Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil.
Department of Clinical Pathology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil.
Thromb Res. 2018 Dec;172:44-50. doi: 10.1016/j.thromres.2018.10.008. Epub 2018 Oct 9.
Tumor necrosis factor-alpha (TNF-α) exerts a critical role in inflammatory events through two distinct receptors, TNFR1 and TNFR2. Platelets have been recognized as important inflammatory cells, but little is known about the effects of TNF-α on the platelet activity.
In the present study we have studied the role of TNF-α on ADP-induced platelet aggregation and its downstream signaling (c-Src and fibrinogen receptor phosphorylation, cytosolic Ca mobilization, cAMP and cGMP levels and cell viability).
Washed rat platelets were incubated with TNF-α (1-3000 pg/ml) for different time-periods (5-60 min) before the addition of ADP (5 μM) to induce platelet aggregation. TNF-α concentration- and time-dependently inhibits ADP-induced aggregation, which was significantly prevented by incubation with the non-selective TNF-α receptor antagonist R7050. TNF-α (300 pg/ml, 30 min) decreases thrombin-induced elevation of cytosolic Ca levels by 2.2- fold compared to untreated platelets. TNF-α decreases the cAMP levels, while significantly increases the intracellular cyclic cGMP levels. However, the pre-incubation of platelets with the guanylyl cyclase inhibitor ODQ, despite decreasing the cGMP levels, does not modify the inhibitory effect of TNF-α on ADP-induced platelet aggregation. Additionally, western blotting analysis showed that TNF-α significantly reduced (Tyr 416)-c-Src and (Tyr773)-β3 subunit of αIIbβ3 integrin phosphorylation. TNF-α does not affect the platelet viability in any condition tested.
Therefore, our results show that TNF-α negatively modulates ADP-induced aggregation via TNFR1/TNFR2 receptors by reducing cytosolic Ca levels and by inhibiting c-Src and fibrinogen receptor activation, which take place through cAMP- and cGMP-independent mechanisms.
肿瘤坏死因子-α（TNF-α）通过两个不同的受体，TNFR1 和 TNFR2，在炎症事件中发挥关键作用。血小板已被认为是重要的炎症细胞，但对 TNF-α 对血小板活性的影响知之甚少。
本研究探讨了 TNF-α 对 ADP 诱导的血小板聚集及其下游信号转导（c-Src 和纤维蛋白原受体磷酸化、细胞浆 Ca 动员、cAMP 和 cGMP 水平和细胞活力）的作用。
用 TNF-α（1-3000 pg/ml）孵育洗涤后的大鼠血小板不同时间（5-60 分钟），然后加入 ADP（5 μM）诱导血小板聚集。TNF-α 浓度和时间依赖性地抑制 ADP 诱导的聚集，这一作用可被非选择性 TNF-α 受体拮抗剂 R7050 显著阻断。与未处理的血小板相比，TNF-α（300 pg/ml，30 分钟）使凝血酶诱导的细胞浆 Ca 水平升高降低了 2.2 倍。TNF-α 降低 cAMP 水平，同时显著增加细胞内环鸟苷酸水平。然而，血小板预先孵育鸟苷酸环化酶抑制剂 ODQ，尽管降低了 cGMP 水平，但不能改变 TNF-α 对 ADP 诱导的血小板聚集的抑制作用。此外，Western blot 分析表明，TNF-α 显著降低了（Tyr416）-c-Src 和（Tyr773）-αIIbβ3 整合素的β3 亚基的磷酸化。在任何测试条件下，TNF-α 均不影响血小板活力。
因此，我们的结果表明，TNF-α 通过 TNFR1/TNFR2 受体负调节 ADP 诱导的聚集，通过降低细胞浆 Ca 水平和抑制 c-Src 和纤维蛋白原受体激活，从而发挥作用，这一过程发生在 cAMP 和 cGMP 非依赖性机制中。
