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Originally Published 25 April 2013
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Targeted Phosphotyrosine Profiling of Glycoprotein VI Signaling Implicates Oligophrenin-1 in Platelet Filopodia Formation

Onno B. Bleijerveld, Thijs C. van Holten, Christian Preisinger, Jasper J. van der Smagt, Richard W. Farndale, Tjitske Kleefstra, Marjolein H. Willemsen, Rolf T. Urbanus, Philip G. de Groot, Albert J.R. Heck, Mark Roest, and Arjen ScholtenAuthor Info & Affiliations
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume 33, Number 7
https://doi.org/10.1161/ATVBAHA.112.300916
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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.

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 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.

Introduction

The response of platelets to vessel injury is essential to prevent bleeding, but hyperreactivity underlies the pathophysiology of various thrombotic diseases. Exposure of the extracellular matrix to flowing blood induces platelet activation, including the release of the contents of α- and δ-granules. In addition, a conformational change in αIIbβ3 increases its affinity for its ligands (eg, fibrinogen) and an active reorganization of the actin cytoskeleton accommodates shape change and the formation of filopodia.1 Collagen, the most abundant matrix protein in the subendothelium, provides a primary activation stimulus and a surface for adhesion.2 Glycoprotein VI (GPVI) is considered the predominant receptor responsible for collagen-induced platelet activation.3,4
The GPVI-mediated signaling pathway is a promising target for novel antiplatelet therapies because individuals with reduced GPVI expression have a mild increase in bleeding tendencies, whereas inhibition of the GPVI pathway may reduce thrombosis risk.2,5–7 Therefore, it is important to improve our knowledge of the GPVI-mediated signaling pathway in platelet activation.
GPVI is a 62-kDa type I transmembrane receptor of the immunoglobulin superfamily of surface receptors, which is exclusively expressed in platelets and megakaryocytes. The signaling capacity of GPVI depends on its association with the Fc receptor γ-chain homodimer. Each Fc receptor γ-chain monomer contains a conserved immunoreceptor tyrosine–based activation motif, which is characterized by 2 conserved YXXL motifs separated by 6 to 12 amino acids.8 On receptor cross-linking by the ligand collagen these 2 conserved immunoreceptor tyrosine–based activation motif tyrosine residues are phosphorylated by the Src family tyrosine kinases, Fyn and Lyn, which localize to a conserved proline-rich region of GPVI.3,9 This phosphorylation then leads to recruitment and activation of the tyrosine kinase Syk, which regulates a complex downstream pathway that involves the adapter proteins LAT, Gads, and SLP-76; the Tec family tyrosine kinases Btk and Tec; the GTP exchange factors Vav1 and Vav3; phosphatidylinositol 3-kinase isoforms; and phospholipase C-γ2.9,10
A handful of proteins that participate in GPVI signaling in human platelets are known, but our understanding of the tyrosine signaling events downstream of GPVI activation is far from complete. This information is considered crucial for understanding the fine molecular details of platelet activation and their clinical implications. Here, we aimed to identify novel GPVI signaling proteins by obtaining site-specific and quantitative information on tyrosine residues being phosphorylated on stimulation. To this end, a quantitative analysis of immunoaffinity-enriched phosphorylated tyrosine peptides11–13 was performed to compare resting and cross-linked collagen-related peptide (CRP-XL)-stimulated human platelets.14 We identified 214 unique phosphotyrosine (pTyr) sites of which 30 showed >2-fold increase in tyrosine phosphorylation after stimulation. Next to expected downstream targets of GPVI, we also detected 3 putatively novel ones. One of these, oligophrenin-1 (OPHN1), is a Rho GTPase–activating protein. Subsequent characterization of platelets obtained from 4 patients with X-linked intellectual disability caused by germline mutations in the OPHN1 gene (OMIM 300486) revealed the specific involvement of OPHN1 in platelet filopodia formation on collagen, substantiating our data obtained from the targeted pTyr proteome profiling approach.

Materials and Methods

Materials and Methods are available in the online-only Supplement.

