Kinetic Analysis of a Unique Direct Prothrombinase, fgl2, and Identification of a Serine Residue Critical for the Prothrombinase Activity

Abstract

fgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this study, the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidyl-l-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent Vmax value of 6 mol/min/mol fgl2 and an apparent Km value for prothrombin of 8.3 μM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent Vmax value to 3670 mol/min/mol fgl2 and decreasing the apparent Km value for prothrombin to 7.2 μM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser89 was critical for the prothrombinase activity of fgl2. Furthermore, fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.

Intact type 1 immunity and immune-associated coagulative responses in mice lacking IFNγ-inducible fibrinogen-like protein 2

“…While studying a lethal coagulopathy induced by hepatitis virus in certain strains of mice, Levy and coworkers (5) identified Fgl2 as the target of a mAb that suppressed the coagulopathy. Follow-up in vitro studies suggested that Fgl2 possesses direct coagulant activity (8). Fgl2-specific antibodies were also found to suppress the coagulationassociated spontaneous abortion that characterizes certain mouse strain breeding combinations (6).…”

“…Given that Fgl2 is IFN␥-inducible (Figs. 1 and 2) and putatively contributes to fibrin formation (5,7,8), we evaluated T. gondii infection in Fgl2-deficient mice. In contrast to IFN␥-or fibrin-deficient mice, which succumb within 2 weeks of infection (12,27), Fgl2-deficient mice survived acute infection by strain ME49 T. gondii.…”

“…Thus, despite its IFN␥ inducibility (Figs. 1 and 2) and putative procoagulant functions (5,7,8), Fgl2 expression is not required to survive the acute phase of T. gondii infection in mice, a model in which both IFN␥ and coagulation play critical protective roles (12,27).…”

“…Functional studies using neutralizing antisera suggested that Fgl2 may also contribute to coagulant activity associated with spontaneous abortion in mice (6). These observations led to a series of publications describing the direct coagulant activity of Fgl2 (5,7,8). In addition, recombinant Fgl2 was recently shown to suppress T cell proliferation and dendritic cell maturation in vitro (9).…”

Global analysis of gene expression inXenopushindlimbs during stage‐dependent complete and incomplete regeneration

“…The prothrombinase fibroleukin (fibrinogen-like protein 2, FGL2) is related to the fibrinogen beta and gamma chains, which can directly cleave prothrombin to thrombin (Chan et al, 2002). FGL2 appears to exist in two forms: a membrane-linked FGL2 with prothrombinase activity, and a soluble FGL2 that has potent immunosuppressive activity .…”