1

Supplementary Material to Vara et al. “Autocrine amplification of

integrin αIIbβ3 activation and platelet adhesive responses by

deoxyribose-1-phosphate” (Thromb Haemost 2013; 109.5)

Materials

Inorganic salts for solution making, common compounds and organic solvents were

purchased from Sigma (Poole, UK). Specialised reagents are listed by application

below.

Platelet isolation and stimulation

Prostaglandin E1: Sigma (Poole, UK), #P5515

Indomethacin: Sigma (Poole, UK), #I7378

Human thrombin: Sigma (Poole, UK), #T6884

Fibrillar collagen I (native collagen fibrils from equine tendons): ChronoLog

(Havertown, US), #385

U46619: Tocris Biosciences (Bristol, UK), #1932

Surface coating and platelet labelling for adhesion experiments

Fibrillar collagen I (native collagen fibrils from equine tendons): ChronoLog

(Havertown, US), #385

Collagen I (from calf skin, solution): Sigma (Poole, UK), #C8919

Human Fibrinogen: Sigma (Poole, UK), #F3879

Calcein Blue: Invitrogen (Paisley, UK), #C1429

2

Mass spectrometry sample preparation

Filtration column Vivaspin 15R 2,000 MWCO Hydrosart: Sartorius Stedim Biotech

(Epsom, UK), # FIL8439

Filtration column Vivaspin 2 2,000 MWCO Hydrosart: Sartorius Stedim Biotech

(Epsom, UK), #FIL8427

Pharmacological tools

2-Deoxy-α-D-ribose 1-phosphate (dRP), bis(cyclohexylammonium) salt: Sigma

(Poole, UK), #D6539

2-Deoxy-D-ribose: Sigma (Poole, UK), #D5899

Apocynin: Santa Cruz Biotechnology (Santa Cruz, US), # sc-203321

Apyrase: Sigma (Poole, UK), #A6535

N-acetyl-L-cysteine (NAC): Sigma (Poole, UK), #A9165

Dihydroethidium (DHE): Invitrogen (Paisley, UK), # D-1168

Mini Complete protease inhibitor cocktail: Roche Applied Science (Burgess Hill, UK),

# 11 836 153 001

Phosphatase inhibitor cocktail I: Sigma (Poole, UK), #P2850

Phosphatase inhibitor cocktail II: Sigma (Poole, UK), # P5726

Kits and antibodies for flow cytometry, pull-down, and immunoblotting

FITC-conjugated anti-human active integrin αIIbβ3 (PAC-1): Becton and Dickinson

(Oxford, UK), # 340507

3

FITC-conjugated anti-mouse active integrin αIIbβ3 (JON/A): EMFRET (Eibelstadt,

Germany), #M023-2

Active Rap1 Pull-Down and Detection Kit: Thermo Scientifics (Rockford, US),

#16120

Phospho-(Ser) PKC Substrate Antibody: Cell Signaling Technology (Danvers, US),

#2261

Anti-actin antibody: Sigma (Poole, UK), #A3853

Anti-phospho-Src (Y416) antibody, clone 9A6: EMD Millipore (Billerica, US), #05-677

Anti-Src antibody (L4A1): Cell Signaling Technology (Danvers, US), #2110

Anti-phospho-p38MAP Kinase antibody (thr180/Tyr182): Cell Signaling Technology

(Danvers, US), #9211

Anti-phospho-ERK1/2 antibody (Thr202/Tyr204): Cell Signaling Technology

(Danvers, US), #9101

Anti-phospho-MLC2 (Ser 19): Cell Signaling Technology (Danvers, US), #3671

Anti-p38 MAPK antibody (C-20): Santa Cruz Biotechnology (Santa Cruz, US), #sc-

535

Anti-ERK2 antibody (C-14): Santa Cruz Biotechnology (Santa Cruz, US), #sc-154

Anti-tubulin antibody (DM1A): Santa Cruz Biotechnology (Santa Cruz, US), #sc-

32293

4

Supplementary figure legends

Suppl. Figure 1: No effect of exogenous dRP on collagen-dependent human platelet

aggregation (A), but potentiation of U46619-dependent aggregation (B). Human

washed platelets were pre-incubated for 5 minutes with vehicle solution (modified

Tyrode’s buffer) or 200 µM dRP. Platelet activation was obtained with either 10 µg/ml

collagen or 100 nM U46619. Aggregation was monitored by turbidimetry for 4 and 10

minutes at 37° under stirring (700 rpm), respectively. Data are representative of 3 or

more independent experiments.

Suppl. Figure 2: Potentiation of thrombin-dependent integrin αIIbβ3 activation by

exogenous dRP. Human and mouse washed platelets were pre-incubated for 5

minutes with vehicle solution (modified Tyrode’s buffer) or 200 µM dRP. Human

platelet activation was obtained with 0.05 unit/ml thrombin (A and B). Integrin αIIbβ3

activation in response to 0.05 unit/ml thrombin without stirring was monitored by flow

cytometry using an activation-dependent FITC-conjugated antibody (Pac-1). A side

scattering (SSC) versus forward scattering (FSC) dot plot is presented of the human

platelet population analysed is shown in (A). The distribution of Pac-1 staining within

the human platelet population is shown for resting platelets, thrombin-stimulated

platelets and thrombin-stimulated platelets pre-incubated with 200 µM dRP (B).