Results

Tyrosine Phosphoproteome Analysis of CRP-XL–Stimulated Platelets

Platelets need to respond rapidly to changes in vascular integrity to prevent excessive blood loss. Signaling pathways leading to platelet activation are therefore rapidly activated on stimulation. To capture most detail, optimal time points of GPVI stimulation for our in-depth targeted and quantitative analysis were evaluated on the kinetics of CRP-XL–dependent platelet activation. To this end, quantification of platelet membrane P-selectin expression, a general marker of activation, was used (Figure 1A). Two time points were selected: 5 minutes to represent the onset and 30 minutes to represent maximal activation. The chosen proteomics approach, which uses specific immune enrichment of peptides carrying a tyrosine phosphorylation is schematically depicted in Figure 1B.11,12,15 After analysis of both the 5- and 30-minute time point, in total 214 pTyr sites on 148 proteins were identified (Table I in the online-only Data Supplement).
Figure 1. Targeted tyrosine phosphorylation profiling in stimulated platelets. A, Platelet stimulation with 2.5 μg/mL cross-linked collagen-related peptide (CRP-XL) as monitored by P-selectin expression on the plasma membrane. B, Overview of the experimental quantitative proteomics workflow. In parallel, resting (left) and CRP-XL–activated (5 and 30 minutes, right) platelets were lysed, proteins extracted and subsequently digested with trypsin. The peptides were differentially labeled using stable isotope dimethyl labeling. For each time point, 2 differentially labeled digests were combined, followed by enrichment of tyrosine-phosphorylated peptides using immobilized phosphotyrosine-specific antibodies. The enriched fraction was analyzed by nanoflow liquid chromatography–mass spectrometry (MS)/MS. C, Proteomics data representation (30-minute experiment is shown as an example). Using MSQuant software, the ratio (heavy/light)=(CRP-XL/Ctrl) was calculated for immunoprecipitated phosphotyrosine (pY)-containing peptides (dark grey dots) and normalized on the ratio of nonphosphorylated peptides (white dots), based on the extracted ion chromatograms of the differentially labeled isotopomers of each peptide. Peptide ratios (2log values) were plotted against peptide abundance (intensity, 10log values).
The quantitative data, based on stable isotope dimethyl labeling, revealed that, as expected, overall protein abundance levels (reflected in the [CRP-XL/Ctrl] ratios of nonphosphorylated peptides) remained identical when comparing resting and activated platelets at both the 5- and 30-minute time point (Figure 1C and Figure IA and IB in the online-only Data Supplement). In contrast, many tyrosine-containing peptides showed >2-fold increased phosphorylation on CRP-XL stimulation (28 unique tyrosine sites on 27 proteins), the majority being detected at both time points (Figure 2 and Figure IC in the online-only Data Supplement). Among these were several expected proteins and tyrosine phosphorylation sites belonging to the presumed core GPVI response proteome (Figure 2 and Figure II in the online-only Data Supplement): one of the Fc receptor γ-chain immunoreceptor tyrosine–based activation motif domains (FCER1G; Tyr65), SYK (Tyr629/Tyr630), GRAP2 (GADS; Tyr45), and other proteins comprising the LAT signalosome.9 Twenty-two (80%) of the regulated tyrosine sites with increased phosphorylation on GPVI activation are novel in platelets (Figure 2, black stars), according to the Uniprot and PhosphoSitePlus human databases and several key references.16–18 Three particular sites were present on proteins not earlier shown to be involved in platelet collagen signaling: the protein tyrosine kinase ABL1/ABL2 (Tyr393/Tyr439), the nonreceptor type protein tyrosine phosphatase 18 (Tyr389), and the Rho GTPase–activating protein OPHN1 (Tyr370).
Figure 2. Tyrosine sites undergoing increased phosphorylation downstream of glycoprotein (GP)VI activation. Tyrosine phosphorylation sites with increased phosphorylation downstream of GPVI in platelets activated with cross-linked collagen-related peptide (CRP-XL). Twenty-eight phosphotyrosine (pTyr) sites on 27 proteins showed at least 2-fold increase in phosphorylation in response to platelet activation through GPVI after 5 minutes (grey bars) and 30 minutes (black bars). A substantial part of these sites belongs to the GPVI core response proteome (Figure II in the online-only Data Supplement). Novel pTyr sites in platelet activation are marked with black stars.

