FXIII-A translocation is partly dependent on STIM1 signaling and cytoskeletal rearrangements. (A-F) Washed human or mouse platelets were unstimulated (Unstim) or stimulated with CVX plus thrombin (IIa) or A23187 for 30 minutes. (A-C) Five-thousand events per sample were analyzed by flow cytometry. (A) Cytograms of human and mouse platelets. Two gates (smaller platelets and/or platelet fragments [left]; intact platelets [right]) were set based on unstimulated platelets. (B) Percentage of smaller platelets and/or platelet fragments for human platelets (n = 3; each dot represents a separate donor). (C) Percentage of smaller platelets and/or platelet fragments for mouse platelets (n = 3; each dot represents a separate mouse). (D-F) Mouse platelet pellet and releasate were separated by centrifugation at 1500g. FXIII-A was visualized by immunoblotting and quantified by densitometry. (D-F) Representative immunoblots (D) and quantitation (E-F) of FXIII-A in the mouse platelet pellet and releasate from 1.5 × 10⁶ platelets. Each lane and dot represents a separate mouse. (G-H) Washed human platelets were unstimulated or stimulated with CVX plus IIa in the absence (buffer, DMSO [vehicle]) or presence of calpeptin (Cpt), rhosin (Rhos), or colchicine (Colch). The platelet pellet and releasate were separated by centrifugation. FXIII-A was quantified by densitometry. (G-H) Representative immunoblot and quantitation of FXIII-A in the human platelet releasate from 1 × 10⁶ platelets (mean ± SEM of 4 separate donors). 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∗). Bars show mean ± SEM. DMSO, dimethyl sulfoxide. Statistical comparison in (H) are with appropriate controls (DMSO or buffer [diluted Tyrodes]).