Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, 3004, Australia.
J Thromb Thrombolysis. 2019 Oct;48(3):430-438. doi: 10.1007/s11239-019-01916-8.
Upon platelet activation, inside-out signals synergistically induced by a variety of agonists and adhesion molecules can enhance the affinity of platelet main integrin, αβ to its ligands. Integrin ligation with fibrinogen induces potent signals which develop platelet function including aggregation, release and spreading of which platelet spreading is considered as a major early consequence of αβ outside-in signaling. Study presented here evaluated platelet spreading on fibrinogen matrix as a marker of platelet activation during storage. PRP-platelet concentrates were subjected to flowcytometry analysis and the expression levels of P-selectin, CD61, GPIbα and active conformation of the αβ (PAC-1 binding) were examined on day 0, 1, 3 and 5 post-storage. Concurrently platelet adhesion and spreading on fibrinogen matrix, glucose concentration and LDH activity were also determined at the same intervals. Results showed significant decreases in platelet spreading on fibrinogen matrix during storage. Spreading was dominant pattern of adhesion till the first day of storage. In 3 day-stored platelets, filopodial or lamellipodial formation was dominant event whereas 5 day-stored platelets simply adhered to fibrinogen with less protrusion formation and partially failed to spread. Compared to simple adhesion, reduction of platelet spreading was also more significantly correlated with the usual markers of platelet storage lesion including P-selectin (r = - 0.88; p < 0.0001) and GPIbα expression (r = 0.76; p = 0.0001), PAC-1 binding (r = 0.66; p = 0.001), glucose concentration and LDH activity. Taken together, we introduced platelet spreading on fibrinogen matrix as a reliable and sensitive marker of platelets functional activity during storage. As a valid marker which is directly and obviously relevant to the platelet functional capacities, the rapid reduction of platelet spreading during storage overshadows other markers of platelet storage lesion while raising serious question about the quality of 5 day-stored platelets.
在血小板激活过程中，各种激动剂和黏附分子协同诱导的内-外向信号可以增强血小板主要整合素 αβ 与配体的亲和力。整合素与纤维蛋白原的结合诱导强烈的信号，这些信号发展血小板功能，包括聚集、释放和扩展，其中血小板扩展被认为是 αβ 内-外向信号的主要早期后果。本研究评估了纤维蛋白原基质上的血小板扩展作为储存过程中血小板激活的标志物。PRP-血小板浓缩物进行流式细胞术分析，在储存后第 0、1、3 和 5 天检测 P-选择素、CD61、GPIbα 和 αβ 的活性构象（PAC-1 结合）的表达水平。同时，在相同时间间隔测定血小板在纤维蛋白原基质上的黏附与扩展、葡萄糖浓度和 LDH 活性。结果表明，储存过程中血小板在纤维蛋白原基质上的扩展显著减少。扩展是储存第一天的主要黏附模式。在 3 天储存的血小板中，丝状伪足或片状伪足形成是主要事件，而 5 天储存的血小板简单地黏附在纤维蛋白原上，突起形成较少，部分不能扩展。与简单黏附相比，血小板扩展减少与血小板储存损伤的常用标志物也具有更显著的相关性，包括 P-选择素（r=-0.88；p<0.0001）和 GPIbα 表达（r=0.76；p=0.0001）、PAC-1 结合（r=0.66；p=0.001）、葡萄糖浓度和 LDH 活性。综上所述，我们将纤维蛋白原基质上的血小板扩展作为储存过程中血小板功能活性的可靠和敏感标志物。作为与血小板功能能力直接相关且明显相关的有效标志物，血小板扩展在储存过程中的快速减少掩盖了血小板储存损伤的其他标志物，同时对 5 天储存血小板的质量提出了严重质疑。
