Crescente Marilena, Pluthero Fred G, Li Ling, Lo Richard W, Walsh Tony G, Schenk Michael P, Holbrook Lisa M, Louriero Silvia, Ali Marfoua S, Vaiyapuri Sakthivel, Falet Hervé, Jones Ian M, Poole Alastair W, Kahr Walter H A, Gibbins Jonathan M
From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
Arterioscler Thromb Vasc Biol. 2016 Jun;36(6):1164-73. doi: 10.1161/ATVBAHA.116.307461. Epub 2016 Apr 14.
Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase 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 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 on activation.
Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy 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 sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy 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 Munc13-4 (absent in platelets from Unc13d(Jinx) 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 soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.
硫醇异构酶在内质网中促进蛋白质折叠，其中几种酶，包括蛋白质二硫键异构酶和内质网蛋白57，会被转运到活化血小板的表面，在那里它们会影响血小板聚集、血液凝固和血栓形成。在本研究中，我们研究了硫醇异构酶在巨核细胞中的合成和运输，确定了它们在血小板中的亚细胞定位，并确定了导致它们在活化时移动到血小板表面的细胞事件。
采用免疫荧光显微镜成像技术，对不同发育阶段的小鼠和人类巨核细胞中的蛋白质二硫键异构酶和内质网蛋白57进行定位。采用免疫荧光显微镜和亚细胞分级分离分析技术，将这些蛋白质在血小板中的定位到一个与已知分泌囊泡不同的区室，该区域与由内质网/肌浆网蛋白钙连蛋白和肌浆/内质网钙ATP酶3定义的细胞内表面膜区域重叠。采用免疫荧光显微镜和流式细胞术，在有或没有肌动蛋白聚合（由拉春库林抑制）的情况下，以及在有或没有由Munc13-4介导的膜融合（Unc13d（Jinx）小鼠的血小板中不存在）的情况下，监测活化血小板中硫醇异构酶的转运。
血小板中的硫醇异构酶在巨核细胞中独立于分泌颗粒内容物进行运输，并集中在血小板外膜内表面附近的一个亚细胞区室中，该区域对应于这些细胞的肌浆/内质网。硫醇异构酶通过一个需要肌动蛋白聚合但不需要可溶性N-乙基马来酰亚胺敏感融合蛋白附着受体/Munc13-4依赖性囊泡-质膜融合的过程被转运到活化血小板的表面。
