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* Multi Organ Transplant Program, Toronto General Hospital and University of Toronto, Toronto, Ontario, Canada;
Department of Pathology, Texas A&M University, College Station, TX 77843; and
McMaster University, Hamilton, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our laboratory has previously demonstrated the pivotal role of a unique
direct prothrombinase, fgl2, in the pathogenesis of fulminant viral
hepatitis secondary to murine hepatitis virus strain-3
(MHV-3)3 infection
(11, 12, 13). The gene fgl2 was originally cloned
from CTLs, and the encoded protein was named as a fibrinogen-like
protein due to homology to the carboxyl terminus of the 
- and
-chains of fibrinogen (14). Several lines of evidence
indicate fgl2 exhibits coagulation activity. First, following MHV-3
infection, mRNA transcripts of fgl2 and
fgl2 protein can be detected predominantly in liver
reticuloendothelial cells, followed by fibrin deposition and widespread
hepatic necrosis (13). We have recently reported a similar
relationship of fgl2 to fulminant hepatitis B virus infection
(1). Second, neutralizing mAb to fgl2 prevents hepatic
fibrin deposition, liver cell necrosis, and the lethality of MHV-3
infection (15). Third, fgl2 is also expressed in a model
of Th1 cytokine-induced fetal loss syndrome in which fibrin deposition
is a prominent feature (16, 17), and neutralizing Ab to
fgl2 prevents cytokine-induced fetal loss (16). Recently,
Ruegg and colleagues (18, 19) have reported the expression
of FGL2 in human peripheral blood T lymphocytes and termed the encoded
protein fibroleukin. Interestingly, FGL2 expressed by T cells as a
secreted form lacks a coagulation activity, and the function for T cell
generated fgl2/fibroleukin remains undefined.
Based on our previous findings, we believe induction of fgl2 represents a novel mechanism whereby macrophages and endothelial cells activate the coagulation cascade. However, the nature of this fgl2 coagulation activity has not been fully defined. In this paper, we generated murine fgl2 protein by a baculovirus expression system and examined the molecular basis of its prothrombinase activity. Using purified fgl2, we assessed the kinetic parameters of thrombin generation. We further demonstrated that phospholipids, calcium, and factor Va were required for the optimal catalytic activity of fgl2. A combination of site-directed mutagenesis and production of truncated proteins identified a domain and a single amino acid residue essential for the coagulation activity of fgl2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Log-phase Spodoptera frugiperda ovarian cells 9 (Sf9) and High Five (H5) cells (Invitrogen, Carlsbad, CA) were maintained in Insect Pathology Laboratory 41 medium supplemented with 10% FBS, 10 µg/ml gentamicin, 3.3 µg/L yeastolate, 2.5 µg/ml fungizone, and 1x lipid concentrate at 27°C as monolayers. Recombinant baculovirus carrying murine fgl2 cDNA was generated in Sf9 cells and fgl2 protein was expressed in H5 cells. Chinese hamster ovary (CHO)-K1 cells were cultured in DMEM supplemented with 2 mM L-glutamine and 10% FBS. All tissue culture reagents were purchased from Life Technologies (Mississauga, Ontario, Canada).
Expression of fgl2 protein
Fgl2 protein with a tandem repeat of six histidine residues followed by an enterokinase cleavage site fused to the N terminus was prepared according to the Invitrogen Insect Expression System protocol. Briefly, a 1.4-kb cDNA encoding murine fgl2 was amplified using the forward primer 5'-TGCCGCACTGGATCCATGAGGCTTCCTGGT-3' (with the methionine start codon underlined) and the reverse primer 5'-TTATGGCTTGAAATTCTTGGGC-3' (nt 1283–1302 relative to the ATG start codon). Amplification was performed for 25 cycles for 2 min at 96°C, 2 min at 55°C, and 3 min at 72°C. The PCR product was cloned into the EcoRI and BamHI sites of the vector pBlueBacHis2A (Invitrogen).
