analysis of protein networks in resting and collagen receptor (gpvi)-stimulated platelet...

DATASET BRIEF

Analysis of protein networks in resting and collagen

receptor (GPVI)-stimulated platelet sub-proteomes

Bernice Wright1, Ronald G. Stanley1, William J. Kaiser1, Davinia J. Mills2 andJonathan M. Gibbins1,3

1 Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences, University of Reading,Reading, Berkshire, UK

2 The Biocentre Facility, University of Reading, Reading, Berkshire, UK3 Blood Transfusion Research Group, King Saud University Riyadh, Saudi Arabia

Received: August 20, 2011

Revised: September 12, 2011

Accepted: September 14, 2011

Proteomics approaches have made important contributions to the characterisation of platelet

regulatory mechanisms. A common problem encountered with this method, however, is the

masking of low-abundance (e.g. signalling) proteins in complex mixtures by highly abundant

proteins. In this study, subcellular fractionation of washed human platelets either inactivated

or stimulated with the glycoprotein (GP) VI collagen receptor agonist, collagen-related

peptide, reduced the complexity of the platelet proteome. The majority of proteins identified

by tandem mass spectrometry are involved in signalling. The effect of GPVI stimulation on

levels of specific proteins in subcellular compartments was compared and analysed using in

silico quantification, and protein associations were predicted using STRING (the search tool

for recurring instances of neighbouring genes/proteins). Interestingly, we observed that

some proteins that were previously unidentified in platelets including teneurin-1 and Van

Gogh-like protein 1, translocated to the membrane upon GPVI stimulation. Newly identified

proteins may be involved in GPVI signalling nodes of importance for haemostasis and

thrombosis.

Keywords:

Cell biology / GPVI-signalling pathway / Platelet signalling / Platelet sub-

proteomes / Protein abundance

Platelets are anucleate, discoid-shaped blood cells that

perform a vital role in the cessation of bleeding. A clear

understanding of platelet regulatory mechanisms is impor-

tant to develop strategies to prevent their inappropriate

activation and, consequently, thrombosis. Platelets express a

number of receptors for exposed or secreted factors present

at sites of injury. Major receptors include glycoprotein (GP)

VI that binds to collagen in exposed subendothelium [1],

and protease-activated receptors 1 and 4 (PAR1 and PAR4)

[2], which are stimulated by thrombin, generated as a

consequence of activation of the coagulation pathways [3].

Proteomics-based studies have in the recent years shed

considerable light on platelet activatory mechanisms

through the identification of novel proteins involved in

platelet signalling pathways initiated by GPVI [4–7] and

PAR1/PAR4 [8, 9].

It is widely recognised, however, that a number of well-

characterised regulatory proteins in platelets are rarely

represented in proteomic data sets. To overcome the

potential masking of low-abundance proteins by highly

abundant proteins, in the present study, sample complexity

was reduced through the separation of platelets into

subcellular compartments.

Abbreviations: CRP, collagen-related peptide; ECM, extracellular

matrix; emPAI, exponentially modified protein abundance index;

GIT2, G protein-coupled receptor kinase-interactor 2; GP,

glycoprotein; PAR, protease-activated receptor; PARD3, parti-

tioning defective 3 homolog; WPL, whole platelet lysate

Correspondence: Dr. Bernice Wright, Institute for Cardiovascular

and Metabolic Research, School of Biological Sciences, Hopkins

Building, University of Reading, Reading, Berkshire, RG6 6UB,

UK

E-mail: [email protected]

Fax: 144-1183787045

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

4588 Proteomics 2011, 11, 4588–4592DOI 10.1002/pmic.201100410

Platelets were isolated by differential centrifugation as

described previously from three different individual blood

donors and each analysed in three technical repetitions [10].

