Reversible Activation of Cellular Factor XIII by Calcium

Factor XIII (FXIII) is a pro-transglutaminase found in the plasma as well as intracellularly in platelets and macrophages. Plasma FXIII is activated by thrombin cleavage (FXIIIa*) and acts in the final stages of blood coagulation cascade. In contrast, the function and activation of cellular FXIII are less characterized. Cellular FXIII relies on a conformational activation of the protein. The nonproteolytic activation of FXIII to FXIIIa degrees induced by Ca2+ alone is well known, but up until now it has been discussed under which conditions the process can be induced and whether it can be reversed. Here, we study the nature of the Ca2+ -induced FXIII activation. Previously used methods to evaluate FXIII activity detect both FXIIIa* and FXIIIa degrees because they rely on occurrence of enzyme activity or on active site Cys-314 solvent accessibility. Therefore, an analytical HPLC method was developed that separates zymogen recombinant FXIII (rFXIII) from rFXIIIa degrees. The data demonstrate that nonproteolytic activation and deactivation are highly dependent on Ca2+ concentration, buffer, and salt components. Moreover, it is established that Ca2+ activation of rFXIII is fully reversible, and only 2-5 mM CaCl2 is sufficient to retain full rFXIIIa degrees activity. However, below 2 mM CaCl2 the rFXIIIa degrees molecule deactivates. The deactivated molecule can subsequently undergo a new activation round. Furthermore, it is demonstrated that thermal stress of freeze-dried rFXIII can induce a new predisposed form that activates faster than nonstressed rFXIII.