Mouse washed platelets from C57BL6\J animals were pre-incubated for 5 minutes

with vehicle solution (modified Tyrode’s buffer) or dRP concentrations varying from

50 to 400 µM (C). Integrin αIIbβ3 activation in response to 0.25 or 1.5 unit/ml

thrombin without stirring was monitored by flow cytometry using an activation-

dependent FITC-conjugated antibody (JON/A). Fluorescence values for the flow

cytometry experiments are fold-increase ratios over basal (no thrombin) and are

5

expressed as mean ± SEM (n=4). Statistical significance was tested by one-way

ANOVA with Bonferroni post-test (* p<0.05).

Suppl. Figure 3: Expression of thymidine phosphorylase (TP) and uridine

phosphorylase (UP) (A) and dRP release in wild type (WT) and TP-/- UP-/- (KO)

platelets (B). Platelet lysates from WT and KO mice were analysed by immunoblot

for the presence of TP and UP (A). Actin immunoblotting was utilised as a loading

control. The data are representative of 3 independent experiments. The release of

dRP by WT ad KO platelet in response to 1 unit/ml thrombin was analysed by direct

injection mass spectrometry as described in the material and methods (B). Data are

specific ion count mean ± SEM from 6 WT and 4 KO animals.

Suppl. Figure 4: Effect of dRP on platelet adhesion on fibrillar collagen I. Human

platelet-rich plasma (PRP) was isolated from anticoagulated blood (citrate) and

incubated for 1 hour with Calcein Blue™ (5 µg/ml) at 37°C. After reconstitution of

whole blood by mixing labelled PRP and the red blood cell fraction, adhesion to

fibrillar collagen I was tested was tested under flow (1000 sec-1) in the absence or

presence of exogenous dRP (200 µM) (A). Whole blood from wild type (WT) and TP-

/- UP-/- mice was also tested for adhesion to fibrillar collagen I at shear rate 1000 sec-

1 (B). Adhered platelets were visualised after 5 minutes of flow by fluorescence

microscopy (human) or by phase contrast microscopy (mouse). Representative

pictures from 3 independent experiments are shown and values of % surface area

coverage (mean ± SEM) are presented. Statistical analysis was performed by t-test.

Suppl. Figure 5: Effect of ROS scavenger N-acetyl-L-cysteine (NAC) on platelet

aggregation (A) and of ADP scavenger apyrase on dRP-dependent potentiation of

aggregation (B). Washed human platelets were pre-incubated with 0, 1, or 10 mM

6

NAC (A) or 0.02 unit/ml apyrase (B) before stimulation with 0.1 unit/ml (A) or 0.05

unit/ml thrombin (B). Aggregation was monitored by turbidimetry for 4 minutes at 37°

under stirring (700rpm). Traces shown here are representative of 3 independent

experiments. The results shown here are representative of at least 3 independent

experiments.

Suppl. Figure 6: Potentiation of basal activity of the kinases PKC, Src, p38-MAPK

and ERKs. Washed human platelets were pre-incubated with 0.5 mM apocynin (A)

or vehicle solution(B-D), then treated for 5 minutes (A) or 0.5, 2 and 5 minutes (B-D)

with 200 µM dRP, and finally stimulated for further 5 minutes with 0.05 unit/ml

thrombin or mock stimulation with vehicle solution. Following platelet lysis, protein

extracts were separated by SDS-PAGE and the membranes were immunostained.

The activity of PKC was assessed by phospho-specific immunoblotting of the PKC

substrates pleckstrin and myosin light chain (MLC) (A), while the activity of the

kinases Src (B), p38-MAPK (C) and ERKs (D) was assessed by immunoblot using a

kinase-specific autophosphorylation antibody. Equal loading was tested by reblotting

for tubulin (A) or for the kinases with a standard antibody (B-D). The immunoblots

presented here are representative of multiple independent experiments.

Suppl. Movie 1: Effect of dRP on human platelet adhesion on fibrinogen. Platelets

were treated as described in supplementary figure 4A. A final concentration of

200 µM dRP was added to the reconstituted blood in the bottom microchannel.

Adhesion to fibrinogen was tested at shear rate 200 sec-1 using a Bioflux200 system

(Fluxion, South San Francisco, US). Platelet adhesion was visualised by

fluorescence microscopy and video clips were obtained by collating pictures taken

7

every 10 seconds for 10 minutes. The results are representative of 3 independent

experiments.

Suppl. Movie 2: Effect of dRP on human platelet adhesion on collagen I. Platelets

were treated as described in supplementary figure 4A. A final concentration of

200 µM dRP was added to the reconstituted blood in the bottom microchannel.

Adhesion to collagen I was tested at shear rate 1000 sec-1 using a Bioflux200 system

(Fluxion, South San Francisco, US). Platelet adhesion was visualised by

fluorescence microscopy and video clips were obtained by collating pictures taken

every 10 seconds for 10 minutes. The results are representative of 3 independent

experiments.

8

Supplementary figures

Supplementary Figure 1

B

A

9

Supplementary Figure 2

Thrombin

Thrombin

+ dRP

Cumulativ

e count

CTRL

Pac-1 staining

B

C

A

Supplementary Figure 2

10

WT KO

TP

UP

Actin

A B

Supplementary Figure 3

11

A

B

Supplementary Figure 4

12

Supplementary Figure 5

A

B

13

Supplementary Figure 6

B

C

A P-pleckstrin

P-MLC

tubulin

dRP

Thrombin

Apocynin - + + - - -

- - - + + +

- - + + + +

D P-ERK

ERK

dRP

Thrombin

- - 5’ 0.5’

- - + - + +

2’ 5’

P-p38MAPK

p38MAPK

dRP

Thrombin

- - 5’ 0.5’

- - + - + +

2’ 5’

P-Src (Y416)

Src

dRP

Thrombin

- - 5’ 0.5’

- - + - + +

2’ 5’