Targeted Phosphotyrosine Profiling of Glycoprotein VI Signaling Implicates Oligophrenin-1 in Platelet Filopodia Formation
SourceArteriosclerosis, Thrombosis, and Vascular Biology, 33, 7, (2013), pp. 1538-1543
Article / Letter to editor
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Primary and Community Care
Arteriosclerosis, Thrombosis, and Vascular Biology
SubjectNCEBP 7: Effective primary care and public health; NCMLS 6: Genetics and epigenetic pathways of disease DCN MP - Plasticity and memory
OBJECTIVE: Platelet adhesion to subendothelial collagen is dependent on the integrin alpha2beta1 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 AND 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 alphaIIbbeta3 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.
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