Impact of genetic and pharmacological targeting of PA pathways on platelet calcium oscillations. (A) Representative images of time-lapse microscopy of migrating human platelets and respective calcium oscillations (green) and PS exposure (annexin V, pink). PH, phase contrast. Bar represents 10 µm. White boxes indicate the area of measurement analyzed in (B). See supplemental Video 2 for corresponding live imaging. (B) Intensity projection for calcium (blue) and PS signal intensity (red) over time as % of maximum intensity for cells 1 to 3. (C) Representative calcium oscillation profiles of migrating platelets from CypD- or TMEM16F-deficient compared with platelets from PF4cre-negative animals. (D) Quantification of mean normalized calcium amplitudes and calcium peak frequency measured from mouse platelets across genotypes. n = 103 individual platelets. One-way ANOVA with Holm-Šídák's multiple comparison test. (E) Representative micrographs and calcium (blue) and PS intensity profiles (mC1, red) derived from live imaging of the 2 CypD-deficient mouse platelets indicated by white boxes. Bar represents 10 µm. Arrows indicate the beginning of contact to collagen fibers. (F) Relative quantification of percentage of supramaximal calcium peaks of all collagen-associated mouse platelets as well as relative quantification of supramaximal calcium peak-positive procoagulant platelets. Individual dots represent percentages derived from individual time-lapse microscopy videos. Platelets were isolated from n = 2 to 3 mice/group. PS staining agent: mC1. One-way ANOVA with Holm-Šídák's multiple comparisons test.