| 摘要: | Phospholipase D (PLD)是phospholipase superfamily 中的一種酵素酶(enzyme) 。PLD 能夠水解phosphatidylcholine (PC) ， 產生phosphatidic acid (PA)和choline。其中PA 為二級傳訊因子(second messenger)，在細胞中被認為是PLD 的主要功能，許多報導已指出 PLD 參與許多生物過程和疾病，如癌症(cancer)、免疫(immunity) 和 阿茲海默症(Alzheimer''s disease)；另外，PLD 在小鼠血小板活化中亦扮演調節的角色，然而，PLD 對人類血小板活化的作用仍不清楚。因此，在本論文我們將研究PLD 是否參與在人體血小板活化和血栓形成。
在本篇研究中，我們研究數據顯示PLD1 抑制劑VU0155069
(VU1，10-25 μM)和PLD2 抑制劑VU0364739 (VU2，10-25 μM)都有
效地抑制各種刺激劑所誘導的人類血小板的凝集，VU1 和VU2 亦會
抑制由thrombin 所誘導的P-selectin 表現和ATP 釋放；而且，VU1 和VU2 能顯著地抑制由thrombin 誘導的p38 MAPK、ERK、JNK 和Akt
的蛋白質磷酸化。另外，由我們研究數據顯示，VU1 和VU2 能顯著
抑制clot retraction、platelet spreading 和adhesion；除此之外，使用一個模擬體內血栓形成的血小板功能分析儀來做檢測，發現同時給予PLD1 和PLD2 抑制劑可以協同地延長阻塞時間，這些結果顯示PLD1和PLD2 對於人類血小板活化是必需的。有趣地，由上述人類血小板所獲得的結果，不同於先前研究的小鼠血小板，這也暗示PLD 可能在人類和小鼠所扮演的角色是有差異的，類似於小鼠先前研究，我們動物實驗結果顯示，PLD1 能顯著地減緩ADP 所誘導的小鼠急性肺血栓形成和中大腦動脈血管阻塞/再灌流模式所誘導的腦損傷；此外，PLD1 和PLD2 這兩個抑制劑皆不會造成出血的副作用。
總結以上結果，本研究首次證實PLD1 和PLD2 兩者能協同地調控人類血小板活化和血栓形成，並得知PLD 在人類和小鼠所扮演的角色是有差異的；此外，我們的研究結果顯示PLD 可以當作一個安全的預防血栓相關疾病的治療標的。
Phospholipase D (PLD) is an enzyme belonging to the phospholipase superfamily. PLD is able to hydrolyze phosphatidylcholine (PC) to produce phosphatidic acid (PA) and choline. PA is the precursor of the second messenger and considered to be the main effector of PLDs function in cells. PLDs have been reported to be involved in many biological processes and diseases including cancer, immunity, and Alzheimer''s disease. PLD also plays a crucial role in regulating platelet activity in mice; however, the
effect of PLD on human platelet activation remains unclear. Thus, this study investigated whether PLD is involved in human platelet activation and thrombus formation.
In this study, our data revealed that the PLD1 inhibitor VU0155069 (VU1, 10-25 μM) and PLD2 inhibitor VU0364739 (VU2, 10-25 μM)inhibited various agonists-induced platelet aggregation in human washed platelets. VU1 and VU2 also inhibited thrombin-induced P-selectin expression and ATP release. Moreover, VU1 and VU2 significantly inhibited the phosphorylation of ERK, JNK, p38 MAPKs, and Akt. In addition, our data revealed that VU1 and VU2 markedly inhibited clot retraction and reduced platelet spreading and adhesion on immobilized fibrinogen. Concurrent inhibition of PLD1 and PLD2 can also synergistically prolong closure time, as detected using platelet function analyzer-100, in a model mimicking in vivo thrombosis, indicating that both PLD1 and PLD2 are essential for human platelet activation. These results obtained for human platelets were different from those previously obtained for mouse platelets, indicating that PLD may have differential in humans and mice. Similar to previous studies in mice, our results revealed that PLD1, but not PLD2, inhibition, markedly alleviate ADPinduced acute pulmonary thrombosis and middle cerebral artery occlusion/reperfusion-induced brain injury in mice. Moreover, both PLD1 and PLD2 inhibition did not exhibit side effect of bleeding.
Taken together, this study for the first time demonstrated that both PLD1 and PLD2 are synergistically involved in platelet activation and thrombus formation in human platelets and that PLD plays differential roles in humans and mice. In addition, our findings indicate that targeting
PLD may provide a safe and alternative therapeutic approach to prevent thromboembolic disorders such as secondary stroke. |
