Abstract
Thrombocytopenia frequently occurs in septic shock patients and is associated with a worsened outcome. Experimental data indicate that platelets may have a protective role during sepsis. Our aims were to i) assess the potential protective role of platelets during septic shock and ii) evaluate the involvement of platelet P2Y1 and P2Y12 purinergic receptors.
We used a model of polymicrobial-induced septic shock in mice by cecal ligation below the ileocecal valve and double puncture (CLP). The impact of moderate to severe thrombocytopenia was evaluated using mice deficient for the thrombopoietin receptor (Mpl-R-/-, 80% reduction in platelet count) and intravenous administration of a platelet specific rat anti-GPIbα monoclonal antibody (RAM.6, 2 mg/kg, 93% reduction in platelet count), respectively. To investigate the role of the P2Y12 receptor, WT mice were treated with clopidogrel, an irreversible inhibitor of the P2Y12 receptor, administered per os at 10 mg/kg, the day before and 2 h prior to surgery. Contribution of the P2Y1 receptor was evaluated using mice deficient for the P2Y1 receptor (P2Y1-/-). Mice underwent sham or CLP surgery (6 animals/group). Twenty hours post-surgery, blood and tissue samples were collected to evaluate critical parameters of septic shock. Mortality was recorded in a second group of animals.
Twenty hours after CLP, mice with either normal or reduced platelet counts displayed signs of septic shock such as apathy, fur ruffling, conjunctivitis and diarrhea. Their mean arterial blood pressure (MAP) fell by 30% indicating systemic hypotension and organ failure characteristic of shock. Mpl-R-/- mice, displaying moderate thrombocytopenia, showed enhanced plasma concentrations of the pro-inflammatory cytokines TNF-α and IL-6, and of myeloperoxidase (MPO), indicating increased systemic inflammation relative to WT mice. These effects were more pronounced in RAM.6-treated mice displaying severe thrombocytopenia. Thrombin-antithrombin (TAT) complexes, reflecting in vivo thrombin generation, were enhanced in RAM.6-treated mice as compared to Mpl-R-/-mice or WT mice. Thrombocytopenia was also associated with increased plasma levels of aspartate aminotransferase (ASAT) relative to mice with normal platelet count, an effect also more pronounced in severely thrombocytopenic mice. These results indicate that depletion of platelets worsened organ injury during septic shock. Finally, severely thrombocytopenic mice succumbed more rapidly than control mice after CLP.
Twenty hours after CLP, clopidogrel-treated mice and P2Y1-/- mice displayed signs of shock attested by a 30% decrease in MAP, similar to that observed in WT mice. Plasma concentrations of TNF-α, IL-6 and MPO were similarly increased in WT, clopidogrel-treated and P2Y1-/- mice. In addition, no differences in TAT levels were observed between the three groups of mice. Finally, the extent of liver damage, as evaluated by measurement of plasma ASAT levels, was similar between clopidogrel-treated mice, P2Y1-/-mice and WT mice.
Overall, these results provide evidence for a beneficial role of platelets during experimental septic shock in mice. However, the platelet ADP receptors do not contribute to this effect, suggesting that anti-platelet therapy with P2Y12 receptor antagonists may not be beneficial in patients with septic shock. The mechanisms by which platelets modulate the host response to septic shock remain to be elucidated.
Disclosures
No relevant conflicts of interest to declare.
IL-1, IL-6, TNF-α, and MIF in relative adrenal insufficiency in septic shock: a piglet model