Feng Dian, Crane Katharine, Rozenvayn Nataliya, Dvorak Ann M, Flaumenhaft Robert
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
Blood. 2002 Jun 1;99(11):4006-14. doi: 10.1182/blood.v99.11.4006.
Morphologic studies have demonstrated a process by which alpha-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for alpha-granule secretion. These observations raise the possibility that morphologic features of alpha-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins-human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca(++)-induced alpha-granule secretion by approximately 50%. Inhibition of alpha-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of alpha-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of alpha-granule secretion that include homotypic alpha-granule fusion and the fusion of alpha-granules with the open canalicular system and plasma membrane.
形态学研究已经证实了一种α-颗粒内容物从血小板中释放的过程。旨在确定这种释放分子机制的研究表明，SNARE蛋白是α-颗粒分泌所必需的。这些观察结果提出了一种可能性，即α-颗粒分泌的形态学特征可能受血小板中SNARE蛋白亚细胞分布的影响。为了评估这种可能性，我们分析了3种功能性血小板SNARE蛋白——人细胞ubrevin、SNAP-23和 syntaxin 2的亚细胞分布。用抗人细胞ubrevin抗体处理经链球菌溶血素O通透的血小板，可使Ca(++)诱导的α-颗粒分泌抑制约50%。抗人细胞ubrevin对α-颗粒分泌的抑制作用可被一种阻断肽逆转。Syntaxin 2和SNAP-23先前已被证明可介导血小板颗粒分泌。通过使用包埋前免疫纳米金法的超微结构研究以及亚细胞组分的免疫印迹分析，确定了这3种SNARE蛋白的亚细胞定位。免疫纳米金定位显示，静息血小板中约80%的人细胞ubrevin定位于血小板颗粒膜。相比之下，SNAP-23主要定位于质膜，而syntaxin 2在α-颗粒、开放小管系统和质膜的膜之间分布更为均匀。因此，这些SNARE蛋白中的每一种在血小板中都有独特的亚细胞分布，并且这些膜区室中的每一个都表现出独特的SNARE蛋白组成。这种分布为α-颗粒分泌的几个特征提供了基础，这些特征包括同型α-颗粒融合以及α-颗粒与开放小管系统和质膜的融合。