Recombinant baculoviruses were generated by cotransfecting Sf9 cells with 2 µg pBlueBacHis2A carrying fgl2 insert plus 1 µg linearized Autographa californica multiple nuclear polyhedrosis virus DNA using the Bac-N-Blue transfection kit (Invitrogen). Putative recombinant viruses were screened for the presence of fgl2 by PCR followed by three rounds of viral plaque purification. The sequence of the recombinant baculovirus containing murine fgl2 cDNA was confirmed by an automated DNA sequencer (model no. 377; PE Applied Biosystems, Foster City, CA).
His-tagged fgl2 protein was purified using a nickel resin column according to Invitrogen’s protocol. Briefly, monolayers of H5 cells were infected with the recombinant baculovirus at a multiplicity of infection of 5. Seventy-two hours later, the infected cells were harvested by centrifugation and lysed in 6 M guanidinium hydrochloride, 20 mM sodium phosphate, and 500 mM NaCl. The soluble material was mixed with 50% slurry of ProBond nickel-nitrilotriacetic acid (Ni-NTA) resin (Invitrogen) for 1 h at 4°C. After washings, bound fgl2 protein was eluted with 8 M urea and 20 mM sodium phosphate (pH 5.3) with 150 mM NaCl. The pH of the eluted protein was adjusted to 7.2 immediately upon elution, and the protein was renatured by dialyzing against urea-saline buffers (150 mM NaCl (pH 7.2)) with successive decreases in urea concentrations (6, 4, 2, and 1 M) and finally against TBS (10 mM Tris, 150 mM NaCl (pH 7.2)). The dialyzed material was concentrated and soluble fgl2 protein was collected by spinning at 14,000 rpm for 10 min to remove insoluble particulates. Protein concentrations were determined by BCA assay (Pierce, Rockford, IL).
Homogeneity of purified soluble fgl2 protein was evaluated by SDS-PAGE and confirmed by Western blot probed with anti-fgl2 Ab as previously described (13). Proteins were stained directly using Coomassie brilliant blue or were transferred to nitrocellulose, and then were probed using polyclonal rabbit anti-mouse fgl2 IgG as the primary Ab. The secondary Ab used for immunoblotting was affinity-purified donkey anti-rabbit IgG conjugated to HRP (Amersham, Little Chalfont, U.K.) and the blot was visualized with an immunochemiluminescent kit (Amersham).
Reconstitution of purified fgl2 protein into phospholipid vesicles
Purified soluble fgl2 protein was incorporated into phospholipid vesicles based on the detergent dialysis technique previously described (20, 21, 22). Briefly, hen egg phosphatidyl-L-serine (PS) and bovine brain phosphatidylcholine (PC) (Sigma-Aldrich, St. Louis, MO) in chloroform were combined to give different ratios of PS:PC (ranging from 0:100 to 40:60) and dried to a thin layer on the walls of a borosilicate glass tube under a stream of dry nitrogen and evacuated for 2 h to remove the resident traces of solvent. The lipid film (16 mg of total lipid content) was resuspended in 3 ml of 20 mM HEPES, 150 mM NaCl, and 80 mM octyl glucoside (pH 7; Sigma-Aldrich), and 20 µM of the lipid mixture was mixed with various concentrations of purified fgl2 protein. The mixed micellar solution was placed in microdialysis chambers (Pierce) and dialyzed against successive changes of HEPES-saline buffer (20 mM HEPES, 0.15 M NaCl (pH 7.2)) for 96 h at room temperature. Following dialysis, samples were centrifuged at 14,000 rpm for 30 min to remove particulates. As a negative control, BSA was also reconstituted into the phospholipid membranes. All the preparations were stored at 4°C and 100 µl of each sample was used to assay for the coagulation activity.
Expression of truncated and mutated fgl2 proteins in CHO cells
The 1.4-kb cDNA encoding the murine
fgl2 was amplified as previously described
and the PCR product was cloned into the mammalian expression vector
pCR3.1-Uni with the TA Cloning kit (Invitrogen) for the expression of
wild-type fgl2 protein. Using primer combinations in
Table I
, various truncated
fgl2 constructs were generated. All truncated
PCR products were amplified with the same 5' primer but various 3'
primers with a stop codon (underlined in Table I) incorporated in the
primer sequence and cloned into the vector pCR3.1-Uni with the TA
Cloning kit. Sequences of the truncated constructs were confirmed by an
automated DNA sequencer.
