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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4198-4206
The Second Exon-Encoded Factor XII Region Is Involved in the
Interaction of Factor XII With Factor XI and Does Not Contribute to
the Binding Site for Negatively Charged Surfaces
By
Franca Citarella,
Giorgio Fedele,
Dorina Roem,
Antonio Fantoni, and
C. Erik Hack
From the Dipartimento di Biotecnologie Cellulari ed Ematologia,
Sezione di Genetica Molecolare, Università di Roma "La
Sapienza," Roma, Italy; the Central Laboratory of The Netherlands
Red Cross Blood Transfusion Service, Amsterdam; and the Department of
Internal Medicine, Free University Hospital, Amsterdam, The
Netherlands.
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ABSTRACT |
Contact system activation, in vitro, is triggered by activation of
factor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been
located within the amino acid residues 1-28 by identifying the epitope
recognized by a monoclonal antibody (MoAb), B7C9, which inhibits
kaolin-induced clotting activity. To further elucidate the role of the
amino terminal binding site in the regulation of FXII activation, we
have characterized a FXII recombinant protein (rFXII- 19) deleted of
the amino acid residues 3-19, which are encoded by the second exon of
FXII gene. A plasmid encoding for rFXII-19 was constructed and
expressed in HepG2 cells by using vaccinia virus. Purified rFXII-19
migrated as a single band of Mr 77,000 on sodium dodecyl sulfate
(SDS)-polyacrylamide gel, did not bind to MoAb B7C9 immobilized on
Protein A-Sepharose, thus confirming that it lacked the epitope for
this MoAb, and had no amidolytic activity towards the chromogenic
substrate S-2302 in the absence of activator. rFXII-19 specific
clotting activity was lower (44%) than that of native FXII. The
activation rate of rFXII-19 by kallikrein in the absence of dextran
sulfate was about four times higher than that of full-length FXII and
was increased in the presence of dextran sulfate. However, rFXII-19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-19 bound to kaolin, which binding was equally well inhibited by either, rFXII-19 or full-length FXII
(IC50 = 7.2 ± 2.2 nmol/L for both
proteins). Accordingly, a synthetic peptide corresponding to FXII amino
acid residues 3-19 did not inhibit the binding of labeled full-length
FXII to kaolin. rFXII-19 generated a similar amount of FXIIa- and
kallikrein-C1-inhibitor complexes in FXII-deficient plasma in the
presence of kaolin, as did full-length FXII; but generated less factor
XIa-C1-inhibitor complexes (50%) than full-length FXII. This impaired
factor XI activation by rFXII-19a was also observed in a purified
system and was independent of the presence of high molecular weight
kininogen. Furthermore, the synthetic peptide 3-19, preincubated with
factor XI, inhibited up to 30% activation of factor XI both in the
purified system as well as in plasma. These results together indicate
that amino acid residues 3-19 of FXII are involved in the activation of
factor XI and do not contribute to the binding of FXII to negatively
charged surfaces.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
HUMAN FACTOR XII (FXII or Hageman
factor), a serine-protease produced by the liver, circulates in plasma
as a single-chain inactive zymogen (molecular weight [Mr]
80,000). On activation, the zymogen is converted to a
two-chain active protease, activated FXII (FXIIa), which can activate
several plasma cascade systems including the contact system, the
intrinsic pathway of coagulation, fibrinolysis, and the complement
system.1-3 Like other serine proteases belonging to plasma
cascade systems, FXII consists of several structural domains, which
starting from the amino terminus are: a leader peptide, a fibronectin
domain type II, an epidermal growth factor-like domain, a fibronectin
domain type I, a second epidermal growth factor-like domain, a kringle
domain, a proline-rich region and the catalytic domain.4-6
Activation of FXII, in vitro, requires interaction with negatively
charged surfaces to induce a conformational change rendering single-chain FXII more susceptible to proteolytic cleavage at amino
acid residues 353-354.7-9 Cleavage at this site yields the
two-chain enzyme FXIIa (Mr 80,000), which consists of a heavy chain (Mr
52,000) and a light chain (Mr 30,000) containing the active
site.10,11 In vitro, proteolytic cleavage of single-chain FXII may occur via autoactivation or, more efficiently, by
kallikrein.12-14 The pathophysiologic activating surface(s)
is still unknown. FXII has been demonstrated to bind to endothelial
cells and to neuthrophils,15-17 but it remains to be
established if these cells may serve as activators of FXII in vivo.
