title: Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome.
abstract: The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure.
authors.affiliation: From the ‡Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
authors.forename: Fiorella A
authors.initials: FA
authors.lastname: Solari
authors.affiliation: §Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
authors.forename: Nadine J A
authors.initials: NJ
authors.lastname: Mattheij
authors.affiliation: From the ‡Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Burkhart
authors.affiliation: §Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
authors.forename: Frauke
authors.initials: F
authors.lastname: Swieringa
authors.affiliation: ¶Arthur Bloom Haemophilia Centre, School of Medicine, Cardiff University, Cardiff, United Kingdom.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Collins
authors.affiliation: §Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
authors.forename: Judith M E M
authors.initials: JM
authors.lastname: Cosemans
authors.affiliation: From the ‡Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany; ‖Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany; **Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, UK.
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
authors.affiliation: §Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; rene.zahedi@isas.de jwm.heemskerk@maastrichtuniversity.nl.
authors.forename: Johan W M
authors.initials: JW
authors.lastname: Heemskerk
authors.affiliation: From the ‡Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany; rene.zahedi@isas.de jwm.heemskerk@maastrichtuniversity.nl.
authors.forename: René P
authors.initials: RP
authors.lastname: Zahedi
mesh_terms: D001778:Blood Coagulation Disorders / Q000097:blood* / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D003435:Crotalid Venoms / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D037181:Lectins, C-Type; D010750:Phosphoproteins / Q000032:analysis* / Q000187:drug effects; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology
chemical_list: D003435:Crotalid Venoms; D037181:Lectins, C-Type; D010750:Phosphoproteins; C028890:convulxin; D015759:Ionomycin; D013917:Thrombin; D002118:Calcium