Platelet factor FXIII-A (FXIII-A) is abundant in healthy platelets and partially colocalizes with actin and actin nodules. Platelets were isolated from whole blood from normal healthy donors and factor XIII (FXIII)–deficient patients. (A) Immunocytochemistry was performed using fluorescein isothiocyanate (FITC) anti–FXIII-A antibody to detect platelet FXIII-A content in fixed, permeabilized platelets that were resting or stimulated with 10-μM thrombin receptor activator peptide 6 (TRAP-6) for 3 minutes. Normal healthy platelets (top panel), and FXIII-A–deficient platelets (bottom panel). Images representative of normal controls (n = 3) and FXIII-deficient patients (n = 2). Scale bar represents 20 μm. (B) Normal healthy platelets were spread on collagen (100 μg/mL) and thrombin (1 U/mL)–coated slides in the presence of a fluorescent probe, N-TAMRA cadaverine (ex 547), to measure FXIII-A activity for 45 minutes. Cells were then fixed, permeabilized, and stained using AF647-phalloidin to detect actin and FXIII-A using FITC anti–FXIII-A antibody. Scale bar represents 5 μm. (C–E) Normal platelets were washed and spread at 37 °C for 60 minutes on either collagen (100 μg/mL) or fibrinogen (100 μg/mL). Platelets were fixed, permeabilized, and stained with stochastic optical reconstruction microscopy (STORM)–appropriate AF532-labeled phalloidin anti–FXIII-A primary antibody with STORM-appropriate AF647-labeled secondary. Platelets were submerged in ONI Imaging Buffer for direct STORM imaging and imaged in Total internal refractive flourescence (TIRF). (C) Representative images of platelets spread on collagen: merged image (left), AF532-phalloidin (middle; green), and AF647-FXIII-A (right; magenta). Scale bar represents 20 μm. (D) Close-up image of a single platelet spread on collagen (left), with close-up images (right) of actin nodules in merged image (top), AF532-phalloidin (middle; green), and AF-FXIII-A (bottom; magenta). Scale bar on left represents 2 μm, and scale bars on right represents 0.5 μm (E) Close-up image of a single platelet spread on fibrinogen (left), with close-up images (right) of actin nodules in merged image (top), AF532-phalloidin (middle; green), and AF-FXIII-A (right, bottom; magenta). Scale bar on left represents 2 μm, and scale bars on right represent 0.5 μm.