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, such that serine residues were
substituted individually by alanines. All sequences of the mutated
constructs were confirmed by an automated DNA sequencer.
CHO-K1 cells were transfected with various plasmid DNA
fgl2 constructs using the Lipofectamine Plus Reagent kit
(Life Technologies). Three days after transfection, the transfected
cells were trypsinized and split onto new plates. After incubation for
24 h to allow the cells attach onto the plate, a neomycin analog,
G418 (800 µg/ml; Life Technologies), was added to the culture and
incubated until the plate became 
70% confluent for 10–14 days.
Colonies were then selected and cultured with G418 (400 µg/ml) to
generate stable cell lines by changing medium every 3–4 days. The
cells were washed briefly with ice-cold PBS and spun at 2500 rpm for 5
min at 4°C. Cell pellets were then frozen immediately in liquid
nitrogen and stored at -80°C until further analysis. Expression of
fgl2 proteins was verified by Western blotting and their
prothrombinase activity was analyzed in a one-stage clotting assay.
One-stage clotting assay
Samples of purified soluble fgl2 protein reconstituted into phospholipid vesicles were analyzed for their ability to accelerate the clotting time of recalcified normal human platelet-poor plasma in a one-stage clotting assay as previously described (23). Phospholipid vesicles alone or reconstituted with BSA were used as negative controls. In addition, stably transfected CHO-K1 cells (2 x 106 cells) subjected to three cycles of freeze/thawing and diluted in 100 µl of RPMI 1640 medium were assayed for their coagulation activity. In brief, samples (100 µl) were mixed with 100 µl of citrated normal human platelet-poor plasma and 100 µl of 15 mM CaCl2 (prewarmed to 37°C), and the time required for the appearance of an insoluble fibrin clot with manual rocking at 37°C was recorded.
Additional one-stage clotting assays were performed with human plasmas deficient in coagulation factor II, VII, or X (Helena Laboratories, Beaumont, TX) to determine the factor dependence of relipidated fgl2 prothrombinase activity. Data were expressed as the mean ± SE of triplicate assays.
Effects of protease inhibitors on fgl2 prothrombinase activity
Assessment of the susceptibility of fgl2 prothrombinase to protease inhibitors was performed as previously described (23). Purified fgl2 protein (150 nM) reconstituted into PCPS (75% PC:25% PS; 20 µM) was incubated with each of the following protease inhibitors: antithrombin III (10 µg/ml) in the presence of heparin (1 U/ml), soybean trypsin inhibitor (10 µg/ml), 4-aminobenzamidine (1 mM), aprotinin (10 U/ml), PMSF (1 mM), and diisopropylfluorophosphate (DFP; 2.5 and 10 mM; Sigma-Aldrich). Following a 15-min incubation with protease inhibitor at 22°C, 10 µl of 125I-labeled prothrombin and 10 µl of 25 mM CaCl2 were added, and the reactions were incubated for an additional 30 min at 37°C. To stop the reaction, 1% SDS and 1% EDTA were added and 125I-labeled prothrombin cleavage was assessed by SDS-PAGE and soft laser scanning densitometry (Zeineh soft laser; Biomed Instruments, Chicago, IL). Human factor Xa, derived from factor X by cleavage with RVV, in the presence of phospholipids was treated identically as positive control. The data were normalized to the maximum amplitude of 125I-labeled prothrombin cleavage when no inhibitor was used (defined as 0% inhibition) to facilitate comparison.
