title: Targeted phosphotyrosine profiling of glycoprotein VI signaling implicates oligophrenin-1 in platelet filopodia formation.
abstract: OBJECTIVE
  Platelet adhesion to subendothelial collagen is dependent on the integrin α2β1 and glycoprotein VI (GPVI) receptors. The major signaling routes in collagen-dependent platelet activation are outlined; however, crucial detailed knowledge of the actual phosphorylation events mediating them is still limited. Here, we explore phosphotyrosine signaling events downstream of GPVI with site-specific detail.
  
  
  RESULTS
  Immunoprecipitations of phosphotyrosine-modified peptides from protein digests of GPVI-activated and resting human platelets were compared by stable isotope-based quantitative mass spectrometry. We surveyed 214 unique phosphotyrosine sites over 2 time points, of which 28 showed a significant increase in phosphorylation on GPVI activation. Among these was Tyr370 of oligophrenin-1 (OPHN1), a Rho GTPase-activating protein. To elucidate the function of OPHN1 in platelets, we performed an array of functional platelet analyses within a small cohort of patients with rare oligophrenia. Because of germline mutations in the OPHN1 gene locus, these patients lack OPHN1 expression entirely and are in essence a human knockout model. Our studies revealed that among other unaltered properties, patients with oligophrenia show normal P-selectin exposure and αIIbβ3 activation in response to GPVI, as well as normal aggregate formation on collagen under shear conditions. Finally, the major difference in OPHN1-deficient platelets turned out to be a significantly reduced collagen-induced filopodia formation.
  
  
  CONCLUSIONS
  In-depth phosphotyrosine screening revealed many novel signaling recipients downstream of GPVI activation uncovering a new level of detail within this important pathway. To illustrate the strength of such data, functional follow-up of OPHN1 in human platelets deficient in this protein showed reduced filopodia formation on collagen, an important parameter of platelet hemostatic function.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Onno B
authors.initials: OB
authors.lastname: Bleijerveld
authors.forename: Thijs C
authors.initials: TC
authors.lastname: van Holten
authors.forename: Christian
authors.initials: C
authors.lastname: Preisinger
authors.forename: Jasper J
authors.initials: JJ
authors.lastname: van der Smagt
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
authors.forename: Tjitske
authors.initials: T
authors.lastname: Kleefstra
authors.forename: Marjolein H
authors.initials: MH
authors.lastname: Willemsen
authors.forename: Rolf T
authors.initials: RT
authors.lastname: Urbanus
authors.forename: Philip G
authors.initials: PG
authors.lastname: de Groot
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Mark
authors.initials: M
authors.lastname: Roest
authors.forename: Arjen
authors.initials: A
authors.lastname: Scholten
mesh_terms: D001792:Blood Platelets / Q000378:metabolism*; D016022:Case-Control Studies; D002648:Child; D003094:Collagen / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000097:blood* / Q000172:deficiency / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000097:blood* / Q000172:deficiency / Q000235:genetics; D006487:Hemostasis; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D013058:Mass Spectrometry; D008661:Metabolism, Inborn Errors / Q000097:blood* / Q000235:genetics; D009687:Nuclear Proteins / Q000097:blood* / Q000172:deficiency / Q000235:genetics; D019007:P-Selectin / Q000097:blood; D010766:Phosphorylation; D010973:Platelet Adhesiveness; D010979:Platelet Function Tests; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D011554:Pseudopodia / Q000378:metabolism*; D015398:Signal Transduction; D013314:Stress, Mechanical; D013997:Time Factors; D014443:Tyrosine
chemical_list: D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; D009687:Nuclear Proteins; C111952:OPHN1 protein, human; D019007:P-Selectin; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; C546937:SELP protein, human; C098341:platelet membrane glycoprotein VI; D014443:Tyrosine; D003094:Collagen
keywords: hemostasis; oligophrenin-1 deficiency; platelet GPVI signaling; proteomics; tyrosine phosphorylation