Factor XIIIa (FXIIIa) introduces covalent ?-glutamyl-?-lysyl crosslinks into the blood clot network. These crosslinks involve both the ? and ? chains of fibrin. The C-terminal portion of the fibrin ? chain extends into the ?C region (210-610). Crosslinks within this region help generate a stiffer clot, which is more resistant to fibrinolysis. Fibrinogen ?C (233-425) contains a binding site for FXIIIa and three glutamines Q237, Q328, and Q366 that each participate in physiological crosslinking reactions. Although these glutamines were previously identified, their reactivities toward FXIIIa have not been ranked. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) methods were thus used to directly characterize these three glutamines and probe for sources of FXIIIa substrate specificity. Glycine ethyl ester (GEE) and ammonium chloride served as replacements for lysine. Mass spectrometry and 2D heteronuclear single quantum coherence NMR revealed that Q237 is rapidly crosslinked first by FXIIIa followed by Q366 and Q328. Both (15)NH4Cl and (15)N-GEE could be crosslinked to the three glutamines in ?C (233-425) with a similar order of reactivity as observed with the MALDI-TOF mass spectrometry assay. NMR studies using the single ?C mutants Q237N, Q328N, and Q366N demonstrated that no glutamine is dependent on another to react first in the series. Moreover, the remaining two glutamines of each mutant were both still reactive. Further characterization of Q237, Q328, and Q366 is important because they are located in a fibrinogen region susceptible to physiological truncations and mutation. The current results suggest that these glutamines play distinct roles in fibrin crosslinking and clot architecture.? 2016 by The American Society of Hematology.