Probing Factor XIII Substrate Specificity for Fibrinogen αC (233-425) Using Maldi-Tof Mass Spectrometry and 2D 15N-1H HSQC NMR Spectroscopy

Abstract

In blood coagulation, the transglutaminase FXIII introduces covalent γ-glutamyl-e-lysinyl crosslinks into the fibrin clot network. The C-terminal portion of the Fibrinogen Aα chain contains the αC region (221-610) that has a flexible αC-connector segment (221-391) and a globular αC domain (392-610). Reactive glutamine (Q) residues from the αC-connector can be covalently cross-linked by activated FXIII (FXIIIa) to reactive lysines (K) located in the C-terminal portion of the αC domain on another fibrin molecule. Studies have shown that αC based crosslinks help generate a clot that has more lateral aggregation, is stronger, and is more resistant to fibrinolysis. Fibrinogen αC (233-425) contains three reactive glutamines (Q237, Q328, Q366) that participate in cross-linking but in-depth kinetic information is not available on individual residues. A combination of mass spectrometry and NMR-based methods is being used to probe for FXIIIa substrate specificity for fibrinogen αC region (233-425). In the MALDI-TOF mass spectrometry assay, FXIIIa-catalyzed cross-linking between reactive glutamines on αC (233-425) and the lysine-mimic glycine ethylester (GEE) are monitored following chymotrypsin and Glu-C protease proteolytic digests. MALDI-TOF MS runs reveal that FXIIIa cross-links GEE to all three reactive glutamines Q237, Q328, and Q366. Q237 is cross-linked first followed by Q328 and Q366. Complementary 2D 15N-1H HSQC experiments show the incorporation of 15NH4Cl or 15N -GEE into three separate glutamines on αC (233-425) in the presence of FXIIIa.Gel electrophoresis studies further reveal that the lysine mimic dansylcadaverine and the Q-containing peptide Dansyl-Ahx-α2-antiplasmin(1-15) can each be crosslinked into αC (233-425).The new knowledge gained about Fibrinogen αC (233-425) and substrate reactivity may later be used to design therapeutic Aα fibrinogens that could influence fibrin clot character.