Hemostasis and Platelet Research Laboratory, Division of Hematology and Central Hematology Laboratory, Lausanne University Hospital (CHUV) and University of Lausanne (UNIL), Lausanne, Switzerland.
Thromb Haemost. 2021 Mar;121(3):309-321. doi: 10.1055/s-0040-171670. Epub 2020 Oct 24.
Procoagulant collagen-and-thrombin (COAT)-activated platelets represent a subpopulation of activated platelets, which retain a coat of prohemostatic proteins and express phosphatidylserine on their surface. Dichotomous intracellular signaling generating procoagulant platelet activity instead of traditional aggregating endpoints is still not fully elucidated. It has been demonstrated that secondary messengers such as calcium and sodium play a critical role in platelet activation. Therefore, we developed a flow cytometric analysis to investigate intracellular ion fluxes simultaneously during generation of aggregating and procoagulant platelets. Human platelets were activated by convulxin-plus-thrombin. Cytosolic calcium, sodium, and potassium ion fluxes were visualized by specific ion probes and analyzed by flow cytometry. We observed high and prolonged intracellular calcium concentration, transient sodium increase, and fast potassium efflux in COAT platelets, whereas aggregating non-COAT platelets rapidly decreased their calcium content, maintaining higher cytosolic sodium, and experiencing lower and slower potassium depletion. Considering these antithetical patterns, we investigated the role of the sodium-calcium exchanger (NCX) during convulxin-plus-thrombin activation. NCX inhibitors, CBDMB and ORM-10103, dose-dependently reduced the global calcium mobilization induced by convulxin-plus-thrombin activation and dose-dependently prevented formation of procoagulant COAT platelets. Our data demonstrate that both NCX modes are used after convulxin-plus-thrombin-induced platelet activation. Non-COAT platelets use forward-mode NCX, thus pumping calcium out and moving sodium in, while COAT platelets rely on reverse NCX function, which pumps additional calcium into the cytosol, by extruding sodium. In conclusion, we described for the first time the critical and dichotomous role of NCX function during convulxin-plus-thrombin-induced platelet activation.
促凝胶原-凝血酶（COAT）激活的血小板代表激活血小板的一个亚群，其保留了一层促凝蛋白，并在其表面表达磷脂酰丝氨酸。产生促凝血小板活性的二分叉细胞内信号而不是传统的聚集终点仍未完全阐明。已经证明，钙和钠等第二信使在血小板激活中起着关键作用。因此，我们开发了一种流式细胞术分析方法，以同时研究聚集和促凝血小板产生过程中的细胞内离子通量。人血小板通过凝血栓蛋白-凝血酶激活。通过特定的离子探针可视化细胞质钙离子、钠离子和钾离子通量，并通过流式细胞术进行分析。我们观察到 COAT 血小板中存在高且持续时间长的细胞内钙离子浓度、短暂的钠离子增加和快速的钾离子外流，而聚集的非 COAT 血小板则迅速降低其钙离子含量，维持较高的细胞质钠离子，并经历较低和较慢的钾离子耗竭。考虑到这些相反的模式，我们研究了钠钙交换器（NCX）在凝血栓蛋白-凝血酶激活过程中的作用。NCX 抑制剂 CBDMB 和 ORM-10103 剂量依赖性地降低了凝血栓蛋白-凝血酶激活诱导的整体钙离子动员，并剂量依赖性地阻止了促凝 COAT 血小板的形成。我们的数据表明，NCX 的两种模式都在凝血栓蛋白-凝血酶诱导的血小板激活后被使用。非 COAT 血小板使用正向模式 NCX，从而将钙离子泵出并将钠离子泵入，而 COAT 血小板依赖于反向 NCX 功能，通过将额外的钙离子泵入细胞质来排出钠离子。总之，我们首次描述了 NCX 功能在凝血栓蛋白-凝血酶诱导的血小板激活过程中的关键和二分叉作用。
