Reversible Activation of Cellular Factor XIII by Calcium

Gunhild Klarskov Kristiansen,Mette Dahl Andersen

doi:10.1074/jbc.m110.174128

Abstract

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° induced 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° 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.

Highlights

Plasma Factor XIII (FXIII) is a heterotetrameric zymogen consisting of two catalytic A subunits and two carrier B subunits
Biophysical characterization has revealed that conformational changes in the secondary structure of rFXIIIa° alter its surface charge and hydrophobicity relative to native recombinant cellular FXIII (rFXIII).3
These changes were utilized in the development of an analytical Anion Exchange HPLC (AIEC) that separated rFXIII and rFXIIIa°. rFXIIIa° was eluted from the column with the activity intact as measured in enzymatic assay

Summary

To whom correspondence should be addressed

In the presence of fibrin and Ca2ϩ, 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*). Several reports have demonstrated that cFXIII is activated without cleavage of the activation peptide, resulting in the FXIIIa° molecule [12,13,14] This nonproteolytic activation of FXIII has been demonstrated to occur in cells e.g. upon platelet activation [12, 13]. These additional Ca2ϩ binding sites were proposed to play a part in the restructuring occurring upon enzyme activation [32]. It is established that Ca2ϩ activation of rFXIII is fully reversible and that the molecule subsequently can undergo a new activation round

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION