Genetic or pharmacological targeting of CypD and TMEM16F reduces procoagulant platelet activation without impairing migratory capacity. (A-B) Representative scatter plots and analyses derived from flow cytometric measurements of stimulated platelets from CypD- (A) or TMEM16F-knockout mice (B) (n = 3-4 mice per group). PS staining agent: mC1. Platelets were stimulated with thrombin (0.1 U/L) and convulxin (0.1 µg/mL) for 30 minutes at room temperature. Student’s t test, two-tailed, unpaired. (C-D) Representative micrographs of isolated murine platelets from platelet-specific CypD- (C) or TMEM16F- (D) knockout mice migrating on an albumin/fibrinogen/collagen I hybrid matrix. Quantification of platelet procoagulant activity and cleared area (as a surrogate for migration length) depicted as SuperPlots, with individual circles indicating individual images and the error bars corresponding to the mean data of 6 images of n = 3 to 4 mice per Cre-positive or -negative group. White dashed lines indicate collagen fibers. White arrowheads indicate migrating platelets with collagen contact but without procoagulant activity. White stars indicate procoagulant platelets. Bar represents 25 (left panel) and 15 µm (right panel). PS staining agent: mC1. Student’s t test, two-tailed, unpaired. (E) Experimental scheme of migration assay on hybrid matrix with targeting of platelet PA-promoting pathways. (F-G) Quantification of procoagulant platelets and cleared area by murine (F) and human platelets (G) treated with inhibitors of CypD (cyclosporine A, CicA, 2 µM) or TMEM16F (niflumic acid [NFA] 10 µM). One-way ANOVA with Holm-Šídák's multiple comparisons test compared with control groups. (H) Representative images of migrating human platelets stained for CD41 and PS (mC1) and incubated with an internally quenched 5-FAM/QXL 520 FRET substrate indicating thrombin activity. White arrowhead indicates a procoagulant, thrombin-positive platelet. Bar represents 10 µm. See supplemental Figure 4R for detailed images. (I) Cell-based quantification of thrombin-positive cells/FOV and the fraction of thrombin-positive cells as percentage of procoagulant platelets. Per condition, >100 cells from at least n = 2 animals were analyzed. One-way ANOVA with Holm-Šídák's multiple comparisons test compared with control groups.