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

Transcription

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 1

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), #D Deoxy-D-ribose: Sigma (Poole, UK), #D5899 Apocynin: Santa Cruz Biotechnology (Santa Cruz, US), # sc 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), # 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), #

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), # 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

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 U 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 activationdependent FITC-conjugated antibody (JON/A). Fluorescence values for the flow cytometry experiments are fold-increase ratios over basal (no thrombin) and are 4

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 5

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 6

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. 7

8 Supplementary figures A B Supplementary Figure 1 8

9 A B Cu mul ativ e cou nt CTRL Thrombin Thrombin + drp Pac-1 staining C Supplementary Figure 2 Supplementary Figure 2 9

10 A TP B UP Actin WT KO Supplementary Figure 3 10

11 A B Supplementary Figure 4 11

12 A B Supplementary Figure 5 12

13 A P-pleckstrin B P-Src (Y416) P-MLC Src tubulin Apocynin drp Thrombin drp Thrombin C P-p38MAPK D P-ERK p38mapk ERK drp Thrombin drp Thrombin Supplementary Figure 6 13