However, artificial negatively charged surfaces such as kaolin, ellagic
acid, sulfatide micelles, and high molecular weight dextran sulfate,
have been extensively used to study activation of FXII and of the
contact system in vitro.14,18-21 The putative surface-binding site of FXII has been identified by mapping the epitope
for monoclonal antibody (MoAb) B7C9, which inhibits kaolin-induced clotting activity of FXII.22 Initial studies with this MoAb pointed to the amino acid residues at positions 134-153 as being the
binding site, which assignment was based on the recognition of the
kallikrein-cleaved FXII fragments by immobilized B7C9.22 Later studies with a FXII cDNA expression library in  gt11, located the epitope for MoAb B7C9, and hence a surface-binding region, between
residues 1 and 28.23 However, there are no experimental data, for example as obtained with recombinant FXII molecules lacking
the surface-binding site or with synthetic peptide mimicking its
sequence, supporting the function of the putative binding site, except
for a study showing that recombinant FXII proteins with gross deletions
of various specific regulatory domains display reduced, but not absent,
binding to negatively charged surfaces.24
To elucidate the role of the amino terminal binding site in the
regulation of FXII activation, we have prepared and characterized a
FXII recombinant protein, rFXII- 19, deleted of the amino acid residues 3-19, ie, the residues encoded by the second exon of FXII
gene. The recombinant FXII protein though lacking (one of) the proposed
binding site for negatively charged surfaces, bound normally to these
surfaces, but showed an impaired clotting activity due to an impaired
capability to interact with FXII substrate factor XI.
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MATERIALS AND METHODS |
General.
Restriction endonucleases, T4 DNA ligase, and the Klenow fragment of
DNA polymerase I were purchased from New England Biolabs GmbH
(Schwalbach/Taunus, Germany). Calf intestine alkaline phosphatase and
T4 polynucleotide kinase were from Boehringer Mannheim (Mannheim, Germany). All of the enzymes were used according to manufacturer's instructions. 125I was from Amersham Intl (Buckinghamshire,
UK). Culture medium and fetal calf serum were obtained from GIBCO
Laboratories (Breda, The Netherlands). Cyanogen bromide
(CNBr)-activated Sepharose 4B, Protein G-Sepharose,
Protein A-Sepharose, and dextran sulfate (Mw 500,000) were from
Pharmacia Fine Chemicals (Uppsala, Sweden), trypsin and soybean trypsin
inhibitor (SBTI) from Sigma Chemical Co (St Louis, MO). Factor XII- and
factor XI-deficient plasma were obtained from George King Biomedical
Inc (Overland Park, KS), hexadimethrine bromide (Polybrene) was
obtained from Janssen Chimica (Beerse, Belgium). The chromogenic
substrates H-D-Pro-Phe-Arg-p-nitroanilide (S-2302) and
pyro-Glu-Pro-Arg-p-nitroanilide (S-2366) were purchased from
Chromogenix AB (Molndal, Sweden). Factor XI was from Kordia Laboratory
Supplies, Leiden, The Netherlands.
Construction of plasmid carrying rFXII-19 sequence.
To obtain a plasmid carrying the sequences encoding for rFXII-19 (a
FXII protein deleted of amino acid residues 3-19, corresponding to the
second exon of FXII gene) the plasmid pBFXII25 was digested with HincII and Nco I restriction endonucleases, the
cDNA fragment comprising nucleotides 48-552 (numbering according to
Citarella et al25) was isolated and digested with the
restriction endonuclease MaeIII to remove a fragment containing
nucleotides 48-126 corresponding to the second exon of FXII gene. The
cDNA fragment containing nucleotides 127-552 was ligated into pBFXII,
HincII-Nco I digested, together with a synthetic
deoxyribonucleotide to restore the correct reading frame and the proper
cleavage site after the leader peptide. The synthetic
deoxyribonucleotide was obtained by annealing the two chemically
synthesized complementary deoxyribonucleotides, 5 -AACACTTTCGATTCCAGTTCTCACT-3 and
3-TTGTGAAAGCTAAGGTCAAGAGTGACAGTG-5. As confirmed by
sequence analysis according to Sanger,26 the FXII cDNA
inserted in pBFXII-19 was 1,768 bp in length and coded for the
complete leader peptide (amino acid residues  19 to +2 ) followed
by amino acid residues 20-596. Recombinant vaccinia viruses carrying
the cDNA sequence coding for rFXII-19 (vFXII.19) were obtained as
described.25
Production of recombinant FXII proteins.