Measurement of rates of thrombin generation
Kinetics of thrombin generation by relipidated fgl2 (150 nM plus 20 µM PL with 25% PS) was measured using a chromogenic substrate of thrombin, Spectrozyme TH (H-D-hexahydrotyrosyl-L-alanyl-L-arginine-p-nitroanalide-diacetate; American Diagnostica, Montreal, Canada). The initial rates of thrombin generation by the reconstituted fgl2 protein with 7 µM prothrombin over a range of CaCl2 concentrations (0–20 mM) were also quantitated. The effect of purified human factor Va (0.5–500 nM; Hematologic Technologies, Essex Junction, VT) on the murine fgl2 prothrombinase activity at 0.7 µM prothrombin was also determined. Kinetics parameters of thrombin generation were obtained by measuring the initial rates of thrombin generation at various concentrations of prothrombin (0.01–250 µM) with a final concentration of 5 mM CaCl2. The effects of factor Va (500 nM) on the Km and Vmax of relipidated fgl2 was determined after incubating with factor Va for 1 min at 37°C and addition of prewarmed prothrombin in the assay buffer. The assay buffer used in all experiments, unless otherwise noted, was 5 mM CaCl2, 10 mM HEPES, and 150 mM NaCl (pH 7.4). The initial rates of activation (<10% of substrate used) were determined. Reactions were monitored over time by quenching aliquots (10 µl) of the reaction in EDTA buffer (120 µl; 10 mM HEPES (pH 7), 100 mM NaCl, 5 mM EDTA, 1 mg/ml BSA, 1 mg/ml polyethylene glycol 8000) and measuring the rate of hydrolysis of Spectrozyme TH (20 µM), and the appearance of the chromophore, p-nitroanilide acetate, was monitored over time at 405 nm. The concentration of thrombin formed was calculated by comparison to a standard curve generated with purified human thrombin (Sigma-Aldrich). Pure prethrombin 1 and prethrombin 2 are known to have negligible amidase activity on Spectrozyme TH. Kinetic values were derived from the least-squares fit of the data based on the Michaelis-Menten equation (SigmaPlot). Rates of thrombin formation were calculated and expressed as moles of thrombin formed per minute per mole of fgl2. Km and Vmax values were determined by statistical analysis of the Lineweaver-Burk plots as described by Eisenthal and Cornish-Bowden (24) using the software SigmaPlot 2001 Enzymatic Kinetics (Sigma-Aldrich).
Flow cytometric analysis of fgl2 expressed on MHV-3 infected peritoneal macrophages
Female BALB/cJ mice, 6–8 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME) and were fed a standard chow diet and allowed to acclimatize for 1 wk before use. Peritoneal exudative macrophages from BALB/cJ mice were harvested in ice-cold PBS 4 days after i.p. injection of 1.5 ml of 5% thioglycolate (Difco, Detroit, MI) as previously described (25, 26). The cells were washed twice in cold PBS and resuspended in RPMI 1640, 2% FBS at 2 x 106 cells/ml. This procedure consistently yielded a >85% macrophage cell population confirmed by staining with anti-Mac1 (CD11b/CD18) Ab (Cedarlane Laboratories, Hornby, Ontario, Canada), with >98% viability by trypan blue exclusion. Cells were incubated for 1 h at 37°C before experimentation. Infection of peritoneal macrophages by MHV-3 was performed as previously described (25, 26). In brief, MHV-3 with a titer of 5 x 106 PFU/ml was used to infect the purified peritoneal macrophages at a multiplicity of infection of 5. After 8 h of infection, infection was stopped by placing the cells on ice.
Expression of fgl2 on peritoneal macrophages was performed by flow cytometry. The MHV-3-infected macrophages were stained with biotinylated polyclonal rabbit anti-fgl2 Ab at 4°C for 30 min in the dark. After two washes, cells were stained with streptavidin-PE (Cedarlane Laboratories) for 30 min at 4°C and finally analyzed using a COULTER Epics-XL flow cytometer (Beckman Coulter, Fullerton, CA).
Statistical analysis
The results were calculated as means ± SEM in all figures
and tables; the means were compared using the analysis of variance by
Student’s t test using the software Statistix 7 (Analytical
Software, Tallahassee, FL). A p value 
0.05
was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine fgl2 protein was generated using the
baculovirus expression system as described above. Fig. 1a shows a SDS-PAGE of
fgl2 baculovirus-infected H5 cells under reducing
conditions followed by Western blot analysis. It can be seen that
Coomassie blue staining (Fig. 1b) showed a dominant band at
the same molecular size of 65 kDa, comparable to the size of the
fgl2 protein previously estimated (13, 25).
