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Redox-dependent regulation of platelets
was tested at concentrations ranging from 1 to 30 μg/mL and both aggregation kinetics (Figure 1A) and oxygen radi- cal generation rates at 10 min from stimulus delivery (Figure 1C) were concentration dependent. Similarly to collagen, a synthetic collagen-related peptide (CRP) that selectively engages GPVI receptor (but not other collagen-binding receptors on platelets), also led to concentration-dependent O2•− formation that was necessary for platelet aggregation
AB
(Online Supplementary Figure S3). Thrombin also showed concentration dependence of both EPR (Figure 1B) and aggregation (Figure 1D) responses between 0.03 and 1 unit/mL. Online Supplementary Table S1 shows the EC50 val- ues of these three agonists for EPR detection of superoxide anion generation and aggregation response.
In order to confirm the chemical nature of the oxygen radicals generated by platelets upon activation and detected
CD
Figure 1. Oxygen radical generation by platelets activated by physiological stimuli col- lagen and thrombin. Washed human platelets were prepared as described. Collagen was test- ed at concentrations ranging from 1 to 30 μg/mL (A and C), while thrombin was tested from 0.03 to 1 unit/mL (B and D). Electron paramagnetic resonance (EPR) was used to measure oxygen radicals formation as 1- hydroxy-3-methoxycarbonyl-2,2,5,5-tetram- ethylpyrrolidine (CMH) oxidation rates [32.7 and 37.3 attomoles per platelet per minute (min), respectively] (A and C) and aggregation was simultaneously assessed by turbidimetry (B and D). For either technique, representative examples are shown in top panels, while quan- tification is shown in the bottom panels. Aggregation curves up to 5 min are shown, while EPR resonance readings were taken after 10 min of stimulation. Examples of EPR traces and aggregation curves are representative of 4 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test. *P<0.05 compared to resting platelets. N=4 for (A-D).
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