Heterogeneity of platelet activation following laser or micropuncture injury. (A) Graphs show total platelet accumulation (red line) and P-selectin expression (green line) following laser (upper) or micropuncture (lower) injury in mouse cremaster muscle arterioles. Data are expressed as mean area ± SEM. N = 21 laser injuries in 6 mice and N = 29 pipette injuries in 7 mice. (B) Two-dimensional confocal images 3 and 20 minutes after laser (upper panels) or micropuncture (lower panels) injury. Platelets are labeled with Alexa-fluor 568 anti-CD41 F(ab)2 fragments (red). Alexa-fluor 647 anti-P-selectin was used to label degranulated platelets (green). Yellow is the merge. (C) Images are 3-dimensional reconstructions (isosurface view) of a series of z-plane images from the same injuries shown in Figure 1B taken 5 minutes post-laser (Ci) or puncture (Cii) injury. P-selectin is shown in green and platelets (CD41) in red. Grid size is 15 µm. Time-lapse videos of the formation and 3D reconstructions of the hemostatic plugs in A–C are provided in supplemental Video 1. (D) P-selectin–positive platelets are stably adherent during hemostatic plug formation in vivo. Embolization area was calculated by summing the total area of CD41 or P-selectin–positive events occurring in a region downstream from the main platelet mass (see supplemental Figure 2 and Supplemental Methods for details). Values are the mean ± SEM of the total CD41 or P-selectin embolus area in the first 250 seconds following injury from n = 5 injuries for pipette injury and n = 5 injuries for laser injury.
