not by thrombin and arachidonic acid. Regarding Ca+2 signaling,PPA45 inhibited Ca+2 mobilization caused by collagen and thrombin, but not that by thapsigargin. We also found that PPA45 did not influence collagen- and thrombin-induced Ca+2 release from internal stores of platelets in the presence of EGTA. However, PPA45 shows inhibitory effect on PMA- and OAG-induced platelet aggregation, and OAG-induced Ca+2 mobilization. This implies that PPA45 inhibited platelet aggregation via downstream target of diacylglycerol ( DAG ) like PKC and affected Ca+2 mobilization through a SOCC-independent channel, TRPC6. Regarding the signaling molecules involved in its mechanism of action, PPA45 inhibited activation of FAK, Akt, PKCα, Fyn, Syk, PLCγ2, p38 and ERK but not c-Src and JNK by collagen. Similarly, PPA45 also inhibited thrombin-induced p-FAK, p-Fyn, p38 and p-ERK expression, except p-JNK and p-c-Src. However, PPA45 does not exhibit marked effect ex vivo and in vivo, and this may be caused by pharmcodynamics and pharmocokinetic factors. In addition, we demonstrated that PPA45 possesses inhibitory effect on the signaling target PKC and the upstream molecules such as Fyn, Syk, MAPKs and PLCγ2, leading to inhibition of platelet aggregation.Taken together, PPA45 has potential as an antithrombotic agent under further optimization of its structure through structure-activity relationship study.