Isolated platelet preparations contained o3% erythrocytes

and o0.02% leukocytes. Platelet activation was induced

with collagen-related peptide (CRP) (5 mg/mL) for 2 min at

371C. Non-stimulated cells were treated in an identical

manner with Tyrode’s HEPES buffer alone. Following cell

lysis, samples were separated into subcellular compart-

ments (Supporting Information Fig. 1) using an ultra-

centrifugation procedure, as described previously [7].

Proteins were identified by tandem mass spectrometry.

Trypsin-digested peptide samples were separated using a

Dionex ultimate RP-LC system (Dionex, Leeds UK) and

analysed using an Esquire high capacity trap (HCT) ion trap

mass spectrometer (Bruker, Daltonics, Coventry, UK). Mass

spectra of peptides were obtained from three technical

repetitions of samples from each of the three platelet

donors. Protein identification was performed in the human

taxonomy selection of the uniprot_sprot and uniprot_

sprot_rev (29 April 2008) databases (Version 2.6) of the

GeneBio PhenyxOnLine proteomics platform (http://

www.genebio.com/products/phenyx/) (Version 2.6). Data

have been deposited in the online PRIDE database [11].

Label-free spectral counting was performed in the Phenyx

Quantitation Module using an exponentially modified

protein abundance index (emPAI) [12] method that facili-

tated comparison of peptides from resting and CRP-stimu-

lated platelet subcellular fractions to estimate changes in

protein abundance upon CRP stimulation. The search tool

for recurring instances of neighbouring genes/proteins

(STRING) (http://string.embl.de/) [13] was used to predict

protein–protein interactions.

A total of 663 proteins were identified (by multiple

peptides) using uniprot_sprot human database of

Figure 1. Numbers of identified proteins and examples of

established and new platelet proteins. A Venn representation

shows the total number of proteins identified (by multiple

peptides) from resting and CRP-stimulated whole platelet lysates

(WPL) and subcellular fractions (A). The numbers of proteins

common to adjacent groups of results are shown and the central

point indicates the number of proteins common to resting and

CRP-stimulated WPL and subcellular fractions. CD109 (Bi),

PARD3 (Bii), ZO-1 (Biii) and Versican core protein (Biv) were

immunodetected in WPL. Identical amounts of total protein

(10 mg) were loaded for resting (R) and CRP-stimulated (S)

samples. Three individual peptide digests of WPL and subcel-

lular fractions from three different blood donors were used to

identify proteins.

Figure 2. Analysis of the proteome of resting and CRP-stimulated

platelets. A wide spectrum of proteins were identified within the

cytosol (CYT), associated with the membrane (MA) and within

the membrane (MEM) of resting (R) and CRP-stimulated (5 mg/

mL) platelets. Platelet activation, neuronal, metabolic, cell

adhesion, cell migration, coagulation and transport, structural

and other non-categorised signalling proteins were identified.

Proteins primarily involved in signalling are labelled - other

signalling. Three individual peptide digests of WPL and each

subcellular fraction from three different blood donors were

analysed using LC-MS/MS and proteins were identified using the

uniprot_sprot database, PhenyxOnLine and characterised using

Uniprot and literature sources.

Proteomics 2011, 11, 4588–4592 4589


PhenyxOnLine (Fig. 1A and Supporting Information

Tables 1 and 2 for complete data set) and 345 of these

proteins were newly identified in platelets (Fig. 1A).

The greatest proportion of proteins (21%) identified

(by multiple peptides) within platelet subcellular regions

are recognised to play signalling roles in platelets or

other cell types (Fig. 2), indicating that the subcellular

fractionation method employed in the present study

ameliorated the masking of low-abundance proteins

(i.e. signalling proteins) by highly abundant proteins (i.e.

structural proteins).