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Initial studies using one-stage clotting analysis showed that the
fgl2 protein expressed by lysates of recombinant
baculovirus-infected H5 cells exhibited coagulation activity with a
dependence on CaCl2. In contrast, uninfected H5
cells and cells infected with the wild-type baculovirus were devoid of
coagulation activity (Table II). To
characterize the molecular basis of fgl2 prothrombinase
activity in detail, we first purified fgl2 protein.
Purified fgl2 protein did not exhibit any measurable
coagulation activity consistent with reports indicating a phospholipid
backbone is required for most clotting enzymes to manifest their
activity (27, 28, 29) (Table II). Table II shows that purified
fgl2 protein reconstituted into phospholipid vesicles
containing 10 or 25% PS had coagulation activity. Similar to data on
the factor Xa/Va complex, phospholipid vesicles consisting of 25% PS
also maximized recovery of prothrombinase activity, whereas no activity
was detected when fgl2 protein was incorporated into
phospholipid vesicles devoid of PS. Unlike factor Xa (30),
soluble purified fgl2 protein exhibited coagulation
activity only after incorporation into PS-containing vesicles, whereas
no activity was detected when fgl2 was mixed with PS
containing vesicles without reconstitution (data not shown).
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).
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Purified fgl2 protein (150 nM) reconstituted into
20 µM PCPS (75% PC:25% PS) exhibited maximal coagulation activity
and was used for kinetic analyses. As shown in Fig. 2, optimal fgl2
prothrombinase activity of this preparation was reached with 5 mM
CaCl2. No prothrombinase activity was detected in
the absence of CaCl2.
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a) and Lineweaver-Burk (Fig. 3b) plots show that relipidated fgl2 behaves
as an enzyme with a Km value for
prothrombin of 8.3 ± 1.1 µM and
Vmax value of 6 ± 0.2 mol of
thrombin generated per minute per mole of fgl2.
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a). The Lineweaver-Burk plot
(Fig. 4b) suggested that factor Va (500 nM) enhances the
catalytic efficiency of fgl2 by increasing the
Vmax value to 3670 ± 87 mol of
thrombin generated per minute per mole of fgl2 and
decreasing the Km value to 7.2 ±
0.8 µM.
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24 kDa was seen when
prothrombin was cleaved by fgl2, as we have previously
reported (Ref. 17 and data not shown). Identification of regions and residues required for fgl2 coagulation activity
To determine the regions of fgl2 protein required
for fgl2 coagulation activity, six truncated proteins
were stably expressed in CHO-K1 cells. The wild-type
fgl2 protein (fgl2-aa1/432) and the
various truncated proteins were expressed at comparable levels as
determined by Western blot analysis (data not shown). One-stage
clotting assays were performed to determine whether the expressed
proteins retained clotting activity. As shown in Fig. 5a, truncated proteins with
deletion of amino acids 387–432 or 337–432 (i.e.,
fgl2-aa1/386 and fgl2-aa1/336,
respectively) retained coagulation activity. Truncated proteins with
amino acids 285–432, 232–432, 182–432, and 136–432 deleted (i.e.,
fgl2-aa1/284, Fgl2-aa1/231, Fgl2-aa1/181, and
Fgl2-aa1/135, respectively) did not exhibit coagulation activity.
Inactive proteins lacked residues 285–335, suggesting that one or more
of these amino acids were critical for fgl2
prothrombinase activity.
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b shows that mutating Ser135 or
Ser425 to alanine did not alter the
fgl2 activity. However, the mutation
Ser89Ala completely abolished the activity,
indicating that Ser89 residue was critical.
To compare fgl2 prothrombinase activity to that of
factor Xa, we examined the effect of protein inhibitors. As shown in
Table IV, fgl2
prothrombinase activity was not inhibited by antithrombin III in the
presence of heparin, soybean trypsin inhibitor, 4-aminobenzamidine,
aprotinin, or PMSF, whereas >80% inhibition of factor Xa activity was
observed in the presence of these protease inhibitors. Furthermore, as
little as 2.5 mM DFP completely inhibited fgl2
prothrombinase activity, whereas 10 mM DFP was required for inhibition
of factor Xa. The data indicated that fgl2 protein
directly cleaves prothrombin to thrombin in a manner that has the
characteristic of a serine protease but differs from the serine
protease activity of factor Xa.
