Tumor necrosis factor alpha has a crucial role in increased reactive oxygen species production in platelets of mice injected with lipopolysaccharide.

Increased reactive oxygen species (ROS) production leads to tissue damage observed in sepsis and lipopolysaccharide (LPS)-exposed animals. LPS stimulates cytokines releasing, including tumor necrosis factor alpha (TNF-α), that is important to ROS production. Platelets, considered inflammatory cells, generate ROS when exposed to LPS in vivo, but not when they are incubated in vitro with this compound. Therefore, we investigated the role of TNF-α on the increased intraplatelet ROS levels after LPS treatment. Mice were injected with LPS (1 mg/kg) or TNF-α (10 ng/kg), and blood was collected to prepare the washed platelets. Animals were treated with infliximab (anti-TNF-α antibody), R-7050 (non-selective TNF-α receptor antagonist) or apocynin (NADPH oxidase inhibitor). At 48 h after LPS or TNF-α injection, the ROS levels in ADP (25 µM)-activated platelets were evaluated by flow cytometry. Our data showed that injection of mice with LPS increased by 4-fold the ROS production (p < 0.05), which was significantly reduced by the treatments with infliximab, R-7050 or apocynin. Injection of mice with TNF-α markedly elevated the ROS formation in platelets (p < 0.05) that was reduced by infliximab, R-7050 or apocynin treatments. In separate experiments, platelets from saline-injected mice were incubated with TNF-α (30 to 3000 pg/mL) in absence or presence of infliximab, R-7050, apocynin or GKT137831 (NOX1/NOX4 inhibitor) before ROS measurements. TNF-α in vitro markedly increased the ROS levels, an effect significantly reduced by all treatments. Therefore, platelets are involved in the oxidative stress induced by LPS through TNF-α action, and NADPH oxidase takes part in this effect.

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)-ß3 subunit of αIIbß3 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.

Cross-talking between lymphocytes and platelets and its regulation by nitric oxide and peroxynitrite in physiological condition and endotoxemia.

AIMS: Cross-talk between platelets and lymphocytes may play a role in different pathological conditions like sepsis. This study aimed to investigate the effect of lymphocytes on platelet aggregation in lipopolysaccharide (LPS)-stimulated and non-stimulated cells. MAIN METHODS: Lymphocytes and platelet-rich plasma (PRP) were obtained from rat arterial blood. Platelets (1.2×108platelets/ml) were incubated with lymphocytes (0.8×106cells/ml) in the presence or not of LPS (100µg/ml), after which ADP (5µM)-induced platelet aggregation was carried out. KEY FINDINGS: Lymphocytes inhibited by 51% the platelet aggregation, which was significantly prevented by the non-selective NO inhibitor l-NAME (300µM) or the selective iNOS inhibitor 1400W (100µM), as well as by the soluble guanylyl cyclase (sGC) inhibitor ODQ (10µM). The platelet inhibition by lymphocytes was accompanied by 2-fold increase of intraplatelet cGMP levels. Next, lymphocytes and platelets were co-incubated with LPS for 6h. In LPS-treated cells, lymphocytes produced a larger inhibition of platelet aggregation (62%), despite the same elevation of cGMP levels (2.2-fold increase). This inhibitory effect was prevented by l-NAME and 1400W, but rather unaffected by ODQ. The peroxynitrite (ONOO-) scavenger -(-)epigallocatechin gallate (ECG, 100µM) abolished the inhibition by lymphocytes on platelet aggregation in LPS-treated cells, but not in non-treated cells. SIGNIFICANCE: Our results show that lymphocytes act to inhibit platelet aggregation via iNOS-derived NO release and cGMP generation. In presence of LPS, ONOO- production accounts for the platelet inhibition.