Fibrinogen is the most abundant protein involved in blood coagulation and has been implicated in cardiovascular disease. During the formation of a blood clot, thrombin initiates fibrin polymerization by cleaving fibrinopeptides A and B from fibrinogen (AαBβγ)2 to form fibrin monomers (αβγ)2. A more stable clot is generated when activated Factor XIII (FXIIIa) introduces covalent γ-glutamyl-ε-lysyl bonds between specific reactive glutamines and lysines in the fibrin clot network. The αC region (Aα221-610) of fibrinogen contains numerous reactive glutamines crosslinked by FXIIIa. However, this region has not been observed by X-ray crystallography. We explored a segment of the αC region—αC (233-425)—as a model protein to understand Factor XIIIa’s substrate specificity. This segment contains a binding site for FXIII located within αC (371-425) and three reactive glutamines (Q237, Q328, and Q366). We hypothesize that Factor XIIIa's specificity towards these reactive glutamines is promoted by the intrinsically disordered nature of the αC region and a proximal binding site for Factor XIII. A combination of MALDI-TOF mass spectrometry and 2D 1H -15N HSQC NMR studies were performed on fibrinogen αC (233-425). Factor XIIIa’s substrate specificity for αC (233-425) was monitored by its ability to crosslink a lysine mimic, glycine ethyl ester (GEE) to the reactive glutamines (Q237, Q328, Q366) over time. A peak height ratio method was used to quantify the rankings. Single site mutations were generated in which each glutamine (Q) in αC (233-425) was replaced with an inactive asparagine (N). Crosslinking for the remaining reactive glutamines was then monitored in a complementary 2D HSQC NMR with 15N-labeled GEE. Fibrinogen αC (233-425) was also expressed in 15N-enriched minimal media for 2D HSQC NMR structural characterization. Results from this project have provided the first ranking of three known reactive glutamines in the αC region of fibrinogen. FXIIIa crosslinks these glutamines to the lysine mimic glycine ethyl ester (GEE) in the order Q237 &gt;&gt; Q366 ≈ Q328. Our work with Q to N mutations in αC (233-425) suggests that FXIIIa does not require crosslinking to occur in a sequential manner. Each glutamine can be cross-linked independently. 2D HSQC NMR experiments with 15N-labeled αC (233-425) confirmed that this protein is intrinsically disordered. The knowledge gained from these studies can be used for subsequent drug designs that are specific for targeting Factor XIII.

Fibrinogen αC (233-425): A model protein for characterizing cross-linking by the transglutaminase factor XIII

253rd National Meeting (2017)

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