ADP, 5-Hydroxytryptamine (5-HT), EDTA and ethidium bromide were from Sigma.

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1 SUPPLEMENTAL MATERIAL Expanded Materials and Methods Materials ADP, 5-Hydroxytryptamine (5-HT), EDTA and ethidium bromide were from Sigma. PAR-4 TRAP (AYPGKF) was custom synthesised by AnaSpec, USA. The FITClabelled antibodies for P-selectin (rat anti mouse) and isotype control (rat IgG1) were from BD Pharmingen. Hirudin was recombinant desulphato-hirudin (Revasc). Fixative solution consisted of saline with 4.6 mmol/l sodium EDTA, 4.5 mmol/lna 2 HPO 4, 1.6 mmol/l KH 2 PO 4 and 0.16% w/v formaldehyde, ph 7.4. Genotyping of P2Y 12 -/- Mice The generation of these P2Y 12 -/- mice was detailed by Foster et al. 1 Genotyping was performed according to a method provided by Schering Plough Research Institute, USA. Briefly, ear clips were taken from each P2Y 12 -/- mouse and DNA was extracted using a Qiagen DNEasy DNA extraction kit. PCR reactions used three primers; one upstream of the insert which was common to both wild-type and the deficient mice, a second primer that binds to a sequence of DNA that is only present within the inserted Neo-gene (P2Y 12 -/- only) and a third primer which binds within the section of the gene that has been deleted (wild-type only). PCR products were visualised by electrophoresis in a 2% (w/v) agarose gel stained with ethidium bromide. Positive controls of DNA from wild-type,

2 heterozygous and P2Y 12 knock out mice were amplified and run alongside each set of samples as was a negative control containing no DNA. Tissue Preparation, Histology and Morphometric Analysis 30 minutes or 3 weeks following surgery, mice were killed by overdose of sodium pentobarbital. The vasculature was immediately flushed with PBS and fixed by intraventricular injection of formalin. The common carotid artery was subsequently harvested, processed and embedded in paraffin wax. 5μm thick sections were cut at 100μm intervals along the entire length of artery and histologically stained with Alcian Blue/Elastic van Gieson to allow measurement of neointima, media, lumen and total vessel areas and corresponding perimeter values via image analysis (Lucia G, Nikon, UK). Perimeter values were subsequently used to correct for vessel distortion during processing. Two methods of data analysis were compared. Firstly, the area values from five artery sections were used, including the section from each artery with the greatest neointima area. Secondly, area values from the section containing the greatest neointimal area were used. Both methods of analysis yielded similar results. The first method was employed for the statistical analysis reported. Immunohistochemistry Paraffin-embedded sections of harvested carotid arteries were stained for α-smooth muscle actin using standard immunostaining techniques. Briefly, sections were dewaxed and rehydrated, endogenous peroxidases blocked by incubation with 3% hydrogen

3 peroxide, and non-specific antibody binding blocked by incubation with a 1:10 solution of normal goat serum, prior to incubation with the primary antibody. Primary antibody (Abcam No , Abcam, Cambridge, UK) was applied for 10 minutes at 22 C. Sections were washed with PBS prior to incubation with biotinylated goat anti-rabbit secondary antibody followed by avidin biotin complex (ABC). Finally, antibody binding was visualized with 3,3 diaminobenzidine tetrahydrochloride (DAB) and counterstained with Carazzi s haematoxylin. Negative controls were performed and consisted of sections incubated with the omission of primary antibody (substituting antibody diluent or the appropriate non-immune IgG in each case). Only negative control sections stained with non-immune IgG gave some slight nonspecific (noncellular, extracellular matrix) staining. Statistical analysis Data were analysed using SPSS software (Version 15.0; SPSS, Inc.) as follows: Platelet P-selectin data (figure 1) were assessed for equality of variance for each concentration of TRAP prior to analysis of the difference between P2Y12 +/+ vs. P2Y12 -/- mice using independent samples t test (saline, 0.3 mmol/l TRAP) or Mann Whitney test (1, 3 and 10 mmol/l TRAP). P values less than 0.01 were considered significant. Ferric chloride injury data for P2Y12 +/+ vs. P2Y12 -/- (figure 3): media and CSA data are presented to show matching of vessel dimensions but were not entered into the statistical analysis of the data. Thrombus area (figure 3A), neointima area (figure 3B) and IMR (figure 3C) were found to have unequal variance between the groups by Levene s test and were compared using the Mann Whitney test. A significance level was

4 assigned to P values less than for thrombus area or neointima area to allow for the inclusion of luminal area in the comparison whilst a significance level of P less than 0.05 was assigned to the IMR analysis which was independent of lumen area. Ferric chloride injury data for P2Y12 +/+ water-treated vs. clopidogrel-treated (figure 4): intima-media ratio data were found to have unequal variance between the groups according to Levene s test and were also analysed using the Mann Whitney test. Significance was assigned to P values less than to allow for comparison of both clopidogrel treatment groups and the individual group comparisons were non-significant according to this criterion. An exploratory analysis comparing control to any clopidogreltreated mice (long and short duration combined) showed significant effect of clopidogrel using the Mann Whitney test. Bone marrow transplant data (figure 5): Levene s testing revealed the groups to have unequal variances and the different groups were compared using the Mann Whitney test with significance assigned to P values less than 0.01 to allow for comparisons between the 4 groups. Wire injury data (figure 6): No neointima formation was seen in the sham operated arteries and a direct comparison between the two active injury groups was made using the Mann Whitney test with significance assigned to P values less than Further details of the analyses are provided in the Appendix.

