Transglutaminase Activities of Blood Coagulant Factor XIII Are Dependent on the Activation Pathways and on the Substrates.

Factor XIII (FXIII) catalyzes formation of γ-glutamyl-ε-lysyl crosslinks between reactive glutamines (Q) and lysines (K). In plasma, FXIII is activated proteolytically (FXIII-A*) by the concerted action of thrombin and Ca2+. Cellular FXIII is activated nonproteolytically (FXIII-A°) by elevation of physiological Ca2+ concentrations. FXIII-A targets plasmatic and cellular substrates, but questions remain on correlating FXIII activation, resultant conformational changes, and crosslinking function to different physiological substrates. To address these issues, the characteristics of FXIII-A* versus FXIII-A° that contribute to transglutaminase activity and substrate specificities were investigated. Crosslinking of lysine mimics into a series of Q-containing substrates were measured using in-gel fluorescence, mass spectrometry, and UV-Vis spectroscopy. Covalent incorporation of fluorescent monodansylcadaverine revealed that FXIII-A* exhibits greater activity than FXIII-A° toward Q residues within Fbg αC (233-425 WT, Q328P Seoul II, and Q328PQ366N) and actin. FXIII-A* and FXIII-A° displayed similar activities toward α2-antiplasmin (α2AP), fibronectin, and Fbg αC (233-388, missing FXIII-binding site αC 389-402). Furthermore, the N-terminal α2AP peptide (1-15) exhibited similar kinetic properties for FXIII-A* and FXIII-A°. MALDI-TOF mass spectrometry assays with glycine ethyl ester and Fbg αC (233-425 WT, αC E396A, and truncated αC (233-388) further documented that FXIII-A* exerts greater benefit from the αC 389-402 binding site than FXIII-A°. Conformational properties of FXIII-A* versus A° are proposed to help promote transglutaminase function toward different substrates. A combination of protein substrate disorder and secondary FXIII-binding site exposure are utilized to control activity and specificity. From these studies, greater understandings of how FXIII-A targets different substrates are achieved.

Evaluating the Effects of Fibrinogen αC Mutations on the Ability of Factor XIII to Crosslink the Reactive αC Glutamines (Q237, Q328, Q366).

Fibrinogen (Fbg) levels and extent of fibrin polymerization have been associated with various pathological conditions such as cardiovascular disease, arteriosclerosis, and coagulation disorders. Activated factor XIII (FXIIIa) introduces γ-glutamyl-ε-lysinyl isopeptide bonds between reactive glutamines and lysines in the fibrin network to form a blood clot resistant to fibrinolysis. FXIIIa crosslinks the γ-chains and at multiple sites in the αC region of Fbg. Fbg αC contains a FXIII binding site involving αC (389-402) that is located near three glutamines whose reactivities rank Q237 >> Q366 ≈ Q328. Mass spectrometry and two-dimensional heteronuclear single-quantum correlation nuclear magnetic resonance assays were used to probe the anchoring role that αC E396 may play in controlling FXIII function and characterize the effects of Q237 on the reactivities of Q328 and Q366. Studies with αC (233-425) revealed that the E396A mutation does not prevent the transglutaminase function of FXIII A2 or A2B2. Other residues must play a compensatory role in targeting FXIII to αC. Unlike full Fbg, Fbg αC (233-425) did not promote thrombin cleavage of FXIII, an event contributing to activation. With the αC (233-425) E396A mutant, Q237 exhibited slower reactivities compared with αC wild-type (WT) consistent with difficulties in directing this N-terminal segment toward an anchored FXIII interacting at a weaker binding region. Q328 and Q366 became less reactive when Q237 was replaced with inactive N237. Q237 crosslinking is proposed to promote targeting of Q328 and Q366 to the FXIII active site. FXIII thus uses Fbg αC anchoring sites and distinct Q environments to regulate substrate specificity.