Protransglutaminase (Factor XIII) Mediated Crosslinking of Fibrinogen and Fibrin*

Abstract

SummaryPlasma factor XIII (plasma protransglutaminase) circulates as an A2B2 tetramer bound to the γ’ variant chains of fibrinogen “2”. During clotting the A subunits of fXIII are cleaved by thrombin to form fXIIIa (transglutaminase) and in the presence of calcium ions, activated A2* subunits dissociate from the B subunits. When purified plasma fXIII or recombinant cellular factor XIII (A2) was incubated with fibrinogen in the presence of calcium ions (≥50 μM) a non-synerizing gel formed concomitant with formation of γ dimers, followed by A γ polymers, and eventually γ trimers and γ tetramers. As is the case of fXIIIa, the fXIII-mediated crosslinking rate was enhanced in the presence of thiols. After an initial lag period, fXIII catalyzed fibrinogen cross-linking at ~75% of the rate of fXIIIa under typical crosslinking conditions (100 Loewy u/ml, 5 mM CaCl2 & 500 μM DTT). Fibrin was crosslinked about 8 times more rapidly by fXIII than was fibrinogen, and after an initial lag period fXIII crosslinked fibrin at nearly the same rate as fXIIIa. Substituting plasma for purified fXIII as the source for fXIII resulted in robust fibrinogen crosslinking activity. In contrast to the high level of fXIII-mediated crosslinking activity observed with fibrinogen or fibrin as substrates, when transglutamination was measured using cadaverine incorporation into casein, fXIII was 30-fold less active than fXIIIa. Thus, factor XIII displays constitutive enzymatic activity with respect to fibrinogen and fibrin. The results further indicate that uncleaved fXIII in plasma provides a potent source of readily available crosslinking activity in clotting blood. Fibrinogen 2, whose γ’chains bind fXIII B subunits, was crosslinked 3.5 times more slowly by fXIII than was fibrinogen 1 (lacking γ’ chains), suggesting that complex formation between fibrinogen 2 and plasma fXIII plays a significant role in down-regulating potential plasma fXIII-mediated crosslinking activity. Since fibrin is a considerably better substrate for fXIII than is fibrinogen, the rate at which crosslinking takes place in a fibrinogen-containing plasma environment is much lower than it would be if fibrin were present.