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MHV-3-infected macrophages of susceptible strain BALB/cJ mice
express fgl2 prothrombinase. Proteins involved in
coagulation are either transmembrane proteins, such as tissue factor
and thrombomodulin, or membrane-associated proteins, such as factors
Xa, IXa, and VIIIa, that can be washed from the cell surface. To
determine whether fgl2 prothrombinase was transmembrane
or membrane associated, we infected peritoneal macrophages of BALB/cJ
mice with MHV-3 and tested for fgl2 on the cell surface
using flow cytometry analysis. The labeling procedure involves
extensive washing. Fig. 6 shows a
representative result. Mean fluorescence intensity of
fgl2 expression detected by specific
anti-fgl2 Ab increased from 0.8 ± 0.05 to
43.8 ± 3.8 after MHV-3 infection. Isotopic control Ab gave mean
fluorescence intensity of 0.5 ± 0.08. We concluded that
fgl2 is a transmembrane protein, unlike factor
Xa.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phospholipid is required for assembly of proteins of the coagulation cascade into functional form. For example, PS-containing membranes enhance the affinity of factor Xa for its cofactor Va by 75- to 800-fold (35). Also, PS is responsible for a 10-fold increase for factor IXa binding to factor VIIIa (29). In addition, PS-containing membranes increase the catalytic efficiency of the factors Va/Xa30, factor VII/tissue factor (35), and the factors VIIIa/IXa complexes (29). Similarly, purified fgl2 protein only exhibited prothrombinase activity when reconstituted into PS-containing vesicles. This supports our previous observation that murine fgl2 coagulation activity was maximal after rearrangement of the negatively charged phospholipid (i.e., PS) from the inner leaflet of the cell membranes to the exterior leaflet during cycles of freeze-thawing of fgl2-expressing cells. In this paper, we report that a similar ratio of PS:PC is crucial for optimal murine fgl2 coagulation activity. Whether relipidation of fgl2 increases binding of fgl2 to prothrombin or enhances catalysis of prothrombin by fgl2 requires further study.
A series of truncated fgl2 proteins were expressed in CHO-K1 cells to determine which region(s) were critical for its activity. Truncation of residues 336–432 did not affect fgl2 prothrombinase activity, whereas deletion of amino acid residues of 285–335 completely abolished the activity. Truncations of murine fgl2 upstream of amino acid residue 284 also resulted in an inactive protein. Deletion of such a large number of amino acid residues might have affected the proper conformation of fgl2. Alternatively, these deletions might have removed an exosite (prothrombin binding site) for the initial interaction of murine fgl2 with prothrombin, distinct from the catalytically active site. A similar phenomenon has been reported in the recognition of prothrombin by the factor Xa/Va complex (36). A third mechanism for the loss of coagulation activity would be the removal of a domain required for a protein-phospholipid interaction. Fourth, the active site of fgl2 could have been deleted. At the present time, we do not have evidence to support any of these possibilities, and additional experiments will have to be performed.
Studies by others have demonstrated that many coagulation proteins, including factors VII, IX, X, XII, and thrombin, have serine protease activity (8, 31, 37). It has been suggested that there are two main types of serine proteases: the trypsin-like and subtilisin-like, which have an identical arrangement of the "classical" catalytic Ser/His/Asp triad but in quite different protein scaffolds. Over 20 families (S1–S27) of serine proteases have now been identified and have been grouped into six clans (clans SA, SB, SC, SE, SF, and SG) on the basis of structural similarity and other functional features (31, 37). In this study, we demonstrated that murine fgl2 protein has the characteristic of a serine protease based on the observation that the prothrombinase activity was inhibited by the serine protease inhibitor DFP. The concentration of DFP required for enzyme inhibition was well within the range that inhibits a number of known serine proteases that participate in the coagulation cascade (28). Moreover, the fact that murine fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, aprotinin, or 4-aminobenzamidine suggests that murine fgl2 is a unique serine protease that behaves differently than other known coagulants.
