title: Exploring the potential of the platelet membrane proteome as a source of peripheral biomarkers for Alzheimer's disease.
abstract: BACKGROUND
  Peripheral biomarkers to diagnose Alzheimer's disease (AD) have not been established. Given parallels between neuron and platelet biology, we hypothesized platelet membrane-associated protein changes may differentiate patients clinically defined with probable AD from noncognitive impaired controls.
  
  
  METHODS
  Purified platelets, confirmed by flow cytometry were obtained from individuals before fractionation by ultracentrifugation. Following a comparison of individual membrane fractions by SDS-PAGE for general proteome uniformity, equal protein weight from the membrane fractions for five representative samples from AD and five samples from controls were pooled. AD and control protein pools were further divided into molecular weight regions by one-dimensional SDS-PAGE, prior to digestion in gel. Tryptic peptides were analyzed by reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Ionized peptide intensities were averaged for each identified protein in the two pools, thereby measuring relative protein abundance between the two membrane protein pools. Log2-transformed ratio (AD/control) of protein abundances fit a normal distribution, thereby permitting determination of significantly changed protein abundances in the AD pool.
  
  
  RESULTS
  We report a comparative analysis of the membrane-enriched platelet proteome between patients with mild to moderate AD and cognitively normal, healthy subjects. A total of 144 proteins were determined significantly altered in the platelet membrane proteome from patients with probable AD. In particular, secretory (alpha) granule proteins were dramatically reduced in AD. Of these, we confirmed significant reduction of thrombospondin-1 (THBS1) in the AD platelet membrane proteome by immunoblotting. There was a high protein-protein connectivity of proteins in other pathways implicated by proteomic changes to the proteins that define secretory granules.
  
  
  CONCLUSIONS
  Depletion of secretory granule proteins is consistent with a preponderance of post-activated platelets in circulation in AD. Significantly changed pathways implicate additional AD-related defects in platelet glycoprotein synthesis, lipid homeostasis, amyloidogenic proteins, and regulators of protease activity, many of which may be useful plasma membrane-expressed markers for AD. This study highlights the utility of LC-MS/MS to quantify human platelet membrane proteins and suggests that platelets may serve as a source of blood-based biomarkers in neurodegenerative disease.
authors.affiliation: Department of Neurology and Center for Neurodegenerative Disease, Emory University School of Medicine, 615 Michael Street NE, Atlanta, Georgia 30322, USA.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Donovan
authors.affiliation: Department of Human Genetics, Emory University School of Medicine, 615 Michael Street NE, Atlanta, Georgia 30322, USA.
authors.forename: Eric B
authors.initials: EB
authors.lastname: Dammer
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, Georgia 30322, USA.
authors.forename: Duc M
authors.initials: DM
authors.lastname: Duong
authors.affiliation: Department of Biostatistics and Bioinformatics, Emory University School of Medicine, 1518 Clifton Road NE, Atlanta, Georgia 30322, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Hanfelt
authors.affiliation: Department of Neurology and Center for Neurodegenerative Disease, Emory University School of Medicine, 615 Michael Street NE, Atlanta, Georgia 30322, USA.
authors.forename: Allan I
authors.initials: AI
authors.lastname: Levey
authors.affiliation: Department of Neurology and Center for Neurodegenerative Disease, Emory University School of Medicine, 615 Michael Street NE, Atlanta, Georgia 30322, USA ; Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, Georgia 30322, USA.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Seyfried
authors.affiliation: Department of Neurology and Center for Neurodegenerative Disease, Emory University School of Medicine, 615 Michael Street NE, Atlanta, Georgia 30322, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Lah
keywords: alpha granule secretion; amyloidogenic protein; blood biomarkers; coagulation; glycoprotein; mass spectrometry; matrix metalloprotease inhibitor; membrane proteomics; platelet activation