Tomasiak Marian, Stelmach Halina, Rusak Tomasz, Ciborowski Michał, Radziwon Piotr
Department of Physical Chemistry, Medical University of Białystok, Białystok, Poland.
Acta Biochim Pol. 2007;54(3):625-39. Epub 2007 Jul 26.
In circulation, platelets may come into contact with both exogenous (cardiac glycoside treatment) and endogenously produced inhibitors of Na+/K(+)-ATPase. We examined whether blocking of platelet Na+/K(+)-ATPase by ouabain results in generation of procoagulant activity. It was shown that an in vitro treatment of platelets with ouabain (20-200 microM for 20 to 60 min) is associated with an intracellular accumulation of sodium (Na+), generation of a weak calcium signal, and expression of procoagulant activity. The ouabain-induced procoagulant response was dose- and time-related, less pronounced than that evoked by collagen and similar to that produced by gramicidin, not affected by EDTA or aspirin, and strongly reduced in the absence of extracellular Na+ or by hyperosmolality. Flow cytometry studies revealed that ouabain treatment results in a unimodal left shift in the forward and side scatter of the entire platelet population indicating morphological changes of the plasma membrane. The shift was dose related, weaker than that evoked by collagen and similar to that produced by gramicidin. Ouabain-treated platelets express phosphatidylserine (PS). The ouabain-evoked PS expression was dose- and time-dependent, weaker than that produced by collagen and similar to that evoked by gramicidin. Electronic cell sizing measurements showed a dose-dependent increase in mean platelet volume upon treatment with ouabain. Hypoosmotically-evoked platelet swelling resulted in the appearance of procoagulant activity. Thromboelastography measurements indicate that, in whole blood, nanomolar (50-1000 nM, 15 min) concentrations of ouabain significantly accelerate the rate of clot formation initiated by contact and high extracellular concentration of calcium. We conclude that inefficiently operating platelet Na+/K(+)-ATPase results in a rise in Na+. An increase in Na+ and the swelling associated with it may produce PS exposure and a rise in membrane curvature leading to the generation of a procoagulant activity.
在循环中，血小板可能会接触到外源性（强心苷治疗）和内源性产生的Na+/K(+)-ATP酶抑制剂。我们研究了哇巴因阻断血小板Na+/K(+)-ATP酶是否会导致促凝活性的产生。结果表明，用哇巴因（20 - 200微摩尔，处理20至60分钟）对血小板进行体外处理，会导致细胞内钠（Na+）的积累、微弱钙信号的产生以及促凝活性的表达。哇巴因诱导的促凝反应与剂量和时间相关，不如胶原蛋白引起的反应明显，与短杆菌肽产生的反应相似，不受乙二胺四乙酸（EDTA）或阿司匹林的影响，并且在没有细胞外钠或高渗状态下会大大降低。流式细胞术研究表明，哇巴因处理会导致整个血小板群体的前向散射和侧向散射出现单峰左移，表明质膜的形态发生了变化。这种移位与剂量相关，比胶原蛋白引起的移位弱，与短杆菌肽产生的移位相似。经哇巴因处理的血小板表达磷脂酰丝氨酸（PS）。哇巴因引起的PS表达与剂量和时间有关，比胶原蛋白产生的表达弱，与短杆菌肽引起的表达相似。电子细胞大小测量显示，用哇巴因处理后平均血小板体积呈剂量依赖性增加。低渗引起的血小板肿胀会导致促凝活性的出现。血栓弹力图测量表明，在全血中，纳摩尔浓度（50 - 1000纳摩尔，15分钟）的哇巴因会显著加速由接触和高细胞外钙浓度引发的凝血形成速率。我们得出结论，血小板Na+/K(+)-ATP酶功能低效会导致Na+升高。Na+的增加及其相关的肿胀可能会导致PS暴露和膜曲率增加，从而导致促凝活性的产生。
