Role of calcium in the conformational dynamics of factor XIII activation examined by hydrogen-deuterium exchange coupled with MALDI-TOF MS

Factor XIII catalyzes formation of γ-glutamyl-ε-lysyl crosslinks within fibrin clots. FXIII A₂ can be activated proteolytically with thrombin and low mM Ca²⁺ or nonproteolytically with high monovalent/divalent cations along with low mM Ca²⁺. Physiologically, FXIII A₂ is poised to respond to transient influxes of Ca²⁺ in a Na⁺ containing environment. A successful strategy to monitor FXIII conformational events is hydrogen–deuterium exchange (HDX) coupled with mass spectrometry. FXIII A₂ was examined in the presence of different cations (Ca²⁺, Mg²⁺, Ba²⁺, Cu²⁺, Na⁺, TMAC⁺, and EDA²⁺) ranging from 1 to 2 mM, physiological Ca²⁺ concentration, to 50–500 mM for nonproteolytic activation. Increases in FXIII solvent exposure could already be observed at 1 mM Ca²⁺ for the dimer interface, the catalytic site, and glutamine substrate regions. By contrast, solvent protection was observed at the secondary cleavage site. These events occurred even though 1 mM Ca²⁺ is insufficient for FXIII activation. The metals 1 mM Mg²⁺, 1 mM Ba²⁺, and 1 mM Cu²⁺ each led to conformational changes, many in the same FXIII regions as Ca²⁺. FXIII could also be activated nonproteolytically with 500 mM tetramethylammonium chloride (TMAC⁺) and 500 mM ethylenediamine (EDA²⁺), both with 2 mM Ca²⁺. These different HDX studies help reveal the first FXIII segments that respond to physiological Ca²⁺ levels.