Key Points• Fibrin aC389-402 binds a cleft in the b-sandwich domain of FXIII-A 2 * exposed only after cleavage of the activation peptide.• Binding of fibrin aC389-402 to FXIII-A 2 * regulates fibrin cross-linking and thus clot stabilization and fibrinolysis.Formation of a stable fibrin clot is dependent on interactions between factor XIII and fibrin. We have previously identified a key residue on the aC of fibrin(ogen) (Glu396) involved in binding activated factor XIII-A 2 (FXIII-A 2 *); however, the functional role of this interaction and binding site(s) on FXIII-A 2 * remains unknown. Here we (1) characterized the functional implications of this interaction; (2) identified by liquidchromatography-tandem mass spectrometry the interacting residues on FXIII-A 2 * following chemical cross-linking of fibrin(ogen) aC389-402 peptides to FXIII-A 2 *; and (3) carried out molecular modeling of the FXIII-A 2 */peptide complex to identify contact site (s) involved. Results demonstrated that inhibition of the FXIII-A 2 */aC interaction using aC389-402 peptide (Pep1) significantly decreased incorporation of biotinamidopentylamine and a2-antiplasmin to fibrin, and fibrin cross-linking, in contrast to Pep1-E396A and scrambled peptide controls. Pep1 did not inhibit transglutaminase-2 activity, and incorporation of biotinyl-TVQQEL to fibrin was only weakly inhibited. Molecular modeling predicted that Pep1 binds the activation peptide cleft (AP-cleft) within the b-sandwich domain of FXIII-A 2 * localizing aC cross-linking Q366 to the FXIII-A 2 * active site. Our findings demonstrate that binding of fibrin aC389-402 to the APcleft is fundamental to clot stabilization and presents this region of FXIII-A 2 * as a potential site involved in glutamine-donor substrate recognition. (Blood. 2013;121(11):2117-2126

A novel class of potent FXIII-A inhibitors containing a (±) cis-bisamido epoxide pharmacophore is described. The compounds display highly potent inhibition of FXIII-A (IC 50 = 5-500 nM) in an in vitro assay. In contrast to other types of previously described covalent transglutaminase inhibitors, the bis-amido epoxides exhibited no measurable reactivity with glutathione, therefore possibly rendering this class of compounds suitable for future in vivo investigations. Additionally, the compounds show selective inhibition for FXIII-A against the cysteine protease, cathepsin S although they proved to have similar potency with a closely related transglutaminase, TGII, to that observed for FXIII-A.