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°i nduced by Ca 2؉ 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 Ca 2؉ -induced FXIII activation. Previously used methods to evaluate FXIII activity detect both FXIIIa* and FXIIIa°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°. The data demonstrate that nonproteolytic activation and deactivation are highly dependent on Ca 2؉ concentration, buffer, and salt components. Moreover, it is established that Ca 2؉ activation of rFXIII is fully reversible, and only 2-5 mM CaCl 2 is sufficient to retain full rFXIIIa°activity. However, below 2 mM CaCl 2 the rFXIIIa°mol-ecule 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. Factor XIII (FXIII)2 (EC 2.3.2.13) is a pro-transglutaminase found in the plasma as well as intracellularly in platelets, monocytes/macrophages, and bone marrow precursors of these cell types. Activated FXIII catalyzes the formation of ⑀-(␥-glutamyl)lysine cross-links between the glutamine ␥-carboxymide group and lysine ⑀-amino-group, resulting in intra-and intermolecular isopeptide cross-links.Plasma FXIII (pFXIII) is a heterotetrameric zymogen consisting of two catalytic A subunits and two carrier B subunits. Activation of pFXIII during the blood coagulation cascade takes place when thrombin cleaves the 37-residue N-terminal activation peptide from the A subunits. In the presence of fibrin and Ca 2ϩ , the FXIII-A subunits undergo conformational changes and are restructured, and the activation peptide and B subunits are released. This results in the thrombin-activated FXIII (FXIIIa*). FXIIIa* is essential for stabilizing the hemostatic clot by cross-linking the fibrin monomers (1-4).In contrast, the function and activation of a cellular FXIII (cFXIII) are less characterized. However, cFXIII, in particular cFXIII from platelets, accounts for approximately half of the FXIII found in the body (5, 6). cFXIII is a homodimeric zymogen consisting solely of catalytic A subunits. cFXIII has been reported to be involved in cytoskeleton remodeling during platelet activation (7,8), as well as other aspects of tissue remodeling in e.g. wound healing and bone development (for review, see Refs. 9 -11).Furt...