Supplementary Appendix 2
Determination of PBR28 binding status
1. P2 membrane preparation
Frozen platelet pellets were thawed on ice. Platelet pellets from the same subject number
(RNR) were pooled and were homogenized (speed 6, 40sec) on ice in 15 mL of buffer (0.32
mM sucrose, 5 mM Tris-HCL, 1 mM MgCl2, pH7.4, 40C). The samples were lightly vortexed
and then homogenised as previously described a further two times. Samples were transferred
to individual centrifuge tubes and centrifuged at 32, 000 x g, 40C for 20min. The supernatant
was discarded and the pellet was resuspended in buffer (50mM Tris-HCL, 1mM MgCl2,
pH7.4, 40C) and centrifuged once more at 32, 000 x g, 40C for 20min. The pellet was
reconstituted in 2-4ml of buffer (50 mM Tris-HCL, 1 mM MgCl2, pH7.4, 40C) and
1mLaliquots frozen at -800C for further analysis.
2. Protein determination
The protein concentration (g protein/ml) of each sample was determined by using a Pierce
BCA Protein Assay Kit, according to the manufacturers 96-microplate instructions.
3. Competition Binding Assay
Aliquots of membrane suspension were thawed on ice and prepared using assay buffer
(50mM Tris-base, 140 mM NaCl, 1.5 mM MgCl2, 5 mM KCL, 1.5 mM CaCl2, pH7.4, 370C)
to a final volume of 15 mL. Membrane suspension was incubated at 370C with circa. 4 nM
[3H]PK11195 to determine the total binding signal. The specific binding component was
determined by incubating membrane suspension at 370C with circa. 4nM [3H]PK11195 and
10 M PK11195. To determine the binding characteristics of PBR28, membrane suspension
was incubated at 370C in the presence of circa. 4 nM [3H]PK11195 and increasing
concentrations of PBR28 (1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 M, 3 M). For
each subject, each point was performed in triplicate with a final assay volume of 500 uL per
well. After incubation, the assays were terminated by filtration through Whatman GF/B
filters, pretreated with 0.05% polyethyleneimine, followed by 3 x 1 mL washes with ice-cold
wash buffer (50 mM Tris-base, 1.4 mM MgCl2, pH 7.4, 40°C). Scintillation fluid (3 mL/vial)
was added and vials were counted on a PerkinElmer Tricarb 2900 liquid scintillation counter.
Data was analysed using GraphPad Prism 5.0 software (GraphPad Software Inc., CA, USA);
one and two site binding model fits were compared using a sum-of-squares F test, the simpler
model was selected unless the P value was less than 0.05.
3. Results
The Ki high (mean±SEM) for HABs was 2.26±0.18 nM; the Ki high and low (mean±SEM)
for MABs was 1.93±0.75 nM and 189.8±14.4 nM respectively (see figure below).
Figure. Competition assay with [3H}PK11195 in the presence of increasing concentrations of
unlabelled PBR28. The lines show individual fits to each subject's data. The nonspecific
binding has been subtracted to leave the specific binding, which has been normalised to 100%
specific binding. Black—high-affinity binder (HAB). Red—mixed-affinity binder (MAB). No
low affinity binders were observed.