Verhoeven A J, Gorter G, Mommersteeg M E, Akkerman J W
Biochem J. 1985 Jun 1;228(2):451-62. doi: 10.1042/bj2280451.
Among the different platelet responses, secretion requires the greatest amount of metabolic energy. The velocities of dense, alpha- and acid hydrolase granule secretion vary in parallel with the increase in energy consumption seen in thrombin-stimulated cells. This covariance is preceded by a phase in which energy consumption is increased without the extracellular appearance of secretion markers. By treating the platelets with thrombin and hirudin we have stimulated the platelets for short intervals and succeeded in separating shape change, single platelet disappearance and secretion to a great extent. In this report we show that the early increase in energy consumption reflects the energy requirement of aggregation but not of shape change. The cost of 100% of single platelet disappearance is 2.8 mumol of ATPeq. X (10(11) platelets)-1. Concurrent analysis of phosphorylation of Mr 20 000 and 47 000 proteins and of 32P-labelled phosphatidylinositol metabolites led to the following observations. Firstly, shape change is neither accompanied by an increase in protein phosphorylation nor by changes in the steady state levels of 32P-labelled phosphatidylinositol metabolites. Secondly, when aggregation occurs both proteins are phosphorylated, but the phosphatidylinositol metabolites do not change. Thirdly, when secretion follows, more phosphorylation of the Mr 47 000 protein occurs and initially only phosphatidic acid accumulates. At a later stage of the secretion responses, more protein phosphorylation and phosphatidic acid accumulation become evident, and are now accompanied by alterations in the steady state levels of 32P-labelled (poly)phosphoinositides. Hence, the early increase in energy consumption coincides with protein phosphorylation and, at a later stage, with alterations in (poly)phosphoinositides metabolites. This demonstrates that metabolic energy is directly involved in stimulus-response coupling in aggregating platelets.
在不同的血小板反应中，分泌需要消耗最多的代谢能量。致密颗粒、α颗粒和酸性水解酶颗粒的分泌速度与凝血酶刺激细胞中能量消耗的增加呈平行变化。这种协方差之前有一个阶段，即能量消耗增加，但分泌标志物未在细胞外出现。通过用凝血酶和水蛭素处理血小板，我们在短时间内刺激了血小板，并在很大程度上成功分离了形状变化、单个血小板消失和分泌。在本报告中，我们表明能量消耗的早期增加反映的是聚集而非形状变化的能量需求。100%单个血小板消失的代价是2.8 μmol ATPeq·X(10¹¹个血小板)⁻¹。对分子量为20000和47000的蛋白质磷酸化以及³²P标记的磷脂酰肌醇代谢物进行同步分析得出以下观察结果。首先，形状变化既不伴随蛋白质磷酸化增加，也不伴随³²P标记的磷脂酰肌醇代谢物稳态水平的变化。其次，当发生聚集时，两种蛋白质都会磷酸化，但磷脂酰肌醇代谢物不变。第三，当随后发生分泌时，分子量为47000的蛋白质磷酸化更多，最初仅积累磷脂酸。在分泌反应的后期，更多的蛋白质磷酸化和磷脂酸积累变得明显，并且此时伴随着³²P标记的（多）磷酸肌醇稳态水平的改变。因此，能量消耗的早期增加与蛋白质磷酸化同时发生，在后期与（多）磷酸肌醇代谢物的改变同时发生。这表明代谢能量直接参与了聚集血小板的刺激-反应偶联。