Identification of platelet proteins by MS was supported

by the detection, by immunoblotting, of selected examples

of known proteins with key roles and newly identified

proteins (identified by multiple peptides) with potential

roles in GPVI-stimulated signalling. CD109 (Fig. 1Bi), a

protein that carries the platelet-specific Gova/b alloantigen

[14], and partitioning defective 3 homolog (PARD3:

Table 1. The abundance of platelet proteins

Protein name Typical function and cellular location Accession no.(SwissProt)

Ratio mean

CYT MEM MA

Adenylyl cyclase-associatedprotein 1 (CAP 1)

Function: Regulates filament dynamics and cell polarity. Location:cytoplasmic face of plasma membrane. Family: Adenylate CAP

Q01518 3.87

b-Parvin Function: Regulation of cell adhesion and cytoskeletonorganization. Location: Cell junctions, focal adhesions andcytoplasmic face of plasma membrane. Family: Parvin

Q9HBI1 1.12

Calmodulin Function: Mediates control of enzymes and other proteins by Ca21.Location: Cytoplasm and cytoskeleton. Family: Calmodulin

P62158 1.51

CD9 antigen Function: Platelet activation and aggregation. Location: integral toplasma membrane. Family: Tetraspanin Transmembrane 4superfamily (TM4SF)

P21926 2.20

Cofilin-1 Function: Controls actin polymerization and depolymerization.Location: Nucleus, cytoplasm and cytoskeleton. Family: actin-binding proteins, actin depolymerizing factor (ADF)

P23528 10.62

G protein-coupled receptorkinase- interactor 2(GIT2)

Function: Associates with paxillin and PIX exchange factors.Location: Nucleoplasm. Family: ADP ribosylation factor.

Q14161 2.63

Protein furry homolog-like Function: Maintains integrity polarised cell extensions/regulatesactin cytoskeleton. Location: Cytoplasm/cytoskeleton. Family:furry Protein

O94915 1.51

SEC14-like protein 1 Function: Golgi secretory function. Location: Golgi apparatusmembrane. Family: Sec 14-like protein

Q92503 1.23

Src Function: Protein kinase cascade and Ras protein signaltransduction. Location: Cytoplasm. Family: Tyrosine proteinkinase

P12931 1.45

Teneurin-1 Function: nervous system development/negative regulation of cellproliferation. Location: cytoplasm. Family: Tenascin

Q9UKZ4 3.04

Thrombospondin-1 Function: Mediates cell-to-cell and cell-to-matrix interactions.Location: External face of plasma membrane, platelet a granulelumen and ECM. Family: Thrombospondin

P07996 2.04 0.37 0.52

Thromboxane-A synthase Function: Prostaglandin biosynthetic process. Location: Integral toplasma membrane. Family: Cytochrome P450

P24557 2.34

Vang-like protein 1 (VanGogh-like protein 1)

Function: Involved in neural tube development. Protein binding.Location: Multi-pass membrane protein. Family: Vang

Q8TAA9 0.53 4.48

Vav Function: Couples tyrosine kinase signals with the activation of theRho/Rac GTPases. Location: Cytoplasmic face of plasmamembrane. Family: Vav

P15498 2.05

Voltage-dependent anion-selective channel protein3

Function: Forms a channel through the mitochondrial outermembrane that allows diffusion of small hydrophilic molecules.Location: Mitochondrion outer membrane. Family: Eukaryoticmitochondrion porin

Q9Y277 1.72

The abundance of platelet proteins changed within cytosolic (CYT), membrane (MEM) and membrane-associated (MA) fractions uponstimulation with CRP (5 mg/mL). Non-highlighted proteins: novel to platelets; highlighted (grey) proteins: established in platelets. The ratioof emPAI scores for resting against stimulated was used as a measure of abundance. Values >1 indicate an increase in protein levels andvalues o1 indicate a decrease in protein levels upon platelet stimulation. Proteins were identified using the uniprot_sprot database,PhenyxOnLine and characterised using Uniprot and literature sources. Three individual peptide digests of WPL and subcellular fractionsfrom three different blood donors were used to identify proteins.

4590 B. Wright et al. Proteomics 2011, 11, 4588–4592


Fig. 1Bii), involved in asymmetric cell division [15], were

detected. PARD3, which is involved in the formation of tight

junctions with junctional adhesion molecule A (JAM-A) [16],

a protein implicated in platelet regulation [17], may perform

a role in platelet function.

