Time after injection (hours) ns ns

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1 Platelet life span (% iotinylated platelets) Time after injection (hours) IgG GPIα GPIβ GPII GPVI Receptor expression (GeoMean, fluorescence inteity) Supplementary figure 1. Platelet life span and surface adhesion receptors expression in unaltered in ζ-deficient mice ζ-wt and ζ-deficient (14-3-3ζ-null) were examined for (a) Platelet life span (n = 4) and () receptor expression [14-3-3ζ-wt n=11; ζ-null n=12], as descried under Materials and Methods. Results were analyzed using two-way ANOVA with Bonferroni s post-hoc test.

2 ai ADP 1 µm ζ-null Maximal aggregation (%) ii No. adherent platelets/field ζ-wt Supplementary figure 2. GPIα-VWF adhesion and platelet aggregation are unaffected in ζ-deficient mouse platelets. (a) Adhesion of ζ-wt or ζ-deficient (14-3-3ζ-null) platelets (2 x 17 ml-1) to human VWF (5 µg ml-1) in the presence of otrocetin (1 µg ml-1) was examined, as descried under Methods. Platelet adhesion was imaged y DIC microscopy (Leica DMIRB, water immersion ojective: x 63, NA: 1.2) and images captured using DVT tools (Pinnacle Systems, USA). (i) Images are taken from one experiment, representative of 5 independent experiments, with the histogram (ii) depicting the numer of adherent platelets per field (mean ± SEM, n=5). () Aggregation of washed platelets in respoe to CRP or ADP was compared etween ζ-wt or ζ-null mice. This histogram depicts the mean +/- SEM of 3 independent experiments. (a,) Results were analyzed using a 2-way ANOVA (Bonferroni s post-hoc testing).

3 c P-sel +ve platelets (% gated) CRP/Thr Iono PAC-1 +ve platelets (% Gated) CRP/Thr Iono # Procoagulant platelets (% total cells/field) * * 9 27 Time (secs) Vehicle RB-11 Supplementary figure 3. The dimer destailiser reduces platelet procoagulant function under flow conditio. Washed platelets were isolated from anticoagulated whole lood from healthy volunteer donors and treated with a dimer destailiser (RB-11, 1 μm) or vehicle (sodium mesylate salt). (a) Washed platelets were perfused into Type I collagen (25 µg ml -1 ) coated microslides at 3 s -1, and allowed to settle for 5 minutes in the asence of flow. Development of procoagulant platelet morphology was assessed under flow conditio (3 s -1 ) over time using DIC microscopy [Leica DMIRB, water immersion ojective: x63, NA 1.2] and images recorded for off-line analysis using Image J. The numer of procoagulant platelets was expressed as a % of the total numer of platelets per field. Results are expressed as the mean ± SEM (n=3), and analysed using a 2-way ANOVA (Bonferroni s post-hoc testing) where *p<.5. () P-selectin and (c) integrin α II β 3 activation in respoe to the indicated concentratio of agonist were quantified through measurement of FITC-conjugated P-selectin (P-sel +ve ) or PAC-1 antiody inding, respectively, as descried under Methods.

4 ANV +ve platelets (% gated) ANV +ve platelets (% gated) ABT-737 Incuation time (min) Ionophore A23187 (µm) Supplementary figure 4. PS exposure in respoe to apoptosis or calcium ionophore are normal in ζ-deficient platelets. Diluted whole lood samples from ζ-wt (lack ars) or ζ-deficient (14-3-3ζ-null, lue ars) mice were treated with (a) ABT-737 [1 µm, indicated times, n=3-6] or () calcium ionophore A23187 [3 min, n=1], in the presence of Alexa-488-laelled Annexin V (ANV). Each figure represents the percentage (%) of platelets positive for Annexin V (ANV+ve platelets). Results are expressed as mean ± SEM from the indicated numer of independent experiments (a: n=3; : n=5), and analysed using a 2-way ANOVA (Bonferroni s post-hoc testing), where p>.5. where

