Activated platelets retain most of their FXIII-A inside the cell, where it is protected from proteolytic degradation. (A) Washed human platelets were unstimulated (Unstim) or stimulated with CVX plus thrombin (IIa) for 30 minutes. Intact (nonpermeabilized [Non-Perm]) or permeabilized (Perm) platelets were labeled with antibodies against CD41/CD61 (surface marker, blue) and FXIII-A (red). A merge of CD41/CD61 and FXIII-A is shown in pink. Platelets were imaged on a Zeiss LSM 900 confocal microscope (UNC Microscopy Service Laboratory) through a Plan-Apochromat 63×/1.40 oil immersion objective. For optimal display, the brightness/contrast was adjusted separately; gray scale images with equivalent settings are shown in supplemental Figure 8. Representative images of experiments performed on platelets from 3 different donors are shown. The white boxes (left images) indicate the selected individual platelets shown on the right images. White arrow points to FXIII-A exposed on the cap-like structure on the platelet surface. (B) Representative immunoblots of FXIII-A from washed human platelets (n = 5 separate donors) that were unstimulated (Unstim) or stimulated with CVX plus thrombin (IIa) in the absence or presence of plasmin (Pm) for up to 30 minutes. The pellet and releasate were separated by centrifugation. For analysis of the pellet, proteins from 2 × 10⁵ platelets were loaded. For analysis of the releasate, supernatant from 1 × 10⁶ platelets were loaded. FXIII-A species are labeled on the right of the immunoblot as ^(O) (representing zymogen FXIII-A or nonproteolytically activated FXIII-A°) and ∗ (representing FXIII-A∗).