title: Platelet glycoproteins associated with aspirin-treatment upon platelet activation.
abstract: Platelet glycoproteins are known to play central roles in hemostasis and vascular integrity and have pathologic roles in vascular occlusive diseases such as myocardial infarction and stroke. Characterizing glycoproteins within and secreted by platelets can provide insight into the mechanisms that underlie vascular pathologies and the therapeutic benefits or failure of anti-platelet agents. To study the impact of aspirin, which is commonly prescribed for primary and secondary cardiovascular prevention, on the platelet glycoproteome, we evaluated washed platelets from ten donors. The platelet glycoproteome, was studied using an iTRAQ in resting and stimulated states and with and without aspirin treatment. Using solid phase extraction of glycosite-containing peptides (SPEG), we were able to identify 799 unique N-linked glycosylation sites (glycosites) in platelets, representing the largest and the most comprehensive analysis to date. We were able to identity a number of glycoproteins impacted by aspirin treatment, which we validated using global proteomics analysis of platelets and their secreted proteins. In our analyses, metallopeptidase inhibitor 1 (TIMP1) was the single most significantly affected glycoprotein by aspirin treatment. ELISA assays confirmed proteomic results and validated our strategy. Functional analysis demonstrated that TIMP1 levels were highly correlated with platelet reactivity in vitro, with a correlation coefficient of -0.5. The release of TIMP1 from platelets, which was previously unknown to be affected by aspirin treatment, may play important roles in hemostasis and/or vascular integrity. If validated, our findings may be useful for developing assays that assess platelet response to aspirin or other anti-platelet therapies.
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Punit
authors.initials: P
authors.lastname: Shah
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Weiming
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Shisheng
authors.initials: S
authors.lastname: Sun
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Jered
authors.initials: J
authors.lastname: Pasay
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Nauder
authors.initials: N
authors.lastname: Faraday
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D001241:Aspirin / Q000494:pharmacology*; D002853:Chromatography, Liquid; D003094:Collagen / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D008297:Male; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism
chemical_list: D006020:Glycopeptides; D010980:Platelet Membrane Glycoproteins; C560822:TIMP1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D003094:Collagen; D001241:Aspirin
keywords: Cardiovascular disease; Glycomics; Glycoproteomics; Metalloproteinase inhibitor; Platelet; TIMP1