5 Calcium flux (fold increase aove asal) 1 a) CRP/Thr Calcium flux (fold increase aove asal) ) CRP/Thr + EGTA Calcium flux (nm) c) Thromin (.5 U/ml) Time (sec) 3 4 Supplementary figure 5. Calcium flux in respoe to agonist is unchanged in ζ-deficient mice. Washed platelets isolated from ζ-wt (lack) or ζ-deficient (14-3-3ζ-null, red) mice were loaded with calcium dyes and calcium flux measured over time using a ratiometric calcium assay, in respoe to CRP (1 µg ml-1)/thromin (1. U ml-1), in the asence (a) or presence () of EGTA (2 mm) or thromin alone (.5 U ml-1) (c), as descried under Methods. Results depict the fold increase in calcium over asal (resting) level, and represent the mean ± SEM from 3 independent experiments, performed in duplicate. Statistical analysis using a 2-way ANOVA demotrated no statistical significance over the time course etween genotypes (p>.5)

6 Intraplatelet metaolic ATP (% resting) 1 5 ** Vehicle RB-11 **** *** Time (minutes) OCR (%asal) Vehicle RB-11 **** *** **** c Reserve capacity (OCR from % of asal) * Time (minutes) Vehicle RB-11 Supplementary figure 6. RB-11 reduces metaolic ATP depletion and mitochondrial oxygen coumption rate. Washed platelets were isolated from anticoagulated whole lood from healthy volunteer donors and treated with a dimer destailizer (RB-11, 1 μm) or vehicle (sodium mesylate salt). Metaolic ATP (a) and oxygen coumption rate (OCR) () following stimulation with CRP/Thromin were quantified, as descried under Methods. OCR has een depicted over the entire 6 minute time course (i), or as a specific change in reserve respiratory capacity, with % OCR increase over asal (ii). Results are expressed as the mean ± SEM (n=3), and analysed using a 2-way ANOVA (Bonferroni s post-hoc testing) where p>.5. These results indicate that treatment of human platelets with the dimer destailizer is coistent with the phenotype of ζ-deficient mouse platelets.

7 ECAR (% asal) Glycolysis Glycolytic capacity Glycolytic reserve ECAR (mph/min) agonist + agonist Non- glycolytic acidification c PK Kinetics 1. min min min OD 57 nm OD 57 nm OD 57 nm d 6-PFK Kinetics 1. min min min OD 57 nm OD 57 nm OD 57 nm Supplementary figure 7. Normal glycolytic capacity in ζ-deficient platelets. (a, ) Washed platelets were isolated from ζ-wt (closed symols) or ζ-deficient (14-3-3ζ-null) mice (open symols). (a, ) Glycolytic capacity was measured in DMEM modified media using the Seahorse XFp analyser, according to manufacturer s itructio. (a) Representative trace identifying the typical pattern of ECAR, depicting asal level, glycolysis (lue), glycolytic capacity (green) and reserve capacity, following the injection of drugs including: 1) vehicle or agonist; 2) Glucose; 3) Oligomycin and 4) 2-DG. () Platelets were assayed for H + production in un-stimulated or CRP/thromin-stimulated (.25 µg ml -1 ;.5 U ml -1, +agonist)) conditio. (c, d) Pyruvate kinase (PK) (c) and 6-Phosphopyruvate kinase (6-PFK) (d) enzyme activity was measured in platelets treated with CRP/thromin (.25 μg ml -1,.5 U ml -1 ) for -3 min, as descried in Methods. Results depict kinetics of enzyme activity in 5 min increments, and represent the mean ± SEM (n=3). 2-way ANOVA statistical analysis (With Bonferroni s Post-hoc testing) was performed, with no statistical significance identified etween ζ-wt and ζ-deficient samples.

8 14-3-3z lysates wt null wt null wt null wt null wt null 1 kda 5 kda 37 kda 2 kda immunolot β/α γ τ ζ Pan c ζ-wt ζ-null IP: GPIα Lysate 1 kda 55 kda 35 kda Pan kda 7 kda 55 kda 35 kda Phospho AMPK β-actin 15 kda 25 kda MW wt null wt null Supplementary Figure 8 - Original immunolot data. Original uncropped immunolot data used in Fig. 3g (a), Fig. 3i () and Fig. 6 (c).