The human hepatoma cell line HepG2, grown to subconfluence in
Dulbecco's modified Eagle's medium containing 10% (vol/vol) fetal
calf serum, was infected with the recombinant viruses carrying the
sequences coding for rFXII (full-length FXII) or rFXII-19 at a
multiplicity ratio of 10 plaque-forming units/cell. Six hours thereafter, medium was replaced with the same medium but without fetal
calf serum, and the infected cells were incubated for 48 hours, before
harvesting medium.
Electrophoretic analysis of immunoprecipitated recombinant proteins.
Vaccinia virus-infected HepG2 cells were labeled with 50 µCi
[L-35S] methionine/mL medium for 6 hours at 37°C.
After radiolabeling, media were harvested and immunoprecipitated with
different MoAbs bound to protein A-Sepharose. The proteins
immunoprecipitated by immobilized MoAbs were electrophoresed on 12%
(wt/vol) sodium dodecyl sulfate (SDS)-polyacrylamide gel under reducing
conditions and visualized by autoradiography using x-ray films.
MoAbs.
Several MoAbs against different regions of FXII were used to purify,
quantify, and characterize the recombinant FXII proteins. MoAbs KOK5
and F1, which recognize epitopes localized in the fibronectin type II
and in the kringle domain, respectively, have been described (manuscript submitted).27 MoAb
B7C922 was kindly provided by Dr Robin A. Pixley (Temple
University, Philadelphia, PA). MoAbs OT2 and F3 are directed against
epitopes localized on the light chain of FXII.24,28 MoAbs
were precipitated from hybridoma-conditioned medium by 50% (wt/vol)
ammonium sulfate, extensively dialyzed against phosphate-buffered
saline (PBS), and affinity-purified on Protein G-Sepharose according to
the manufacturer's instructions. MoAbs were biotinylated as
described.29
Purification of proteins.
Recombinant rFXII-19 protein was purified from HepG2
cell-conditioned medium by affinity chromatography using MoAb OT2
coupled to CNBr-activated Sepharose 4B according to a procedure
described earlier.24 The purified rFXII-19 preparation
was analyzed by electrophoresis on 10% to 15% SDS-polyacrylamide
gels.
FXII and prekallikrein were immunopurified from 150 mL of human
citrated plasma.27,30 Prekallikrein was converted into kallikrein as described.27 High molecular weight kininogen
(HK), prepared according to Kerbiriou and Griffin,31 was a
gift from Dr B.N. Bouma (Academic Hospital, Utrecht, The Netherlands).
All of the protein preparations used were more than 95% homogeneous as
determined by SDS-polyacrylamide gel electrophoresis.
The amount of FXII and rFXII-19 present in purified preparations was
assessed both by measuring the absorbance at 280 nm using the
absorbance coefficient A 1%cm 14.2 and by
enzyme-linked immunosorbent assay (ELISA) as described.24 rFXII-19 preparations contained less than 0.1% of native FXII as
was assessed using a sensitive sandwich ELISA with MoAb B7C9 as the
capturing antibody and biotinylated MoAb F3 as the detecting antibody.
Purified FXII and rFXII-19 were labeled with 125I by the
chloramine-T method. Free label was removed by dialysis against PBS (10 mmol/L sodium phosphate, 140 mmol/L NaCl, pH 7.4), containing 0.1%
(wt/vol) Tween-20. The specific activity of the labeled preparations was 4.2 × 107 cpm per µg of FXII and 8.6 × 107 cpm per µg of rFXII-19, respectively. Labeled
proteins were electrophoresed on SDS-polyacrylamide gel and visualized
by autoradiography to assess the quality of preparation.
Peptides.
Synthetic peptides were synthesized at the synthetic peptides facility
of the Amsterdam-Leiden Institute for Immunology by Dr J.W. Drijfhout
(Department of Immunohaematology and Blood Bank, University Hospital
Leiden, Leiden, The Netherlands). Synthetic peptides were made on
Abimed 422 multiple peptide synthesizer (Abimed, Langenfeld, Germany)
as described.32 The peptides were isolated and purified by
repeated ether precipitations, dissolved in 10% acetic acid, and
lyophilized. The amino acid content was checked by reverse phase
high-performance liquid chromatography (HPLC). Finally, the peptides
were resuspended in distilled water and stored in aliquots at
70°C. The concentration of synthetic peptides was determined
by BCA protein assay reagent (Pierce, Rockford, IL).
