Lipopolysaccharide treatment reduces rat platelet aggregation independent of intracellular reactive-oxygen species generation

Abstract

High production of reactive-oxygen species (ROS) by blood cells is involved in damage of the vascular endothelium and multiple organ dysfunction in sepsis. However, little is known about the intraplatelet ROS production in sepsis and its consequences on platelet reactivity. In this study, we evaluated whether the treatment of rats with lipopolysaccharide (LPS) affects platelet aggregation through intraplatelet ROS generation. Rats were injected with LPS (1 mg/kg, i.p.), and at 2 to 72 h thereafter, adenosine diphosphate (ADP) (3-10 µM) induced platelet aggregation was evaluated. Production of ROS in platelets was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA). Treatment of rats with LPS time-dependently inhibited ADP-induced platelet aggregation within 72 h. The inhibitory effect of LPS on platelet aggregation was further increased when the platelets were incubated with polyethylene glycol-superoxide dismutase (PEG-SOD; 30 U/mL), polyethylene glycol-catalase (PEG-CAT; 1000 U/mL) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI; 10 µM). The ROS production in non-stimulated platelets did not differ between control and LPS-treated rats. However, in ADP-activated platelets, generation of ROS was increased by 3.0- and 7.0-fold, as evaluated at 8 and 48 h after LPS injection, respectively. This increased ROS production was significantly reduced when platelets were incubated in vitro with DPI, PEG-SOD or PEG-CAT. In contrast, treatment of rats with N-acetylcysteine (150 mg/kg, i.p.) significantly reduced the inhibitory effect of LPS on platelet aggregation, and prevented the increased ROS production by in vivo LPS. Our results indicate that the increased intraplatelet ROS production does not contribute to the inhibitory effect of LPS on platelet aggregation; however, the maintenance of redox balance in LPS-treated rats is fundamental to restore the normal platelet response in these animals.

Low‐grade endotoxemia and platelet activation in cirrhosis

“…Regarding platelet function recent studies have negated the existence of a thrombocytopathy and demonstrated, conversely, that LC is associated with platelet hyperfunction; the underlying mechanism, however, is still to be elucidated. We speculated that, as for clotting system, low‐grade endotoxemia might represent a trigger for platelet activation as studies have demonstrated, even if not univocally, that bacterial lipopolysaccharides (LPS) triggers platelet activation . To explore this issue, we measured serum levels of LPS and two markers of platelet activation, soluble cluster of differentiation 40 ligand (sCD40L) and soluble p‐selectin (sPs), in a population with LC and in a control group.…”

“…We speculated that, as for clotting system, low-grade endotoxemia might represent a trigger for platelet activation as studies have demonstrated, even if not univocally, that bacterial lipopolysaccharides (LPS) triggers platelet activation. (10)(11)(12)(13) To explore this issue, we measured serum levels of LPS and two markers of platelet activation, soluble cluster of differentiation 40 ligand (sCD40L) and soluble p-selectin (sPs), in a population with LC and in a control group. Furthermore, we performed ex vivo study to evaluate platelet aggregation (PA) in response to subthreshold concentrations (STCs) of common agonists in platelets from cirrhosis patients and controls.…”

Signalling pathways involved in p47phox‐dependent reactive oxygen species in platelets of endotoxemic rats

“…Rats were injected intraperitoneally (ip) with saline (300 μL) or LPS (1 mg/kg), and 48 hours thereafter, arterial blood was collected, and washed platelets were obtained. This time of 48 hours post‐LPS injection was selected based on our previous experience showing a significant inhibition of ADP (10 μmol/L)‐induced platelet aggregation . Briefly, washed platelets were pre‐incubated for 15 minutes at 37°C with PP2 (inhibitor of Src family protein tyrosine kinases; 10 μmol/L), wortmannin (PI3K inhibitor; 100 nmol/L), API‐1 (Akt inhibitor; 20 μmol/L), GF109203X (PKC inhibitor; 10 μmol/L), ODQ (sGC inhibitor; 25 μmol/L), Rp‐8‐Br‐cGMPS (PKG inhibitor; 5 μmol/L) or vehicle (1% DMSO).…”

“…However, the signalling pathways that lead to intraplatelet ROS release are not completely understood. A study presented by our group showed that activated platelets of LPS‐treated rats release NADPH oxidase‐dependent ROS . Recently, we showed that PKG‐mediated inhibition of platelet aggregation by LPS treatment is modulated upstream by PKC and Akt .…”

“…Platelets are essential for primary hemostasis and take a significant part in sepsis pathophysiology . Animals treated with LPS exhibit marked thrombocytopenia and decreased platelet aggregation . Platelets express NADPH oxidase of both NOX1 and NOX2 isoforms, and generate ROS that interfere in the platelet function .…”