BLOC-1调控血小板致密颗粒发生与血小板功能

Platelets are nonnuclear blood cells derived from megakaryocytes, which only exist in mammalian, and play important roles in hemostasis and thrombosis. Platelet dense granule deficiency can cause coagulation dysfunction. It has been found that BLOC-1 (Biogenesis of Lysosome-Related Organelles Complex-1) is involved in vesicle trafficking required for dense granule formation. The deficiency of dysbindin, one of the subunits of BLOC-1, leads to defects in platelet dense granules, but so far the molecular mechanism is unclear. In our previous study, through the quantitative analysis of platelet protein levels of dysbindin mutant sdy mice, we found that the expression level of TMEM163 was significantly lower than control group, and showed comparable changes with platelet dense granule membrane protein CD63. Immunofluorescent staining showed that TMEM163 was colocalized with another dense granule membrane protein VMAT2, suggesting that TMEM163 is an unreported dense granule membrane protein. The phenotype of dense granule biogenesis in TMEM163 knockout MEG-01cells was similar to dysbindin knockout MEG-01 cells with the differentiation progressed, suggesting that TMEM163 plays an important role in the biogenesis of platelet dense granules. In this study, we will explore the molecular mechanism of the BLOC-1 in regulating the biogenesis of platelet dense granules through mediating the trafficking of TMEM163 into platelet dense granules at cellular and molecular levels, and elucidate the pathological mechanism of the disorders with defects in platelet dense granule biogenesis such as HPS syndrome (Hermansky-Pudlak syndrome).

血小板是来源于巨核细胞的无核血细胞，在凝血、血栓形成中发挥重要生理作用。血小板致密颗粒缺陷会造成凝血障碍。BLOC-1参与囊泡运输过程并在致密颗粒形成中发挥重要作用。BLOC-1亚基dysbindin缺失会导致血小板致密颗粒发生缺陷，但分子机制不清楚。我们前期研究对dysbindin突变的sdy小鼠血小板蛋白水平定量分析发现，在sdy小鼠血小板中TMEM163表达比对照组显著降低，并与血小板致密颗粒膜蛋白CD63表达水平呈一致性变化。免疫荧光显示TMEM163与VMAT2共定位，暗示其是未报道的致密颗粒膜蛋白。敲除TMEM163的MEG-01细胞分化时，致密颗粒发生表型与dysbindin敲除的细胞类似，暗示其在血小板致密颗粒发生中有重要作用。本课题将从小鼠、细胞、分子层次解析BLOC-1通过运输TMEM163调控血小板致密颗粒发生的分子机制，进一步阐明HPS等致密颗粒发生障碍的病理机制。

溶酶体相关细胞器（LROs）是一类分泌细胞器，内部富含很多离子如钙离子等而且能够通过离子转运蛋白或者离子通道来维持细胞器内的离子平衡。之前的研究证明LROs的生物学发生和分泌与离子平衡之间有非常密切的关系。一类常染色体隐性疾病HPS（Hermansky-Pudlak syndrome）综合征具有眼皮肤白化、凝血障碍等表型，这类疾病被证明有多个LRO的功能缺陷，通常是黑素小体和血小板致密颗粒（DGs）。然而，导致凝血障碍的DG缺陷目前还没有明确的分子机制阐述。本课题首先通过定量蛋白组学，鉴定到一个之前未报到的血小板锌离子转运蛋白TMEM163，其表达量在BLOC-1（sdy和pa）、BLOC-2（ru）、AP-3（pe）缺陷的HPS小鼠模型以及HPS患者（HPS2、HPS3、HPS5、HPS6或HPS9）中显著降低。之后在构建的TMEM163敲除小鼠模型中，发现其血小板存在类似的DG发生缺陷和较高的细胞内锌离子堆积，这一表型在sdy小鼠（BLOC-1复合物其中一个亚基dysbindin缺陷）中也存在。同时，TMEM163敲除会导致血小板DG分泌功能缺陷进而影响血小板功能。此外，在血小板前体细胞系MEG-01中，TMEM163定位于DG以及晚期内体（可认为是DG前体）上，这一转运过程是需要BLOC-1复合体介导的。综上，我们的结果表明，TMEM163对于DG的生物发生至关重要，BLOC-1复合体是TMEM163向DG前体转运所必需的。本课题发现TMEM163缺失会导致细胞内锌离子稳态的破坏，说明锌离子对于LROs的功能非常重要，为HPS综合征的发病机制提供了新的解释，也为以后的治疗提供潜在的靶点。