Clotting assay for FXII proteins.
FXII-specific coagulant activity was determined by a modification of
the activated partial thromboplastin time as described.24 To study the effect of MoAb B7C9 on the clotting activity, the purified
proteins (400 nmol/L) were preincubated 15 minutes with serial
dilutions of MoAb B7C9 in PBS after which three dilutions of the
mixture were assayed for the remaining clotting activity.
FXII binding to kaolin.
Binding of labeled FXII proteins to kaolin was determined as
described33 with minor modifications. Briefly, 50 µL of
fivefold dilutions of nonlabeled proteins in PBS 0.1% (wt/vol)
Tween-20 (PT) were added to 50 µL of 125I-FXII or
125I-rFXII-19 (0.025 pmol/mL) in 0.5 mL polypropylene
tubes and then incubated with 50 µL of kaolin (0.032 mg/mL in PT) for
10 minutes at room temperature. The tubes were centrifuged for 2 minutes at 10,000g, and the pellets were counted for
radioactivity. Binding was expressed as the percentage of the total
counts added. Under these experimental conditions, no significant
binding of labeled proteins to the tubes (less than 0.5%) was
observed. The efficiency of the inhibition of the binding was analyzed
by computing the regression line of the binding curve and establishing
the amount of the competitor able to inhibit the binding of the tracer molecule to 50% (IC50).33
FXII activation by kallikrein.
The kinetics of activation of FXII proteins by kallikrein were
determined as described.14 Briefly, 25 µL of FXII (125 nmol/L) were added to 25 µL of dextran sulfate (DS500) (50 µg/mL)
and 25 µL of buffer to yield final concentrations of 50 mmol/L
Tris-HCl, 150 mmol/L NaCl, 0.1% (wt/vol) Tween-20, pH 7.8, in Dynatech
(Plochingen, Germany) microplates (96 wells). After a 5-minute
preincubation at 37°C, 25 µL of prewarmed kallikrein (12.5 nmol/L) were added to start the reaction. After various time intervals,
the amount of FXIIa formed was determined from the rate of hydrolysis
of the chromogenic substrate S-2302: 50 µL of assay mixture (S-2302 1 mmol/L, SBTI 0.1 mg/mL in Tris 50 mmol/L, NaCl 150 mmol/L, pH 7.8) were
added to the wells and the increase in absorbance at 405 nm was
recorded at time intervals of 2 minutes by a Bio-Kinetics Reader
(Biotek Instruments Inc) set at 37°C. Under these
conditions, the rates of increase in absorbance were constant for at
least 10 minutes and were used to calculate the amount of FXIIa on the basis of a calibration curve obtained with known amounts of fully activated FXII. In control experiments, it was established that the
amount of SBTI added was sufficient to prevent conversion of the
chromogenic substrate by kallikrein at a concentration of 100 nmol/L.
FXII autoactivation.
Autoactivation of FXII proteins was studied in a 96-well microtiter
plate: 25 µL of FXII proteins (400 nmol/L), 25 µL of twofold serial
dilutions of DS500 and 50 µL of buffer yielding final concentrations of 50 mmol/L Tris-HCl, 50 mmol/L NaCl, 0.1% (wt/vol) Tween-20, pH 7.8, were incubated for 60 minutes at 37°C. Thereafter, 50 µL of assay
mixture (see above) were added to the wells; the increase in absorbance
at 405 nm was recorded at time intervals of 2 minutes and the amount of
FXIIa calculated as described above.
Activation of contact system in plasma.
