Fig. 2: MTH1 deficiency impairs GPCR-dependent platelet aggregation, granule secretion, calcium mobilization and mitochondrial ATP production.

Washed platelets (200 × 10⁹/l) from MTH1^fl/fl or MTH1^-/- mice were stimulated with CRP (0.1 μg/ml) (mean ± SE, n = 3 independent isolated platelets) (a), thrombin (0.01 U/ml) (mean ± SE, n = 4 independent isolated platelets) (b), U46619 (0.3 μM) (mean ± SE, n = 3 independent isolated platelets) (c) followed by analysis of platelet aggregation and ATP release (reflecting dense granule secretion) in a Lumi-Aggregometer Model 700 (two-tailed unpaired Student’s t test). Washed platelets were stimulated with CRP or thrombin to measure integrin αIIbβ3 activation (presented by JON/A binding) (mean ± SE, n = 4 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test) (d) and phosphatidylserine exposure (presented by Annexin-V binding) (mean ± SE, n = 3 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test) (e) by flow cytometry or calcium mobilization using Fluo-4 AM by a microplate reader (mean ± SE, n = 3 independent isolated platelets, two-tailed unpaired Student’s t test) (f). g Platelet aggregation in response to thrombin (0.01 U/ml) after addition of apyrase (0.25 U/ml) or ADP (1 μM) (mean ± SE, n = 3 independent isolated platelets, one-way ANOVA with Tukey multiple comparisons test). h Mitochondrial ATP production was measured using the Biotracker dye in thrombin-stimulated platelets (mean ± SE, n = 4 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test).