Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity.
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 V(max) value of 6 mol/min/mol fgl2 and an apparent K(m) value for prothrombin of 8.3 microM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent V(max) value to 3670 mol/min/mol fgl2 and decreasing the apparent K(m) value for prothrombin to 7.2 microM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser(89) 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.
Chan
Camie W Y
CW
Multi Organ Transplant Program, Toronto General Hospital and University of Toronto, 621 University Avenue 10th Floor, Room 116, Toronto, Ontario M5G 2C4, Canada.
Chan
Matthew W C
MW
Liu
Mingfeng
M
Fung
Laisum
L
Cole
Edward H
EH
Leibowitz
Julian L
JL
Marsden
Philip A
PA
Clark
David A
DA
Levy
Gary A
GA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001777:Blood Coagulation / Q000235:genetics; D016466:CHO Cells; D002118:Calcium / Q000502:physiology; D006224:Cricetinae; D004789:Enzyme Activation / Q000235:genetics; D005260:Female; D005340:Fibrinogen / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D017737:Macrophages, Peritoneal / Q000201:enzymology / Q000378:metabolism / Q000821:virology; D051379:Mice; D008807:Mice, Inbred BALB C; D006517:Murine hepatitis virus / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics / Q000821:virology; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
C490637:Fgl2 protein, mouse; D010743:Phospholipids; D011994:Recombinant Proteins; D012694:Serine; D005340:Fibrinogen; D013925:Thromboplastin; D013917:Thrombin; D002118:Calcium