FXIII-A₂ stabilizes FXIII-B₂ in circulation. (A) rFXIII-B₂ was incubated in normal, pooled human plasma for 24 hours at 37°C in the absence or presence of rFXIII-A₂ and visualized by immunoblotting with anti-FXIII-B antibody. Representative blot of N = 2 experiments. (B) Fibrinogen, FXIII-A, and FXIII-B antigen were visualized in plasma from wild-type, Fibγ^390-396A, or afibrinogenemic (Fga^–/–) mice by immunoblotting using anti-fibrinogen, anti–FXIII-A, and anti–FXIII-B antibodies. (C) Schematic of in vitro FXIII-A₂B₂ activation with thrombin (IIa) and CaCl₂ to separate FXIII-A and -B subunits before infusion. Reactions were performed in the presence of the transglutaminase inhibitor T101 to inhibit activated FXIII-A₂^∗ (FXIII-A₂^∗-I [inhibited dimeric or monomeric FXIIIa]), and thrombin was quenched with hirudin before infusion into F13a1^–/– mice via the tail vein. (D) Representative immunoblot of FXIII-A and -B antigen in mice infused with complexed (FXIII-A₂B₂) or uncomplexed FXIII-B₂ at the indicated times after infusion. Subunits were visualized with anti–FXIII-A and -B antibodies. (E) FXIII-B antigen over time, after infusion, was visualized for each condition (solid line, complexed; dashed line, uncomplexed) using immunoblot with anti–FXIII-B antibody and quantified by densitometry. Data were quantified relative to FXIII-B antigen at the 0 hour timepoint immediately after infusion. Dots show mean ± standard deviation, N = 1-11/time point. (F) Relative FXIII-B antigen for each condition at the 6 hour timepoint. Dots represent individual mice, bars show mean ± standard deviation. Groups were compared using a 2-tailed, unpaired t test, ∗∗ P < .01. A.U., arbitrary units.