Animals
The study protocol was approved by the Institutional Animal Care and Use Committee at the University of California, Davis. Thirty clinically healthy staff- or student-owned dogs greater than 5 kg were used in this study and 8 dogs were enrolled for each experiment. Dogs were deemed to be clinically healthy by physical examination performed by the corresponding author and a complete blood count using an automated hematology analyzer (Coulter ACT diff, Beckman-Coulter Inc., Miami, FL) and blood smear evaluation. Dogs were not enrolled in the study if they were vaccinated 30 days prior to enrollment, on any concurrent medications, or had any abnormalities on hematological examination. Eight dogs were enrolled.
Generation of gel-filtered platelets
Whole blood (4 to 6 ml) was drawn from either the jugular or cephalic vein using a 22 gauge needle connected to a 6 ml syringe before transferring into 3.2% sodium citrate tubes. Blood tubes were gently inverted 2 to 3 times and carefully inspected for clots. Citrated blood was transferred to polypropylene tubes and platelet rich plasma was generated by centrifugation (300 x g, 5 min, no brakes) at room temperature. Platelets were separated from plasma by gel-filtration over a Sephrose 2B column at 37 °C and eluted with filtered Tyrodes-HEPES buffer (pH 7.4, 5 mM dextrose, 0.5% canine serum, without divalent cations) [31]. Gel-filtered platelets were observed to determine if they exhibited the “swirling” characteristic found in truly discoid resting platelets [31, 32]. Isolated platelets that failed to display swirling movements were not included in the study. Platelet count was obtained using an automated analyzer (Coulter ACT diff, Beckman-Coulter Inc., Miami, FL) and confirmed by bloodsmear evaluation. All experiments were carried out in a sterile manner.
Detection of platelet TLR4 and P-selectin surface expression by flow cytometry
Platelet count was adjusted to a concentration of 1 × 107 cells/ml with Tyrodes-HEPES (pH 7.4, 5 mM dextrose, 0.5% canine serum, without divalent cations) and platelets were either unstimulated (resting) or stimulated with 0.1 unit/ml bovine-derived alpha-thrombin (Haematologic Technologies, Inc., Essex Junction, VT) or ADP (1 μM or 10 μM) for 15 min at 37 °C [33]. Surface TLR4 expression was assessed using a biotinylated mouse anti-human monoclonal antibody to TLR4 (1:400, Clone: HTA 125, BioRad Laboratoires, Herculues, CA), known to cross react with canine TLR4 [34]. Following 45 min of incubation at 37 °C, platelets were incubated with streptavidin conjugated to phycoerythrin cy7 (1:200, Invitrogen, Carlsbad, CA) for an additional 45 min at 37 °C. For the detection of P-selectin (CD62p), platelets were either resting or first primed with 10 μM ADP (15 min, 37 °C) prior to treatment with 1, 5, or 10 μg/ml LPS from Escherichia coli 0111:B4 (EMD Millipore, Temecula, CA) for 30 min at 37 °C. Unstimulated and thrombin-activated (0.1 U/ml) platelets served as negative and positive controls, respectively. Following activation, platelets were incubated with fluorescein isothiocyanate-conjugated rat anti-mouse monoclonal antibody to P-selectin (CD62p) (1:200, Clone:RB40.34, BD Biosciences, San Diego, CA) for 45 min at 37 °C. Platelets were identified by forward and side scatter properties as well as the platelet integrin, β3a (CD61), using a mouse anti-human monoclonal antibody conjugated to allophycocyanin (1:1000, Clone:VI-PL2, eBioscience, San Deigo, CA) (45 min, 37 °C). Cells were fixed in 0.1% paraformaldehyde and analyzed using a 5-color flow cytometer (Beckman-Coulter FC500, Beckman-Coulter Inc., Miami, FL) within 4 h. Gating boundaries were established by fluorescence-minus-one controls. Anti-mouse compensation beads (BD Biosciences, San Diego, CA) conjugated to matched experimental fluorochromes were used for compensation and compensation matrices were calculated using commercially available software (FlowJo, Tree Str Inc., Ashland, OR). TLR4 and P-selectin expression was measured as perecent positive events or fold change in mean fluorescence intensity (MFI) between activated platelets and resting platelets using the following formula:
$$ \mathrm{MFI}\ \mathrm{fold}\ \mathrm{change}=\left({\mathrm{Log}}_{10}\ {\mathrm{MFI}}_{\mathrm{Activated}}\right)-\left({\mathrm{Log}}_{10}\ {\mathrm{MFI}}_{\mathrm{Resting}}\right) $$
The platelet and platelet-derived microvesicle (PDMV) gates were determined as previously described using 0.5 μm and 3 μm calibration beads [35, 36]. In brief, PDMV were quantified based on either the number of CD62P-positive events or MFI fold change in CD62P. Flow cytometry data were analyzed using commercially available software (FlowJo, Tree Str Inc., Ashland, OR).
