Ranking reactive glutamines in the fibrinogen αC region that are targeted by blood coagulant factor XIII

Abstract

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

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

“…Proteolysis of fibrinogen by GoF ADAMTS‐13 removes the α‐chain residues 241–476 and 518–584 (Fig. 2C), which are required for α‐chain association 34.…”

Characterizing reactive glutamines in fibrinogen and elucidating factor XIII substrate specificity.

“…In our previous study (Chapter 3), a mass spectrometry and a complementary NMR spectroscopy assay were developed to monitor and rank the extent of crosslinking for three known reactive glutamines (Q237, Q366, and Q328) within a segment of the αC region (αC 233-425). 108 Fbg αC (233-425) was previously used to localize and characterize a Factor XIII binding site within the fibrinogen Aα chain. 72 A residue within this αC region, Aα E396, was identified to be critical for the binding of Factor XIII.…”

“…More recently, research findings have suggested that the alpha chain of fibrinogen contributes unique roles to the overall clot structure and stability. 86,108,109 The main focus of this dissertation has been to understand fibrin crosslinking at a molecular level.…”

“…108 For this assay, Factor XIII (500 nM final) was incubated with Fbg αC WT or E396A, GEE, and MALDI kinetic buffer for 5 min at 25ºC to promote…”

“…For these experiments, the pre-activated Factor XIII activation strategy described in Chapter 3 was used. 108 Briefly, Factor XIII A 2 (500 nM) was preactivated in the presence of thrombin (8.4 U/ml) and calcium (4 mM).…”