horseradish peroxidase-labeled anti-mouse secondary antibody were procured from

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1 SUPPORTING INFORMATION Characterization of anti-platelet properties of silver nanoparticles Siddhartha Shrivastava, Tanmay Bera, Sunil K. Singh, Gajendra Singh, P. Ramachandrarao and Debabrata Dash 1. Materials Mouse monoclonal antibody against phosphotyrosine (clone 4G10) and horseradish peroxidase-labeled anti-mouse secondary antibody were procured from Upstate biotechnology and Bangalore Genei, respectively. Phycoerythrine-labeled antibody against P-selectin, Fura-2 AM and Super Signal West Pico chemiluminescent substrate were the products of BD Pharmingen, Molecular probes and Pierce, respectively. PVDF membrane was from Millipore. Phalloidin-FITC, human thrombin, apyrase, EGTA, sodium orthovanadate, acetylsalicylic acid, bovine serum albumin (fraction V), cytochalasin D, Triton X-100, protease inhibitors, and other reagents were procured from Sigma. Collagen was from Nycomed and ADP was purchased from Chronolog Corporation. Reagents for electrophoresis were purchased from Sisco Research Laboratory and Merck India. All other reagents were of analytical grade. Milli- Q grade deionized water (Millipore) was used for preparation of solutions.

2 2. Synthesis and characterization of silver nanoparticles. Silver nanoparticles were synthesized following the procedure described in our last report (Ref 1). In short, g AgNO 3 was dissolved in deionized water along with sodium hydroxide (0.01M) and liquid ammonia (2 %) to form a 0.01 M solution of stable soluble complex of silver ions. D-glucose and hydrazine (each at 0.01 M concentration) were added to the solution of silver ions to ensure its complete reduction at a final concentration of M. The ph of solution was adjusted to 7.4 with citric acid. The final solutions were carefully stored in the glass vials at 4 o C for further characterization. The solution containing nanoparticles was sonicated (Labsonic 2000 U, B. Braun) for about two minutes and passed through filters of 0.2 µm pore size (Sartorius) before each experiment. Reduction of silver ions was reflected in change in the color of solution and appearance of a hump at 400 nm due to surface plasmon resonance (SPR) of nanoparticles (Figure S1a). The sharp peak of SPR could be attributed to a narrow size distribution of the particles formed. The particles showed hardly any change in the λ max even after a month of aging time. Symmetric spectra along with the electron microscopic findings (Figure 1) were suggestive of monodispersed spherical particles. Selected area electron diffraction (SAED) pattern from these particles matched the crystallographic planes of the face centered cubic (fcc) silver particles (Figure S1b). The particle size distribution was also quite narrow (Fig. S1c), with an average of nm.

3 Synthesis parameters like ph and concentration of the reactants were varied to obtain nano silver of different sizes (30-35, and nm). Figure S1: Characterization of silver nanoparticles. (a) Optical spectra of silver before (1) and after reduction (2). Inset shows the corresponding change in color. (b) Electron diffraction pattern showing various crystallographic plans. (c) Particle size distribution showing preponderance of particles in the size range of nm. Ref 1. Shrivastava, S., Bera, T., Roy, A., Singh, G., Ramchandrarao, P., & Dash, D. Characterization of enhanced antibacterial effect of novel silver nanoparticles. Nanotechnology, 2007, 18, Synthesis and characterization of gold nanoparticles Traditional Turkevich method was followed with slight modifications for synthesis of gold nanoparticles (Ref 2). In brief, 50 ml of 0.2 mm chloroauric acid was heated to boiling, to which 1.2 ml of trisodium citrate (25 mm) was added. The pale yellow solution gradually turned into ruby red and it was the quenched into ice bath to obtain the

4 nanoparticles of gold. The ph of the solution was subsequently adjusted with sodium hydroxide and citric acid. Concentration of trisodium citrate was varied to obtain gold nanoparticles of different sizes. Final solutions were carefully stored in glass vials at 4 o C for further characterization. Solution containing nanoparticles was sonicated (Labsonic 2000 U, B. Braun) for about two minutes and passed through filters of 0.2 µm pore size (Sartorius) before each experiment. Surface plasmon resonance (SPR) of the nanoparticle sols was obtained using UV-visible spectrophotometer. Morphological analysis was performed by dropping the solution on to carbon-coated copper grids and carefully drying them before subjecting to transmission electron microscopic analysis. Ref 2. A. Sugunan and J. Dutta, Novel Synthesis of Gold Nanoparticles in Aqueous Media, In print, Proceedings of Materials Research Society (MRS) Fall 2005, Boston, USA 4. Effect of silver nanoparticles on aggregation of platelets from diabetic platelets.