Immunofluorescence microscopy of platelet TLR4
Gel-filtered platelets (1 × 108/mL) were either unstimulated or activated with 0.1 unit/ml thrombin or 10 μM ADP. Following activation, platelets were fixed in 1% paraformaldehyde for 15 min at room temperature and concentrated onto microscope slides using a cytocentrifuge (Cytospin 4, ThermoScientific Inc., Grand Island, NY) at 1500 rpm for 5 min. Platelets were then washed 3 times in phosphate buffered saline (pH 7.4) and were either unpermeabilized or permeabilized with 0.1% NP40 (Surfact-AMPs™ NP-40, Pierce, Rockford, IL) for 2 min at room temperature. After washing, cells were blocked with 10% bovine serum albumin (1 h, 37 °C), and subsequently incubated with biotinylated mouse anti-human monoclonal antibody to TLR4 (25 μg/ml, Clone:HTA125, BioRad Laboratories, Hercules, CA) (1 h, 37 °C), followed by streptavidin conjugated to Alexa Fluor 555 (1:200 in 2% BSA, S21381, ThermoFisher Scientific, Waltham, MA) (1 h, 37 °C). P-selectin (CD62P) and integrin β3a (CD61) were detected by incubating cells with rat anti-mouse CD62P monoclonal ab (10 μg/ml, Clone:RB40.34, BD Biosciences, San Diego, CA) and mouse anti-human monoclonal antibody (1 μg/ml, Clone:VI-PL2, eBioscience), respectively, followed by incubation with Alexa Fluor 488-conjugated goat anti-rat IgG and Alexa Fluor 405-conjugated goat anti-mouse IgG (eBioscience, ThermoFisher Scientific, Waltham, MA) overnight at 4 °C. The final dilution of all the secondary antibodies used was 1:50 diluted in 5% goat serum. After washing, a #1.5 glass coverslip was placed on the cells with an anti-fade mounting medium (ProLong Gold, ThermoFisher Scientific, Waltham, MA) and cured overnight at 4 °C. Interfrence controls were prepared by excluding incubation with the primary antibodies in the second immuno-labelling step.
Fluorescent images were acquired using a combination of confocal and super-resolution stimulated emission depletion (STED) microscopy (Leica TCS SP8 STED 3x, Leica Microsystems, Buffalo Grove, IL). Imaging powers of STED wavelengths were set to 20 to 50% of excitation wavelengths. The following imaging sequence was performed to avoid photobleaching: 1. CD61 was first detected by confocal microscopy excited with 405 nm with 1–6 nsec HyD gating; 2. Alexa Fluor 555 (TLR4) was then excited with 555 nm and 660 nm STED depletion laser; 3. Alexa Fluor 488 (P-selectin) was excited with 488 nm and 592 nm STED depletion laser with 1.2–6 nsec HyD gating. All images were acquired at 100x magnification with pixel dwell time of 800 nsec; pixel size (20 to 25 nm) was optimized at four times the image format of 512 × 512 pixels. Deconvolution of microscope images was performed using commercially available software (Huygens Professional, 18.10, Sceintific Folume Imaging) and analyzed using publically available software (FIJI, NIH).
Thromboxane B2 ELISA
Resting or ADP-primed platelets (10 μM ADP,15 min, 37 °C) were treated with 5 μg/ml E.coli 0111:B4 LPS (EMD Millipore, Temecula, CA) for 30 min at 37 °C. Platelets pre-treated with 100 μM acetylsalicytic acid (ASA) (Sigma-Aldrich, St. Louis, MO) (30 min, 37 °C) served as negative control. Immediately following stimulation, samples were centrifuged (1500 RPM, 18 °C, 10 min, no brakes) and platelet supernatant was collected, flash frozen in liquid nitrogen and stored at − 80 °C until analysis. Concentration of thromboxane B2 (TxB2) in the platelet supernatant was measured using a commercial ELISA kit (Enzo Life Sciences Inc., Farmingdale, NY).
Platelet TLR4 inhibition
Gel-filtered platelets, corrected to 1 × 107 cells/ml with Tyrodes-HEPES, were either incubated with preservative-free function blocking antibody to TLR4 (50 μg/ml, Clone:HTA125, BioRad Laboratories, Hercules, CA) or equivalent concentration of mouse IgG2a (Clone: OX-34Mouse IgG2a, BioRad Laboratories, Hercules, CA) as isotype control for 20 min at room temperature. An additional population of platelets were incubated with 50 μg/ml ultrapure LPS from Rhodobacter sphaeroides (LPS-RS) (InvivoGen, San Diego, CA), a TLR4 antagonist, for 1 h at 37 °C. Following TLR4 blocking, platelets were either unstimulated (resting) or stimulated with 10 μM ADP (15 min, 37 °C) before treatment with 5 μg/ml LPS (30 min, 37 °C). CD62P and CD61 were labeled as described above in room temperature.
Inhibition of platelet cyclooxygenase-1
Platelets (1 × 107 cells/ml) were first treated with 100 μM ASA or an equal volume of deionized water as vehicle control, for 30 min at 37 °C. Resting or ADP-stimulated (10 μM, 15 min) platelets were then treated with 5 μg/ml LPS for 30 min at 37 °C. Surface P-selectin and CD61 were labeled and detected by flow cytometry as described above.
Statistical analysis
Normality of data was tested using Shapiro-Wilk normality test or visual inspection of normal quartile plots. Non-parametric data were presented as median and interquartile range (IQR) and parametric data were presented as mean ± standard deviation. Normally distributed and paired data were analyzed using t-tests while nonparametric and paired data were analyzed using Wilcoxon signed-rank test. Unpaired data was measured using either Mann-Whitney U test or unpaired t test. One-way repeated measures ANOVA was used for comparing the means of dose-response studies followed by post-hoc analysis using Dunnet test. Data that violated the assumption of sphericity were analyzed using Greenhouse-Geisser correction. Correlation and correlation coefficiency were calculated using Pearson correlation. An alpha-priori of < 0.05 was considered statistically significant. Interindividual variability was calculated as the ratio between the standard deviation of a group and its means and expressed as coefficient of variation (CV). Data were analyzed using commercially available software (Prism 7.0,GraphPad Software, La Jolla, CA).
