Abstract Number: OC 42.1
Meeting: ISTH 2021 Congress
Background: Transglutaminase Factor XIII (FXIII) inhibits fibrinolysis by crosslinking reactive glutamines and lysines between fibrin and other proteins. Fibrinogen αC (233 – 425) contains three reactive glutamines (Q237, Q328, and Q366) and a FXIII binding site (αC 389 – 402) where E396 was reported as instrumental for FXIII binding. Intriguingly, crosslinking is reduced but not abolished in the absence of E396. Further examination of αC 389 – 402 and its importance to FXIII activity is therefore warranted.
Aims: This project sought to elucidate key features of Fbg αC (389 – 402) that promote FXIII crosslinking activity in Fbg αC. Mutants involving αC E395, E396, and the full αC binding region were examined.
Methods: Fbg αC (233 – 425) species with substitutions E396A, E396D, E395A, E395K, E395S, as well as a mutant without the binding region (αC 233 – 388, “389 Stop”), were recombinantly expressed and purified. Glycine ethyl ester (GEE) crosslinking to αC reactive glutamines was monitored over time by mass spectrometry. Assays were performed in triplicate and reported as mean ± SD.
Results: Fbg αC “389 Stop” exhibited reduced Q237-GEE crosslinking at 15 minutes by 4-fold (P ≤ 0.001) compared to WT. In comparison, Fbg αC E396A diminished Q237-GEE crosslinking by ~2-fold (P ≤ 0.01) at 15 minutes with respect to WT. Fbg αC E395A, E396D, and E395K had no significant effects on the amount of Q237-GEE crosslinking (P > 0.05), whereas E395S significantly increased the crosslinking by ~2-fold (P ≤ 0.001) at 15 minutes when compared to WT.
Conclusions: Fbg αC 389 – 402 boosts FXIIIA* activity, but is not required for crosslinking. The electrostatic contribution of αC E396 has a greater impact on FXIII activity than E395, but does not fully account for the binding region’s enhancement of crosslinking. The αC anchoring role is proposed to be regulated by αC sequence and FXIII structure.
To cite this abstract in AMA style:Ablan FD, Hindi M, Maurer M. Role of the Coagulation Factor XIII Binding Site on Crosslinking of Fibrinogen αC (233 – 425) [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/role-of-the-coagulation-factor-xiii-binding-site-on-crosslinking-of-fibrinogen-%ce%b1c-233-425/. Accessed September 24, 2021.
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