Abstract：
Platelet concentrates (PC) are used to treat patients with thrombocytopenia and hemorrhage, but there is still the demand to find the optimal strategy for temperature-dependent storage of PC. Recently, we could show that cold storage for 1 h (short-term refrigeration) is sufficient to induce enhanced platelet responsiveness. The aim of this study was to investigate effects of cold storage on collagen-dependent activating signalling pathways in platelets from apheresis-derived PC (APC). APC on day 1 or day 2 of storage, were either continuously kept at room temperature (RT, 22 °C), or for comparison, additionally kept at cold temperature (CT, 4 °C) for 1 h. CD62P expression was determined by flow cytometry. Western Blot technique was used to analyze collagen-induced phosphorylation of p38, ERK1/2 or Akt/PKB and its inhibition by prostaglandin E1 (PGE1) or nitric monoxide donor. Adhesion of platelets on collagen-coated surfaces and intracellular phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was visualized by immune fluorescence microscopy. CD62P expression was increased after short-term refrigeration. CT exposition for 1 h induced an elevation of basal ERK1/2 phosphorylation and an alleviation of PGE1- or DEA/NO-suppressed ERK1/2 phosphorylation in APC on day 1 and 2 of storage. Similar, but more moderate effects were observable for p38 phosphorylation. Akt/PKB phosphorylation was increased only in APC on day 2. Refrigeration for 1 h promoted platelet adhesion and reduced basal VASP phosphorylation in adherent platelets. The attenuation of inhibitory signalling in short-term refrigerated stored platelets is associated with enhanced reactivity of activating signalling pathways, especially ERK1/2. Functionally, these processes correlate with increased adhesion of refrigerated platelets on collagen-coated surfaces. The results help to further optimize temperature-dependent strategies for platelet storage.
摘要：
血小板浓缩物（PC）用于治疗血小板减少症和出血的患者，但是仍然需要找到PC温度相关存储的最佳策略。最近,我们可以证明，冷藏1小时（短期冷藏）足以诱导增强的血小板反应性。这项研究的目的是研究冷藏对来自单采术衍生的PC（APC）的血小板中胶原蛋白依赖性激活信号通路的影响。APC在储存的第1天或第2天，要么连续保持在室温下(RT，22°C),或者为了比较，额外保持在低温下(CT，4°C）持续1小时。通过流式细胞术确定CD62P表达。WesternBlot技术用于分析胶原蛋白诱导的p38，ERK1/2或Akt/PKB磷酸化及其被前列腺素E1（PGE1）或一氧化氮供体的抑制作用。通过免疫荧光显微镜观察血小板在胶原包被表面上的粘附和血管扩张剂刺激的磷蛋白（VASP）的细胞内磷酸化。短期冷藏后CD62P表达增加。在储存的第1天和第2天，CT暴露1小时会导致基础ERK1/2磷酸化升高，并减轻APC中PGE1-或DEA/NO抑制的ERK1/2磷酸化。类似，但对于p38磷酸化可观察到更温和的影响。Akt/PKB磷酸化仅在第2天在APC中增加。冷藏1小时可促进血小板粘附，并降低粘附血小板的基础VASP磷酸化。短期冷藏储存血小板中抑制性信号的减弱与激活信号通路的反应性增强有关。尤其是ERK1/2。功能上,这些过程与冷冻血小板在胶原涂层表面上的粘附增加有关。结果有助于进一步优化血小板储存的温度依赖性策略。
