Migrating platelets turn procoagulant upon sensing collagen. (A) Experimental setup of hybrid matrices mimicking the inflamed endothelium, with black lines corresponding to collagen fibers. (B) Representative confocal micrograph of human migrating platelets (CD41, white) with or without contact to collagen fibers. White arrowheads indicate procoagulant platelet formation with PS positivity (mC1) and secretion of microvesicles after sensing collagen; dashed white lines indicate collagen fibers. Bar represents 10 µm. (C-E) Representative micrographs of human platelets migrating on an albumin/fibrinogen matrix (upper panel) or a hybrid matrix containing albumin, fibrinogen, and collagen I (lower panel, dashed white lines). Quantification of procoagulant platelet activation on the respective matrix of freely migrating vs collagen-sensing platelets after 45 minutes (fixed time point) or over a period of 1 hour (time course experiment). PS staining agent: mC1. Bar represents 10 µm. Student’s t test, two-tailed, unpaired; one-way ANOVA with Holm-Šídák's multiple comparisons test compared with t = 0 minutes for time course experiment (right panel, E). (F) Quantification of procoagulant platelet activation and migrating platelets of PF4cre-Arpc2^fl/fl Cre-positive mice and Cre-negative littermates. Student’s t test, two-tailed, unpaired. (G) Relative velocity plots of tracked human platelets from live-imaging data. Absolute velocities were normalized to peak velocity to allow for interplatelet comparisons. Blue lines indicate the onset of procoagulant platelet activation. (H) Quantification of absolute velocity and Euclidean distance of migrating human platelets from live-imaging data. Individual dots represent n = 3 individuals per experimental group, with n > 30 individual platelets analyzed per n. Student’s t test, two-tailed, unpaired.