5 Figure S2: Effect of silver nanoparticles on aggregation of platelets from diabetic patients. Tracings 1 and 2 represent platelets pretreated with either buffer (control) or silver nanoparticles (50 µm), respectively. The curves were representative of 3 different experiments. 5. Effect of silver nanoparticles on secretion of the contents of dense bodies and alpha granules Platelet stimulation is associated with secretion of the contents of dense bodies and alpha granules into the surrounding medium. Thrombin enhanced release of ATP/ADP from platelet dense bodies by 8±2- or 6±0.6-folds when platelets were either stirred (aggregated) or not stirred (activated), respectively (Figure S3). Pretreatment of platelets with silver nanoparticles (50 µm) significantly inhibited (by 34±3%, P=0.0021, n=5) release of adenine nucleotides under stirred condition (Figure S 3, columns 2 and 3). On the contrary, ATP secretion was only marginally suppressed (by 7±0.7%, P=0.0004, n=5) by nano silver when platelets were activated without stirring (Figure S3, columns 4 and 5). As platelet aggregation involves ligation of surface integrin-α IIb β 3, these

6 observations reflect putative modulation of integrin-mediated pathway by silver nanoparticles. Figure S3: Effect of nano silver on release of adenine nucleotides from stimulated platelets. Platelets were pretreated with either vehicle (columns 2 and 4) or silver nanoparticles (50 µm) (columns 3 and 5) followed by exposure to thrombin. Columns 2 and 3, aggregated platelets (stirred); columns 4 and 5, activated platelets (non-stirred). Column 1, resting platelets (no thrombin addition). The result was representative of 5 independent experiments (mean ± SD). Exposure of P-selectin (CD62P), a marker for alpha granule contents, was also studied subsequently using a fluorescently labeled antibody. Thrombin treatment significantly enhanced surface expression of P-selectin under non-aggregating condition (Figure S4). However, consistent with observations above, preincubation with silver nanoparticles (50 µm) had no inhibitory effect on surface externalization of P-selectin in these platelets (Figure S4).

7 Figure S4: Flow cytometric analysis of P-selectin exposure in platelets in presence or absence of silver nanoparticles in thrombin stimulated platelets. a, resting platelets; b, activated platelets, and c, activated platelets in presence of silver nanoparticles. Data are from a single experiment representative of five different experiments. 6. Suppression of actin polymerization by nano silver As platelet stimulation is associated with extensive reorganization of actin-based cytoskeleton, we investigated whether exposure to nano silver modulated actin polymerization in these cells. Thrombin evoked nearly 1.5-fold increase in platelet F- actin (filamentous actin) content, which was significantly suppressed (by 21±7%, P=0.043, n=3) upon preincubation with silver nanoparticles (50 µm) (Fig. S5; columns 2 and 3). Pretreatment with cytochalasin D (10 µm), a barbed end capping agent for actin

8 filaments, reduced platelet F-actin by almost similar extent (Fig. S5; column 4), raising the possibility that of nano silver could interfere in barbed end polymerization. Figure S5: Effect of nano silver on platelet F-actin content. Phalloidin-FITC-labeled platelets were preincubated with either vehicle (column 2), nano silver (50 µm) (column 3), or cytochalasin D (10 µm) (column 4), followed by stimulation with thrombin. Column 1, resting platelets. The result was representative of 3 independent experiments (mean ± SD). 7. Effect of silver and gold nanoparticles of different sizes on platelet aggregation

9 Figure S6: Effect of silver nanoparticles of different sizes on platelet aggregation. Tracings 2-4 represent platelets pretreated with silver nanoparticles (50 µm) of sizes 13-15, 30-35, and nm, respectively. Tracing 1 represent platelets pretreated with buffer (control). Figure S7: Effect of gold nanoparticles of different sizes on platelet aggregation. Tracings 2-4 represent platelets pretreated with gold nanoparticles (50 µm) of sizes 13, 20 and 29 nm, respectively. Tracing 1 represent platelets pretreated with buffer (control).