Characterization of OPHN1-Deficient Platelets

Deficiency of OPHN1 (OPHN1−/y) is associated with a rare form of X-linked mental retardation known as oligophrenia, a syndrome characterized by defects in neuronal dendrite formation and synaptic plasticity.19,20 Despite the fact that oligophrenia is a rare disorder, we were able to obtain blood from 4 OPHN1-deficient patients. As far as we know, no bleeding disorders are reported in relation to loss of OPHN1. In line, the patients did not have a bleeding phenotype, and there were no indications of thrombotic complications. Although each patient had a different gene variant, Western blotting confirmed the absence of OPHN1 in the platelets of each patient (Figure 3A), whereas 2 control individuals showed robust expression of OPHN1 in their isolated platelet lysates (apparent molecular mass 91 kDa). The mean platelet count±SD (434±56×109/L), mean platelet volume±SD (7.3±0.6 fL), and the expression of the platelet surface receptors GPIbα, GPIX, β1-integrin, and the β3-integrin were within the normal range in OPHN1−/y platelets (Figure 3B).
Figure 3. Expression levels of oligophrenin-1 (OPHN1) and several platelet receptors in OPHN1−/y patients and controls. A, Washed platelets from 4 OPHN1−/y patients and 2 healthy controls were lysed and analyzed by Western blotting using goat antioligophrenin-1 polyclonal antibody (green) and sheep antiglycoprotein (GP) VI polyclonal antibody (red). B, Platelets from 4 OPHN1−/y patients (black bars) and 5 controls (white bars) were incubated with antibodies against β3, α2, GPIb, and GPIX and expression was analyzed by flow cytometry. Data are expressed as mean median relative fluorescence units (MFI)±SD.

OPHN1-Deficient Platelets Are Hemostatically Normal

To determine whether the absence of OPHN1 affects platelet function, we assessed the response of OPHN1-deficient platelets to stimulation of P2Y12, PAR-1, and GPVI (Figure III in the online-only Data Supplement). OPHN1-deficient platelets showed no significant differences in P-selectin expression or αIIbβ3 activation compared with healthy controls.
We then assessed the influence of OPHN1 on platelet adhesion to collagen under conditions of high shear flow (1600/s; Figure IV in the online-only Data Supplement) and found that OPHN1−/y platelets adhered and formed aggregates on a collagen-coated surface to a similar extent as healthy controls. Moreover, the absence of OPHN1 did not affect clot retraction in thrombin-stimulated platelet-rich plasma (Figure V in the online-only Data Supplement).

OPHN1-Deficient Platelets Show Defective Filopodia Formation

Because deficiency of OPHN1 is reported to be associated with decreased neuronal dendrite formation,20,21 we looked into the role of OPHN1 in platelet spreading using real-time microscopy. Because OPHN1 phosphorylation was increased on stimulation of the collagen-dependent activation pathway, we also studied platelet spreading on a mixture of the collagen peptides that bind GPVI (CRP-XL) and α2β1 (GFOGER).22 CRP-XL is a potent activator of platelets and causes rapid aggregate formation. Because this obscures the spreading process, we prevented aggregate formation with 0.2 mmol/L of RGD peptide, thereby blocking αIIbβ3-ligand interactions. Under these conditions, OPHN1−/y platelets showed equal lamellipodia formation but significantly less filopodia formation during spreading (Figure 4A and 4B, Movies I and II in the online-only Data Supplement). OPHN1−/y platelets form filopodia (OPHN1−/y, 100±SEM 0%; controls, 99±SEM 1%; not significant) and spread normally on fibrinogen (OPHN1−/y, 68±SEM 11%; controls, 83±SEM 7%; not significant), which is mainly αIIbβ3-dependent. In addition, we did not observe differences in filopodia length between OPHN1−/y platelets and control platelets spreading on surfaces coated with CRP-XL and GFOGER (Figure 4C) or on fibrinogen-coated surfaces (data not shown).
Figure 4. Filopodia formation in platelets of oligophrenin-1 (OPHN1)−/y patients and controls. OPHN1 deficiency is associated with decreased filopodia formation before spreading on cross-linked collagen-related peptide (CRP-XL)/GFOGER-coated coverslips. A,Platelet-rich plasma of 4 OPHN1−/y patients (black bars) and 5 healthy controls (white bars) containing RGD was perfused over CRP-XL/GFOGER-coated cover glasses at 25/s for 20 minutes. Pictures were taken every 10 seconds. Filopodia formation and subsequent lamellipodia formation were counted and expressed as a percentage of total quantified platelets. Data are shown as mean percentage±95% confidence interval. Differences between patients and controls were significant (*Wilcoxon rank P<0.05). B, Snapshots of a platelet from an OPHN1−/y patient (top) and a healthy control (bottom) adhering and spreading at increasing time points (t0 to t5, corresponding to ≈0 [adhesion], 100, 200, 300, 400, and 500 s, respectively), perfused over CRP-XL/GFOGER. C, Filopodia length of OPHN1−/y and control platelets perfused over CRP-XL/GFOGER-coated coverslips as described for A.

