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Coagulation transglutaminase factor XIII (FXIII) exists in circulation as heterotetrameric proenzyme FXIII-A2B2. Effectively all FXIII-A2B2 circulates bound to fibrinogen, and excess FXIII-B2 circulates in plasma. The motifs that mediate interaction of FXIII-A2B2 with fibrinogen have been elusive. We recently detected reduced binding of FXIII-A2B2 to murine fibrinogen that has γ-chain residues 390-396 mutated to alanines (Fibγ390-396A). Here, we evaluated binding features using human components, including recombinant fibrinogen variants, FXIII-A2B2, and isolated FXIII-A2 and -B2 homodimers. FXIII-A2B2 coprecipitated with wild-type (γA/γA), alternatively-spliced (γ′/γ′), and αC-truncated (Aα251) fibrinogens, whereas coprecipitation with human Fibγ390-396A was reduced by 75% (P < .0001). Surface plasmon resonance showed γA/γA, γ′/γ′, and Aα251 fibrinogens bound FXIII-A2B2 with high affinity (nanomolar); however, Fibγ390-396A did not bind FXIII-A2B2. These data indicate fibrinogen residues γ390-396 comprise the major binding motif for FXIII-A2B2. Compared with γA/γA clots, FXIII-A2B2 activation peptide release was 2.7-fold slower in Fibγ390-396A clots (P < .02). Conversely, activation of recombinant FXIII-A2 (lacking FXIII-B2) was similar in γA/γA and Fibγ390-396A clots, suggesting fibrinogen residues γ390-396 accelerate FXIII-A2B2 activation in a FXIII-B2–dependent mechanism. Recombinant FXIII-B2 bound γA/γA, γ′/γ′, and Aα251 with similar affinities as FXIII-A2B2, but did not bind or coprecipitate with Fibγ390-396A. FXIII-B2 also coprecipitated with fibrinogen from FXIII-A–deficient mouse and human plasmas. Collectively, these data indicate that FXIII-A2B2 binds fibrinogen residues γ390-396 via the B subunits, and that excess plasma FXIII-B2 is not free, but rather circulates bound to fibrinogen. These findings provide insight into assembly of the fibrinogen/FXIII-A2B2 complex in both physiologic and therapeutic situations. [ABSTRACT FROM AUTHOR]