Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases.
Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases.
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DOI：
10.1161/atvbaha.116.307461
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发表时间：
2016-06
期刊：
影响因子：
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通讯作者：
Gibbins JM
中科院分区：
文献类型：
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作者：
Crescente M;Pluthero FG;Li L;Lo RW;Walsh TG;Schenk MP;Holbrook LM;Louriero S;Ali MS;Vaiyapuri S;Falet H;Jones IM;Poole AW;Kahr WH;Gibbins JM
巯基异构酶促进内质网中的蛋白质折叠，并且这些酶中的几种，包括PDI和ERp 57，被动员到活化的血小板的表面，在那里它们影响血小板聚集、血液凝固和血栓形成。在这项研究中，我们首次研究了巨核细胞中巯基异构酶的合成和运输，确定了它们在血小板中的亚细胞定位，并确定了活化后它们向血小板表面移动的细胞事件。免疫荧光显微镜（IFM）成像用于定位在小鼠和人类巨核细胞在不同的发育阶段的PDI和ERp 57。IFM和亚细胞分级分离分析被用来本地化这些蛋白质在血小板的一个区室不同的已知的分泌囊泡，重叠的内质网/肌浆网蛋白钙连接蛋白和SERCA 3定义的内细胞表面膜区域。IFM和流式细胞术用于监测在存在和不存在肌动蛋白聚合（由latrunculin抑制）的情况下，以及在存在或不存在由Munc 13-4介导的膜融合（在Unc 13 dJinx小鼠的血小板中不存在）的情况下活化血小板中的巯基异构酶动员。血小板携带的巯基异构酶被独立于巨核细胞中的分泌颗粒内容物进行运输，并且在与这些细胞的肌浆网/内质网对应的血小板外膜的内表面附近的亚细胞区室中浓缩。巯基异构酶通过需要肌动蛋白聚合而不是SNARE/Munc 13-4依赖的囊泡-质膜融合的过程被动员到活化的血小板表面。
Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including PDI and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation and thrombus formation. In this study we examined for the first time the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets and identified the cellular events responsible for their movement to the platelet surface upon activation. Immunofluorescence microscopy (IFM) imaging was used to localize PDI and ERp57 in murine and human megakaryocytes at various developmental stages. IFM and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and SERCA3. IFM and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin), and in the presence or absence of membrane fusion mediated by Munc 13-4 (absent in platelets from Unc13dJinx mice). Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes, and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not SNARE/Munc 13-4-dependent vesicular-plasma membrane fusion.