Discussion

To study the nature of GPVI signaling specifically in human platelets, we used anti-pTyr immunoprecipitation of peptides, directly from primary human platelet digests. The quantitative proteomics data show immediately that GPVI signaling was rapidly engaged because of the highly increased phosphorylation of the immunoreceptor tyrosine–based activation motif domain at Tyr65 after 5 minutes. In addition, the phosphorylation of other known downstream targets was prominent (Syk, GADS, etc), confirming the validity of our approach.
García et al16 have used pTyr immunoprecipitation at the protein level to identify several proteins that are implicated in GPVI signaling in human platelets. In our study, immunoprecipitation of tyrosine phosphorylation at the peptide level combined with stable isotope labeling-based quantitation adds much additional detail. For instance, we were able to identify the specific phosphorylation sites on the earlier implicated proteins (DOK2 [Tyr299], MAPK14 [Tyr182], and nonreceptor type protein tyrosine phosphatase 6/SHP-1 [Tyr64]), and quantified their relative upregulation on GPVI stimulation. The 3 novel platelet proteins with increased tyrosine phosphorylation downstream of GPVI seem valid novel additions to the downstream GPVI signaling cascades. ABL1 (Tyr393) and ABL2 (Tyr439; the observed tyrosine-phosphorylated peptide is present in both isoforms) regulate cytoskeletal reorganization in several myeloid cell types23 and known ABL interactors such as Src family kinases, GADS, NCK1, and SLP-76 are also found regulated in this study. Given the importance of cytoskeletal rearrangement in platelet activation, the presence of ABL and its phosphorylation in platelets are not unexpected.
Nonreceptor type protein tyrosine phosphatase 18 is a member of the PEST family of protein tyrosine phosphatases. Little is known about its biological function, although overexpression studies suggested a role in neurite outgrowth and actin cytoskeleton reorganization.24 Nonreceptor type protein tyrosine phosphatase 18 is regulated by tyrosine phosphorylation, including the GPVI downstream target site discovered in the present study (Tyr389).
Our attention was drawn to the potential impact of OPHN1 deficiency on platelet function. In patients with OPHN1 mutations, loss or dysfunction of OPHN1 is associated with reduced dendritic spine and filopodia length of neurons, the molecular explanation of their neurological phenotype.20,21 In neurons, OPHN1 localizes to filopodia, lamellipodia, and stress fibers to regulate the actin cytoskeleton.20,25,26 To determine the platelet phenotype, here a thorough functional screen of the platelets of patients with oligophrenia was performed. On the basis of several standard functional tests, OPHN1-deficient platelets seemed hemostatically normal, with no exception for the clot retraction assay, a measure for actin cytoskeleton contractility in which we expected to see differences because of the strong effects on filopodia length in neurons. The single phenotype we found was reduced filopodia formation of platelets during spreading on a collagen-like surface, but not on fibrinogen, indicative of the specific function of OPHN1 in human platelets.
Recently, Elvers et al27 reported a study on the presence of OPHN1 in human and murine platelets and its Rho-GTPase activity toward RhoA, Cdc42, and Rac1 in an A5-Chinese hamster ovary cell culture model system. They showed that on platelet spreading on fibrinogen, OPHN1 colocalized with actin in filopodia, the actin ring, and lamellipodia. In addition, OPHN1 colocalized with Rac1 and Cdc42 in the late phase of platelet spreading on fibrinogen, whereas RhoA colocalization was observed independent of activation and spreading. In our experiments, we could not confirm a role for OPHN1 in platelet spreading on fibrinogen. Given the data of Elvers et al,27 OPHN1 may have a redundant role in platelet spreading on fibrinogen in human platelets, which becomes apparent when overexpressed.
On collagen, we found a more pronounced role for the Rho GTPase–activating protein because its absence leads to reduced filopodia formation before spreading. In the experiments of Elvers et al,27 overexpression of OPHN1 in A5-Chinese hamster ovary cells inhibited lamellipodia formation. This may be consistent with the observed phenotype in this article (Figure 4) because the balance toward lamellipodia formation by absence of OPHN1 may overrule the formation of filopodia, causing platelets to spread without the formation of filopodia.
In conclusion, we identified 28 pTyr sites on 27 proteins, which undergo >2-fold increase in phosphorylation on GPVI activation in human platelets. We discovered 3 novel factors that are involved downstream of GPVI signaling after platelet activation, one of which was OPHN1. In response to GPVI stimulation, OPHN1 becomes phosphorylated at Tyr370 and plays a role in the formation of filopodia during platelet spreading on collagen.

