Targeted phosphotyrosine profiling of glycoprotein VI signaling implicates oligophrenin-1 in platelet filopodia formation.
Arterioscler Thromb Vasc Biol
; 33(7): 1538-43, 2013 Jul.
Article
en En
| MEDLINE
| ID: mdl-23619296
ABSTRACT
OBJECTIVE:
Platelet adhesion to subendothelial collagen is dependent on the integrin α2ß1 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. APPROACH ANDRESULTS:
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 αIIbß3 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.Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Seudópodos
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Plaquetas
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Proteínas Nucleares
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Glicoproteínas de Membrana Plaquetaria
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Transducción de Señal
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Proteínas Activadoras de GTPasa
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Proteínas del Citoesqueleto
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Errores Innatos del Metabolismo
Tipo de estudio:
Observational_studies
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Prognostic_studies
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Qualitative_research
Límite:
Child
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Humans
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Male
Idioma:
En
Revista:
Arterioscler Thromb Vasc Biol
Asunto de la revista:
ANGIOLOGIA
Año:
2013
Tipo del documento:
Article
País de afiliación:
Países Bajos