The dimeric FXIII-A(2), a pro-transglutaminase is the catalytic part of the heterotetrameric
coagulation FXIII-A(2)B(2) complex that upon activation by calcium binding/thrombin
cleavage covalently cross-links preformed fibrin clots protecting them from premature
fibrinolysis. Our study characterizes the recently disclosed three calcium binding
sites of FXIII-A concerning evolution, mutual crosstalk, thermodynamic activation
profile, substrate binding, and interaction with other similarly charged ions. We
demonstrate unique structural aspects within FXIII-A calcium binding sites that give
rise to functional differences making FXIII unique from other transglutaminases. The
first calcium binding site showed an antagonistic relationship towards the other two.
The thermodynamic profile of calcium/thrombin-induced FXIII-A activation explains
the role of bulk solvent in transitioning its zymogenic dimeric form to an activated
monomeric form. We also explain the indirect effect of solvent ion concentration on
FXIII-A activation. Our study suggests FXIII-A calcium binding sites could be putative
pharmacologically targetable regions.