Acknowledgments

We thank A.D. Barendrecht, C.A. Koekman, B. Rutten, Dr Angelis, and Z. Iqbal for technical assistance, and Dr Watson for critically reviewing our article.

Significance

Central to their hemostatic function, platelets are capable of rapidly adhering to exposed subendothelial collagen. The immunoglobulin glycoprotein (GP) VI is the major receptor mediating platelet activation by collagen, and the GPVI signaling pathway is considered a promising target for novel antiplatelet therapies. As site-specific knowledge of phosphorylation-based signaling downstream of GPVI is still limited, it is important to improve our molecular knowledge of GPVI signaling. In a quantitative phosphoproteomics approach using immunoprecipitation of tyrosine-phosphorylated peptides and mass spectrometry, we quantitatively assessed the specific tyrosine residues that become increasingly phosphorylated at the onset of human platelet activation through GPVI. Among an interesting set of novel players, oligophrenin-1 was identified as a novel signaling protein downstream of GPVI in human platelets. Functional characterization of platelets deficient in oligophrenin-1, in essence a human knockout, implicates a role for this protein in filopodia formation on collagen, an important parameter of platelet hemostatic function.

Supplemental Material

File (atv201828_supplemental_material1.pdf)
File (atv201828_supplemental_material4.pdf)

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Go to Arteriosclerosis, Thrombosis, and Vascular Biology
Go to Arteriosclerosis, Thrombosis, and Vascular Biology

Molecular model depicting the interaction of thrombospondin-1 TSR2 domain with the CLESH domain of CD36. TSR2 residues that bind to the CD36-CLESH are colored according to identification by NMR (cyan), mutagenesis (green) or both (orange). CD36-CLESH is colored violet with TSR2-interacting residues shown as sticks. (See pages 1655-1662.)

Arteriosclerosis, Thrombosis, and Vascular Biology
Volume 33 • Number 7 • July 2013
Pages: 1538 - 1543
PubMed: 23619296

Copyright

© 2013 American Heart Association, Inc.

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History

Received: 29 November 2012
Accepted: 10 April 2013
Published online: 25 April 2013
Published in print: July 2013

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Keywords

  1. hemostasis
  2. oligophrenin-1 deficiency
  3. platelet GPVI signaling
  4. proteomics
  5. tyrosine phosphorylation

Authors

Affiliations

Onno B. Bleijerveld*
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Thijs C. van Holten*
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Christian Preisinger
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Jasper J. van der Smagt
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Richard W. Farndale
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Tjitske Kleefstra
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Marjolein H. Willemsen
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Rolf T. Urbanus
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Philip G. de Groot
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Albert J.R. Heck
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Mark Roest
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Arjen Scholten
From the Biomolecular Mass Spectrometry and Proteomics and Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Netherlands Proteomics Centre, Utrecht, The Netherlands (O.B.B., C.P., A.J.R.H., A.S.); Departments of Clinical Chemistry and Haematology (T.C.v.H., R.T.U., P.G.d.G., M.R.) and Medical Genetics (J.J.v.d.S.), University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (R.W.F.); Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (T.K., M.H.W.). O.B.B. is currently affiliated with the Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
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Notes

*
These authors contributed equally.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.300916/-/DC1.
Correspondence to Mark Roest, PhD, Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands (e-mail [email protected]); or Arjen Scholten, PhD, Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute of Pharmaceutical Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands (e-mail [email protected]).

Disclosures

None.

Sources of Funding

This research was performed within the framework of the Center for Translational Molecular Medicine (www.ctmm.nl), project CIRCULATING CELLS (grant 01C-102), and was supported by The Netherlands Heart Foundation. The Netherlands Proteomics Center embedded in the Netherlands Genomics Initiative is kindly acknowledged for financial support (A.J.R. Heck, A. Scholten). The Cardiovascular Focus en Massa Programma at Utrecht University is acknowledged for additional financial support (A. Scholten).

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Targeted Phosphotyrosine Profiling of Glycoprotein VI Signaling Implicates Oligophrenin-1 in Platelet Filopodia Formation
Arteriosclerosis, Thrombosis, and Vascular Biology
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