The α-granule proteome: novel proteins in normal and ghost granules in gray platelet syndrome

Abstract

Background: 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.

Objectives: 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).

Methods: 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.

Results: 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.

Conclusions: 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.

Trial registration: ClinicalTrials.gov NCT00001846 NCT00369421.

Figure 1. Blood smears stained with Wright-Giemsa stain

(A) Normal control. (B) GPS. The normal smear shows typical darkly-stained platelets whereas the patient smear shows enlarged, gray platelets, indicative of GPS (inset).

Figure 2. SDS-PAGE separation of fraction 6 proteins from control and GPS sucrose gradients

Gels were loaded with 40 μg total protein and stained with Coomassie Brilliant Blue. Proteins were reduced, alkylated, and digested with trypsin. Peptides were extracted and analyzed by LC-MS/MS. Each gel band represents many proteins; among them are labeled some relevant platelet α-granule proteins that contribute to the signal. Labels show soluble biosynthetic and endocytosed proteins detected in the control sample. Some proteins were severely reduced or undetected in the corresponding GPS band (e.g. vWF, thrombospondin) whereas other proteins were only moderately affected (e.g. serum albumin, fibrinogen) in the GPS band. See also Supplemental Table 2 and Methods.

Figure 3. Average number of normalized peptide hits (NPH) from several known and new α-granule proteins identified in fraction 6 from control and GPS platelets

Compared with control, the NPH from soluble, biosynthetic proteins was markedly decreased or undetected whereas the NPH from soluble, endocytosed proteins was somewhat decreased in GPS fraction 6. The NPH from membrane-bound proteins was similar in GPS fraction 6 compared with control, though Glut-3 and P-selectin were slightly decreased in GPS. MS analysis revealed additional proteins with decreased NPH in GPS compared with control. These proteins follow NPH trends similar to biosynthetic proteins and are potentially new α-granule proteins.

Figure 4. Glut-3 immunolabeling and semi-quantitative analysis

Frozen thin-sections of resting platelets immediately were fixed from whole blood. Immunolabeling for Glut-3 using 10nm protein-A gold. (A) Control platelets. The majority of Glut-3 was confined to the α-granule membrane, with low levels also present on OCS and PM. (B) GPS platelets. Glut-3 was predominantly expressed on the cell surface and in the open canalicular system (OCS) Occasional putative α-granules with less densely-packed cargo (stars, inset) were also positive for Glut-3. (C) Semi-quantitative analysis of the Glut-3 distribution over α-granules, plasma membrane, and OCS structures in control and GPS platelets. Glut-3 is predominantly associated with the cell surface and OCS structures in GPS platelets. Bars, 200nm. % au = % gold; alpha = α-granule; OCS = open canalicular system; PM = plasma membrane.

Figure 5. Immunolabeling of Control and GPS platelets with known α–granule proteins

Frozen thin-sections of resting platelets immediately fixed from whole blood. (A, C, E) Control platelets. (B, D, F) GPS platelets. (A,B) Immunolabeling for albumin using 10nm protein gold. Inset in B: clathrin-coated OCS structure (arrowhead) containing albumin. (C,D) Immunolabeling for fibrinogen in clathrin-coated residual α-granule structures. Albumin and fibrinogen are still found in GPS platelets associated with residual α-granules and OCS-like structures. (E,F) Immunolabeling for βTG in control and GPS platelets. βTG was significantly reduced in GPS and only associated with residual α-granules. Bars, 200nm.

Figure 6. Simultaneous demonstration of LTBP-1 and Glut-3

(A, C) Control platelets. (B, D) GPS platelets. (A, B) Immunogold labeling using mouse anti-LTBP-1 antibody and 10nm protein-A gold. LTBP-1 is localized in normal platelet α-granules but is absent in GPS. (C, D) Immunogold double-labeling of Glut-3 using 10nm protein-A gold (arrowheads) and LTBP-1 (arrows) using 15nm protein A-gold. α, α-granules; LTBP-1 is localized within Glut-3-positive α-granules in control platelets but it is absent from residual Glut-3 positive OCS-like membrane structures (marked with an asterisk) in GPS platelets. Bars, 200 nm.