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 a21 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 aIIb3 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 followup of OPHN1 in human platelets deficient in this protein showed reduced filopodia formation on collagen, an important parameter of platelet hemostatic function. © 2013 American Heart Association, Inc.