Department of Physical Chemistry, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland.
Thromb Res. 2013 Jan;131(1):e17-25. doi: 10.1016/j.thromres.2012.11.010. Epub 2012 Nov 30.
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) (IC(50)=100μM) and in whole blood (IC(50)=200μM) dose-dependently. In a reconstituted system (washed platelets+fibrinogen), CRR was inhibited by 5-100nM ONOO(-) (IC(50)=25nM). 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(-) (5-20nM) produced a drop of the mitochondrial transmembrane potential (ΔΨ(m)). Blocking of mitochondrial energy production resulted in a reduction of CRR, whereas inhibition of glycolysis failed to affect CRR. ONOO(-), up to 300μM, failed to affect coagulation in platelet free plasma. Fibrinolysis of platelet-fibrin clots was enhanced by ONOO(-) (25-300μM), cytochalasin B and following the reduction of platelet energy production. Fibrinolysis of plasma clots was resistant to ONOO(-) treatment up to a concentration of 500μM. Tromboelastometry (ROTEM) measurements performed in PRP show that inhibition of platelet energy production or treatment with ONOO(-) (100-300μM) 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.
我们使用猪血液来检验过氧亚硝酸盐（ONOO(-)）是否可能通过抑制血小板能量产生来影响血小板-纤维蛋白凝块的形成、凝块回缩率（CRR）和纤维蛋白溶解。结果发现，ONOO(-)浓度依赖性地降低富含血小板的血浆（PRP）（IC(50)=100μM）和全血（IC(50)=200μM）中的 CRR，并且增大最终凝块的大小。在重组系统（洗涤血小板+纤维蛋白原）中，5-100nM 的 ONOO(-)（IC(50)=25nM）抑制 CRR。PRP 中降低 CRR 的 ONOO(-)浓度抑制血小板耗氧量，增加乳酸生成并降低 PRP 来源凝块中的总 ATP 含量。在洗涤血小板中，ONOO(-)（5-20nM）导致线粒体跨膜电位（ΔΨ(m)）下降。阻断线粒体能量产生导致 CRR 降低，而抑制糖酵解未能影响 CRR。高达 300μM 的 ONOO(-)未能影响无血小板血浆中的凝血。血小板-纤维蛋白凝块的纤维蛋白溶解被 ONOO(-)（25-300μM）增强，细胞松弛素 B 也增强了纤维蛋白溶解，并且降低了血小板的能量产生。高达 500μM 的 ONOO(-)处理不影响血浆凝块的纤维蛋白溶解。在 PRP 中进行的血栓弹性测定（ROTEM）测量表明，抑制血小板能量产生或用 ONOO(-)（100-300μM）处理会降低 MCF、alpha 角和 MCE 参数。用细胞松弛素 B 阻断血小板收缩装置会导致 CRR 和 ROTEM 变量（MCF、alpha 角、MCE）降低。我们得出结论，生理相关浓度的 ONOO(-)可能通过抑制血小板线粒体能量产生来抑制凝块回缩、降低凝块稳定性并加速其溶解。
