Platelet activity is negatively modulated by tumor necrosis factor alpha through reductions of cytosolic calcium levels and integrin alphaIIbbeta3 phosphorylation.

INTRODUCTION: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. OBJECTIVES: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 Ca²⁺ mobilization, cAMP and cGMP levels and cell viability). METHODS AND RESULTS: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. CONCLUSION: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.

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