Peroxynitrite - altered platelet mitochondria-A new link between inflammation and hemostasis

Using porcine blood, we tested the hypothesis that peroxynitrite (ONOO−) may affect platelet-fibrin clot formation, clot retraction rate (CRR) and fibrinolysis through the inhibition of platelet energy production. It was found that ONOO− reduces CRR and enlarges final clot size in platelet rich plasma (PRP) (IC50 = 100 μM) and in whole blood (IC50 = 200 μM) dose-dependently. In a reconstituted system (washed platelets + fibrinogen), CRR was inhibited by 5-100 nM ONOO− (IC50 = 25 nM). Concentrations of ONOO− reducing CRR in PRP, inhibited platelet oxygen consumption, augmented lactate production and decreased total ATP contents in clots derived from PRP. In washed platelets ONOO− (5-20 nM) produced a drop of the mitochondrial transmembrane potential (ΔΨm). Blocking of mitochondrial energy production resulted in a reduction of CRR, whereas inhibition of glycolysis failed to affect CRR. ONOO−, up to 300 μM, failed to affect coagulation in platelet free plasma. Fibrinolysis of platelet-fibrin clots was enhanced by ONOO− (25-300 μM), cytochalasin B and following the reduction of platelet energy production. Fibrinolysis of plasma clots was resistant to ONOO− treatment up to a concentration of 500 μM. Tromboelastometry (ROTEM) measurements performed in PRP show that inhibition of platelet energy production or treatment with ONOO− (100-300 μM) diminishes MCF, alpha angle and MCE parameters. Blockage the platelet contractile apparatus by cytochalasin B resulted in reduction of CRR and ROTEM variables (MCF, alpha angle, MCE). We conclude that physiologically relevant ONOO− concentrations may inhibit clot retraction, reduce clot stability and accelerate its lysis through the inhibition of platelet mitochondrial energy production.