Previously uncharacterised proteins included ZO-1

(Fig. 1Biii) and Versican core protein (Fig. 1Biv). ZO-1, typi-

cally involved in the formation of tight junctions [18], has also

been reported to co-distribute with junctional adhesion

molecule A during the formation of tight junctions [19].

Versican core protein that maintains integrity of the extra-

cellular matrix (ECM) [20] may therefore influence platelet

interactions with ECM proteins, for example, collagen.

Well-recognised platelet proteins including Src, Vav, CD9

and calmodulin demonstrated activation-dependent changes

in subcellular localisation (Table 1: known proteins-high-

lighted and Supporting Information Table 3 for full data

set). Newly identified platelet proteins and characterised

proteins distributed and changed in relative abundance

together in different subcellular locations upon CRP

stimulation (Table 1: novel proteins non-highlighted and

Supporting Information Table 3). Examples include the

neuronal proteins, Van Gogh-like protein 1 and teneurin-1

that have not been previously identified in platelets.

Van Gogh-like protein 1 decreased in the cytosol (emPAI

ratio: 0.53) and both teneurin-1 and Van Gogh-like protein 1

increased (emPAI ratio: 3.04 and 4.48, respectively) in the

membrane fraction. These data suggest that upon CRP

stimulation, Van Gogh-like protein 1 and teneurin-1 may be

involved in signalling functions, which require recruitment

to the platelet membrane.

Another previously uncharacterised protein, G protein-

coupled receptor kinase-interactor 2 (GIT2), increased

(emPAI ratio: 2.63) in the platelet cytosol upon stimulation

of these cells with CRP. Levels of this protein were elevated

as Src, which has been previously shown to associate with

GIT family proteins [21], was also increased (emPAI ratio:

1.45) in the membrane fraction. Potential predicted asso-

ciations between the novel protein, GIT2, and the well-

characterised platelet proteins Src and integrin b1 (a2b1)

(Supporting Information Fig. 2), suggest a functional

complex. These interactions may be of relevance in platelets

as lamellipodia formation during platelet spreading medi-

ated through integrin a2b1 has been shown to be Src-

dependent [22] and GIT2 has been reported to repress

lamellipodia extension [23].

Protein network analysis based upon proteins identified

in this study predicted correctly the presence (detected by

immunoblotting) of FKBP1B (Supporting Information

Fig. 3B) (involved in catalysis of the cis–trans isomerisation

of peptidyl prolyl bonds in peptides and proteins [24]), a

protein previously unidentified in platelets. This protein was

not detected in contaminating levels of leukocytes

(Supporting Information Fig. 4) and displayed limited cross-

reactivity with FKBP family members including FKBP6

(Supporting Information Fig. 5). FKBP1B is shown within a

cluster of proteins (Supporting Information Fig. 3A)

predicting associations between a number of members of

the FKBP family (FKBP4, FKBP5, FKBP6 and FKBP12) and

known platelet proteins including inositol 1,4,5 trispho-

sphate receptor (IP3R), glucocorticoid receptor and heat

shock protein 90 a.

This protein data set highlights the value of sample

simplification prior to MS and the application of this

approach to investigation of the subcellular distribution of

novel signalling proteins within platelets (GIT2, teneurin-1,

Van Gogh-like protein 1) following GPVI activation.

Previously unidentified signalling protein clusters predicted

through network analysis of the identified proteome may

represent key uncharacterised regions within the GPVI

pathway that regulate haemostasis and thrombosis.

Protein data from this study has been deposited in the onlinePRIDE database (accession numbers 18827–18850).

Financial support: Medical Research Council (MRC) andBritish Heart Foundation (BHF). Technical assistance: Dr.Laurence Bindschedler (University of Reading, Biocentre Facility).

The authors have declared no conflict of interest.

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