5 Additional Results Confirmation of Bone Marrow Transplantation Efficiency Blood from selected bone marrow transplant mice was profiled for P-Selectin expression in response to PAR-4 TRAP (see methods) to provide evidence of efficient bone marrow transplantation. Two types of chimeric mice; P2Y +/+ 12 mice with P2Y -/- 12 bone marrow (- /- to +/+), and P2Y -/- 12 mice with P2Y +/+ 12 (+/+ to -/-) bone marrow were screened. -/- to +/+ mice exhibited little response to PAR-4 TRAP at all doses apart from 10 μm, thus mirroring P2Y 12 deficient mice, whereas +/+ to -/- mice exhibited a dose dependent response to PAR-4 TRAP which was maximal at 3 μm, thus mirroring P2Y 12 wild type animals (Figure 1). This observation provides evidence of normal platelet function with regard to P-Selectin expression (in response to TRAP) and thus provides confirmation of successful bone marrow transplantation. References 1. Foster CJ, Prosser DM, Agans JM, Zhai Y, Smith MD, Lachowicz JE, Zhang FL, Gustafson E, Monsma FJ, Jr., Wiekowski MT, Abbondanzo SJ, Cook DN, Bayne ML, Lira SA, Chintala MS. Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs. J Clin Invest. 2001;107:

6 Figures Figure 1. P-Selectin expression (median fluorescence) in response to PAR-4 TRAP in platelets from +/+ to -/- (, n=5) and -/- to +/+ (, n=4) mice. Median CD62P Fluorescence Saline 1 mm 3 mm 10 mm [TRAP] mm

7 APPENDIX Statistical analysis results Figure 1 TRAP 1mM P2Y12 +/+ vs -/- VAR00004 N Mean Rank Sum of Total 12 VAR00004 Mann-Whitney U Wilcoxon W Z.000 Asymp. Sig. (2-tailed) (a) TRAP 3mM P2Y12 +/+ vs -/- VAR00005 N Mean Rank Sum of Total 12 VAR00005 Mann-Whitney U.000 Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

8 TRAP 10mM P2Y12 +/+ vs -/- VAR00006 N Mean Rank Sum of Total 12 VAR00006 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

9 Figure 3A. Thrombus area P2Y12 +/+ vs -/- N Mean Rank Sum of Total 10 Mann-Whitney U.000 Wilcoxon W Z Asymp. Sig. (2-tailed) (a) Lumen area P2Y12 +/+ vs -/- N Mean Rank Sum of Total 10 Mann-Whitney U.000 Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

10 Figure 3B Neointima area P2Y12 +/+ vs -/- N Mean Rank Sum of Total 8 Mann-Whitney U.000 Wilcoxon W Z Asymp. Sig. (2-tailed) (a) Lumen area P2Y12 +/+ vs -/- N Mean Rank Sum of Total 8 Mann-Whitney U.000 Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

11 Figure 3C Intima:media ratio P2Y12 +/+ vs -/ Total N Mean Rank Sum of Test Statistics b Mann-Whitney U.000 Wilcoxon W Z Asymp. Sig. (2-tailed) a a. Not corrected for ties. b. Grouping Variable:

12 Figure 4A. Neointima Water vs clopidogrel long VAR00007 N Mean Rank Sum of Total 9 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) b Grouping Variable: VAR00007 Water vs clopidogrel short VAR00007 N Mean Rank Sum of Total 10 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) b Grouping Variable: VAR00007

13 Water vs clopidogrel groups combined - Mann Whitney test VAR00008 N Mean Rank Sum of Total 14 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) b Grouping Variable: VAR00008

14 Figure 4B. Intima:media ratio Mann Whitney test for water vs. both clopidogrel treated groups VAR00008 N Mean Rank Sum of Total 14 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) b Grouping Variable: VAR00008 Water vs clopidogrel - long VAR00007 N Mean Rank Sum of Total 9 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) b Grouping Variable: VAR00007

15 Water vs clopidogrel short VAR00007 N Mean Rank Sum of Total 10 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) b Grouping Variable: VAR00007

16 Figure 5A. Neointima. Mann Whitney tests -/- to +/+ vs +/+ to -/- N Mean Rank Sum of Total 19 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) -/- to +/+ vs +/+ to +/+ N Mean Rank Sum of Total 17 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

17 -/- to +/+ vs -/- to -/- N Mean Rank Sum of Total 16 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) +/+ to -/- vs +/+ to +/+ N Mean Rank Sum of Total 20 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

18 +/+ to -/- vs -/- to -/- N Mean Rank Sum of Total 19 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) +/+ to +/+ vs -/- to -/- N Mean Rank Sum of Total 17 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

19 Figure 5B. Intima:media ratio Mann Whitney tests -/- to +/+ vs +/+ to -/- N Mean Rank Sum of Total 19 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) -/- to +/+ vs +/+ to +/+ N Mean Rank Sum of Total 17 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

20 -/- to +/+ vs -/- to -/- N Mean Rank Sum of Total 16 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) +/+ to -/- vs +/+ to +/+ N Mean Rank Sum of Total 20 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

21 +/+ to -/- vs -/- to -/- N Mean Rank Sum of Total 19 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a) +/+ to +/+ vs -/- to -/- N Mean Rank Sum of Total 17 Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) (a)

22 Figure Total N Mean Rank Sum of Test Statistics a Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed).023 a. Grouping Variable: