Key Points• FXIIIa exhibits a preference for Q237 in crosslinking reactions within fibrinogen aC (233-425) followed by Q328 and Q366.• None of the reactive glutamines in aC 233-425 (Q237, Q328, and Q366) are required to react first before the others can crosslink.Factor XIIIa (FXIIIa) introduces covalent g-glutamyl-«-lysyl crosslinks into the blood clot network. These crosslinks involve both the g and a chains of fibrin. The C-terminal portion of the fibrin a chain extends into the aC region (210-610). Crosslinks within this region help generate a stiffer clot, which is more resistant to fibrinolysis. Fibrinogen aC (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 N-GEE could be crosslinked to the three glutamines in aC (233-425) with a similar order of reactivity as observed with the MALDI-TOF mass spectrometry assay. NMR studies using the single aC 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. (Blood. 2016;127(18):2241-2248
apparent that conformational reorganization coupled to the ionization of the buried group is a major determinant of these pK a values. Specifically, the creation of charge in hydrophobic environments can trigger a shift from the fully folded state to local or partially unfolded states in which the charge can gain access to water or to an environment where the charge can be solvated. These alternative conformational states are not normally populated owing to the large free energy difference between the alternative and fully-folded native states; however, the partially unfolded states can become the new ground state under pH conditions where the internal group is charged. If the ionization of an internal group promotes the transition to a new conformational state then its pK a should be sensitive to the global thermodynamic stability (DG) of the protein because this determines the energy gap between the ground and the alternative states. This was tested by measuring the pK a of two internal Lys residues in variants of staphylococcal nuclease with thermodynamic stabilities ranging from 8.4 to 13.8 kcal/mol. The magnitude of the shift in the pK a of the internal Lys residues was found to be sensitive to the DG of the protein confirming that the pK a values of these Lys residues are determined by the probability of structural reorganization more than by local dielectric properties of their microenvironments. These observations imply that structure-based pK a calculations for buried groups and other electrostatic processes in hydrophobic environments require accurate treatment of conformational reorganization, which remains an extremely challenging proposition.
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