Activity and Substrate Specificity of Factor XIII Resulting from Different Activation Pathways

Abstract

Factor XIII (FXIII) is a transglutaminase that catalyzes the formation of a calcium-dependent isopeptide bond between an acyl donor and a primary amine substrate. Plasma FXIII A2B2 is proteolytically activated by the concerted action of thrombin and low mM Ca2+ (FXIIIA∗) whereas cellular FXIII A2 is activated non-proteolytically by high mM Ca2+ concentration (FXIIIA°). Previous studies demonstrated that these activation pathways result in FXIIIA species that differ conformationally and functionally. The activities and substrate specificities of FXIIIA∗ and FXIIIA° were further examined using monodansyl cadaverine (MDC) incorporation and UV-spectrophotometric assays. In the MDC assay, reactive glutamines within fibrinogen αC (233-425) (Q237, Q328, Q366) were crosslinked by FXIII (A∗ versus A°) to MDC (amine substrate), and results were evaluated by SDS-PAGE. The assay revealed that with αC WT, Q328P (Seoul II), and Q237only, FXIIIA∗ had more transglutaminase activity than FXIIIA°. In contrast, αC E396A and αC 389stop (233-388) exhibited no substantial differences in activities between FXIIIA∗ and A°. This observation may be due to loss of αC E396 and surrounding residues from the putative FXIII anchoring site αC (389-403). From the UV-spectrophotometric assay, kinetic parameters for FXIII A∗ and FXIIIA° were determined using glutamine donor peptides (K9 (1-10), α2 AP (1-15) and α2 AP (1-15, Q4S)) crosslinked with the amine DMPDA. Km values for FXIIIA∗ were modestly lower than FXIIIA° for K9 (1-10) and α2 AP (1-15, Q4S) and higher for α2 AP (1-15). The smaller kinetic differences observed with peptides may be due to amino sequence or the need for larger FXIII substrates that better mimic physiological targets. Overall, FXIIIA activity can be a function of its activation pathway, in addition to substrate size and sequence.