Examination of the amino acid sequence of murine fgl2 protein shows the presence of serine protease clan SE motifs SXXK with serine residues at positions 89, 135, and 425. Proteases such as Escherichia D-Ala-D-Ala peptidase A, Streptomyces R61 D-Ala-D-Ala peptidase, and Actinomadura R39 peptidase have been categorized into the clan SE family of serine proteases (31). No homology at the amino acids level besides the SXXK motif is found between fgl2 protein and other members of the clan SE family. By site-directed mutagenesis, we have shown here that serine 89 is essential for murine fgl2 prothrombinase activity. Results obtained using the truncated murine fgl2 proteins are consistent with the site-directed mutagenesis results. Removal of residues 336–432 does not seem to be important for the expression of the clotting activity, consistent with the finding that mutation of serine 425 did not abolish the expression of coagulation activity. The mutation of serine 89 suggests the importance of the residue for the prothrombinase activity; however, as mentioned previously, the mechanism of the effect has not been determined. A similar case is observed for factor Xa when arginine 347 is mutated to asparagine, resulting in a selective reduction in the factor Va affinity of factor Xa (38).
Expression of murine fgl2 protein with a coagulation activity on activated macrophages and endothelial cells has been reported. However, Marazzi et al. (19) have recently reported that murine fgl2 protein is spontaneously secreted in vitro by freshly isolated human CD4+ and CD8+ T lymphocytes, but the secreted protein was devoid of coagulation activity. Thus, bioactive murine fgl2 protein can express either as a transmembrane form or as a soluble form under different circumstances, but coagulation activity of murine fgl2 requires phospholipid. The role of the soluble form of fgl2 protein lacking coagulation activity remains undetermined.
In this paper we have defined a unique coagulant, fgl2. From the hemostasis perspective, murine fgl2 bypasses both the intrinsic and extrinsic coagulation pathways to trigger prothrombin activation directly without the need of additional coagulation factors. However, similar to factor Xa, factor Va enhances the coagulation activity of murine fgl2. From the immunologic perspective, murine fgl2 results in direct generation of thrombin, which plays a significant role in inflammation by amplifying the inflammatory response. For instance, thrombin causes endothelial cells to produce IL-8, which induces endothelial P-selectin expression and early neutrophil adhesion and activation (39, 40). Also, thrombin is a potent chemotaxin for monocytes and polymorphonuclear cells, resulting in influx of massive infiltrates to the site of inflammation, which is the hallmark of diseases in which murine fgl2 has been implicated in the pathogenesis (1, 2, 16). The characterization of fgl2 and identification of serine 89 residue accounting for its prothrombinase activity will allow the development of novel strategies to prevent fulminant liver failure, cytokine-induced fetal loss, and other diseases in which fgl2 plays a pathogenic role.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gary A. Levy, Multi Organ Transplant Program, Toronto Hospital, 621 University Avenue, 10th Floor, Room 116, Toronto, Ontario M5G 2C4, Canada. E-mail address: gl
3 Abbreviations used in this paper: MHV-3, murine hepatitis virus strain-3; Sf9, Spodoptera frugiperda ovarian cell 9; H5, High Five; CHO, Chinese hamster ovary; PS, phosphatidyl-L-serine; PC, phosphatidylcholine; DFP, diisopropylfluorophosphate; Ni-NTA, nickel-nitrilotriacetic acid.
Received for publication January 8, 2002. Accepted for publication March 13, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and chains. Proc. Natl. Acad. Sci. USA 84:1609.
-independent IL-8 secretion and E-selectin expression. J. Immunol. 158:5435.[Abstract]
B and potentiates endothelial cell activation by TNF. J. Immunol. 159:5620.[Abstract]This article has been cited by other articles:
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 G. E. Olson, V. P. Winfrey, S. K. NagDas, and M. H. Melner Region-specific Expression and Secretion of the Fibrinogen-related Protein, fgl2, by Epithelial Cells of the Hamster Epididymis and Its Role in Disposal of Defective Spermatozoa J. Biol. Chem., December 3, 2004; 279(49): 51266 - 51274. [Abstract] [Full Text] [PDF]
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