Section on Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA.
J Thromb Haemost. 2010 Aug;8(8):1786-96. doi: 10.1111/j.1538-7836.2010.03932.x. Epub 2010 May 27.
Deficiencies in granule-bound substances in platelets cause congenital bleeding disorders known as storage pool deficiencies. For disorders such as gray platelet syndrome (GPS), in which thrombocytopenia, enlarged platelets and a paucity of α-granules are observed, only the clinical and histologic states have been defined.
In order to understand the molecular defect in GPS, the α-granule fraction protein composition from a normal individual was compared with that of a GPS patient by mass spectrometry (MS).
Platelet organelles were separated by sucrose gradient ultracentrifugation. Proteins from sedimented fractions were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis, reduced, alkylated, and digested with trypsin. Peptides were analyzed by liquid chromatography-tandem MS. Mascot was used for peptide/protein identification and to determine peptide false-positive rates. MassSieve was used to generate and compare parsimonious lists of proteins.
As compared with control, the normalized peptide hits (NPHs) from soluble, biosynthetic α-granule proteins were markedly decreased or undetected in GPS platelets, whereas the NPHs from soluble, endocytosed α-granule proteins were only moderately affected. The NPHs from membrane-bound α-granule proteins were similar in normal platelets and GPS platelets, although P-selectin and Glut3 were slightly decreased, consistent with immunoelectron microscopy findings in resting platelets. We also identified proteins not previously known to be decreased in GPS, including latent transforming growth factor-β-binding protein 1(LTBP1), a component of the transforming growth factor-β (TGF-β) complex.
Our results support the existence of 'ghost granules' in GPS, point to the basic defect in GPS as failure to incorporate endogenously synthesized megakaryocytic proteins into α-granules, and identify specific new proteins as α-granule inhabitants.
血小板颗粒结合物质的缺乏会导致先天性出血性疾病，称为储存池缺陷。对于诸如灰色血小板综合征 (GPS) 等疾病，观察到血小板减少、血小板增大和α-颗粒稀少，仅定义了临床和组织学状态。
为了了解 GPS 的分子缺陷，通过质谱 (MS) 比较正常个体的α-颗粒级分蛋白组成与 GPS 患者的蛋白组成。
通过蔗糖梯度超速离心分离血小板细胞器。用十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离沉降部分的蛋白质，还原、烷基化并用胰蛋白酶消化。通过液相色谱-串联 MS 分析肽段。Mascot 用于肽/蛋白鉴定和确定肽假阳性率。MassSieve 用于生成和比较简约的蛋白质列表。
与对照相比，GPS 血小板中可溶性、生物合成的α-颗粒蛋白的归一化肽命中（NPH）明显减少或无法检测，而可溶性、内吞的α-颗粒蛋白的 NPH 仅受到中度影响。正常血小板和 GPS 血小板的膜结合α-颗粒蛋白的 NPH 相似，尽管 P-选择素和 Glut3 略有减少，与静息血小板的免疫电子显微镜观察结果一致。我们还鉴定了以前未知在 GPS 中减少的蛋白质，包括潜伏转化生长因子-β结合蛋白 1（LTBP1），转化生长因子-β（TGF-β）复合物的一个组成部分。
我们的结果支持 GPS 中存在“幽灵颗粒”的观点，指出 GPS 的基本缺陷是未能将内源性合成的巨核细胞蛋白纳入α-颗粒，并确定特定的新蛋白为α-颗粒居民。
