Crosslinking kinetics of the human transglutaminase, factor XIII[A(2)], acting on fibrin gels and gamma-chain peptides

Factor XIII is the terminal enzyme of the coagulation cascade which serves to rapidly crosslink the adjacent gamma-chain C-termini of fibrin clots. In vivo, this process is initiated by the proteolytic action of thrombin which simultaneously converts both soluble fibrinogen to fibrin and activates zymogen FXIII; fibrin then spontaneously polymerizes to form a gel which activated FXIII stabilizes through crosslinking. Due to the kinetic complexity and the difficulty of investigating gel phase reactions, methods employing pre-activation of recombinant human Factor XIII (rFXIII[A'(2)]) were developed to effectively decouple these reactions. By utilizing these methods, the kinetic parameters of gamma-chain crosslinking in fibrin gels could be determined by both initial rate and integrated rate techniques under physiologically relevant conditions. The crosslinking of the gamma-chain of fibrin gels could be described by apparent Michaelis kinetics with K-m(app) = 6.2 mu M, k(cat) = 1872 min(-1), and K-sp = 302 min(-1) mu M(-1) for a fibrin gamma-chain monomer of M(r) = 170 000 Da. In contrast, both the crosslinking rates of alpha-chains within fibrin gels (K-sp = 0.38 min(-1) mu M(-1): Bishop et al. (1993)) and the crosslinking of a soluble synthetic peptide containing the unique gamma-chain fibrin crosslinking site (K-sp = 0.030 min(-1) mu M(-1)) could not be shown to saturate and gave apparent first-order rates with respect to rFXIII[A'(2)]. These observations coupled with the large differences in the turnover rates (similar to 10(4)) suggest two likely mechanisms for FXIII[A'(2)]-substrate interactions: (1) random (or independent) binding of non- or weakly interacting substrate pairs imposes a high entropic barrier (i.e., Delta G(binding)) to the formation of a productive catalytic complex, e.g., for soluble gamma-chain peptides and the flexible alpha-chains within fibrin, and (2) binding to an oriented substrate pair effectively lowers the entropic barrier to formation of a Michaelis complex and thus greatly enhances the rate of catalysis, e.g., for gamma-chain pairs within the fibrin fibrils.