The capability of rFXII-19 protein to activate contact system was
assessed by measuring the generation of FXIIa-C1-inhibitor, kallikrein-C1-inhibitor, and factor XIa-C1-inhibitor complexes in
FXII-deficient plasma reconstituted with either native FXII or the
recombinant protein. To this, 20 µL of serial dilutions of FXII
proteins in PT were added to 40 µL of FXII-deficient plasma and then
incubated with 20 µL of kaolin (5 mg/mL in PT) for 20 minutes at
37°C. The reaction was stopped by addition of 120 µL of stop
solution (PBS, 0.1 mg/mL SBTI, 0.05% [wt/vol] Polybrene), followed
by centrifugation for 2 minutes at 10,000g to discard the
kaolin pellet. The amount of FXIIa-C1-inhibitor and
kallikrein-C1-inhibitor complexes generated in EDTA-plasma was
determined by ELISA developed as a modification of previously described
radioimmunoassays (Minnema M, et al, manuscript
submitted). Sample dilutions were tested in duplicate and
results were calculated by comparison with an inhouse standard that
consisted of normal pooled human plasma fully activated by DS500 (50 µg/mL). Generation of factor XIa-C1-inhibitor complexes was
determined by an ELISA using kaolin-activated reference plasma.34
To study the effect of the synthetic peptide 3-19 on the activation of
factor XI in plasma, 10 µL of serial dilutions of peptides in PT (16 mmol/L to 0.2 mmol/L) were incubated with 10 µL of purified factor XI
(160 nmol/L) overnight at 4°C. Thereafter, 40 µL of factor
XI-deficient plasma were added and contact activation was started by
adding 20 µL of kaolin (5 mg/mL). After a 20-minute incubation at
37°C, the reaction was stopped by adding 120 µL of stop solution
followed by centrifugation to discard the kaolin pellet and the amount
of protease-C1-inhibitor complexes generated was measured.
Factor XI activation by FXII proteins.
The rate of factor XI activation by FXIIa was determined as
described35 with minor modifications. Briefly, FXII and
rFXII-19 (50 nmol/L) were first activated by incubation with DS500
(1 µg/mL) in 50 mmol/L Tris-HCl, 50 mmol/L NaCl, 0.1% (wt/vol)
Tween-20 for 90 minutes at 37°C, after which time the activation of
the enzymes was tested (to be the same) by measuring the amidolytic activity toward the chromogenic substrate S-2302. A total of 25 µL of
the mixture 1 to 20 diluted (containing 2.5 nmol/L FXIIa [rFXII-19a] and 0.05 µg/mL of DS500) were added to 25 µL of
purified human factor XI (40 to 80 nmol/L) preincubated 5 minutes at
room temperature with 25 µL of HK (16 to 33 nmol/L, respectively) in Dynatech (Plochingen, Germany) microplates (96 wells). The buffer final
concentrations were 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% (wt/vol)
Tween-20, pH 7.8. After a 15-minute incubation at 37°C, the
reaction was stopped by adding 25 µL of MoAb OT2 (100 nmol/L) (which
blocks the amidolytic activity of FXIIa30) and the rate of
factor XI activation was measured by adding 50 µL of the chromogenic
substrate S-2366 (1 mmol/L) and recording the increase in absorbance at
405 nm at time intervals of 5 minutes. Factor XIa generation was
quantitated from a standard curve prepared using purified factor XIa.
In control experiments, it was established that the amount of MoAb OT2
added was sufficient to prevent conversion of the chromogenic substrate
by FXIIa at a concentration of 10 nmol/L.
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RESULTS |
Production and characterization of recombinant FXII proteins.
Production, purification, and a partial functional characterization of
the recombinant full-length FXII (rFXII) have been detailed in previous
publications by this laboratory.24,25 The recombinant
deletion mutant rFXII-19 was produced by infecting HepG2 cells with
recombinant vaccinia viruses carrying the FXII cDNA deleted of the
sequences of the second exon encoding for amino acid residues 3-19. rFXII and rFXII-19 were labeled in vivo and were immunoprecipitated
from the culture media with two protein A Sepharose-bound MoAbs
directed against the heavy chain region of FXII. As shown in
Fig 1, both proteins were
immunoprecipitated with MoAb KOK5 and rFXII-19 showed the expected
molecular size (Mr, approximately 77,000). However, rFXII-19 was not
recognized by MoAb B7C9, which is in agreement with a previous report
showing the amino acid residues 1-28 to contain the epitope for MoAb
B7C9.23 The recombinant protein rFXII-19 was purified
from the culture medium of infected HepG2 cells by
immunoaffinity-chromatography. Purified rFXII-19 was homogeneous and
consisted of a single peptide chain as assessed by SDS-polyacrylamide
gel electrophoresis (not shown). Purified rFXII-19 did not show a
catalytic activity towards the chromogenic substrate S-2302 before
activation. On activation by limited proteolysis with
trypsin,24 rFXII-19 showed a specific amidolytic
activity (A min 1nmol1 = 15.1 ± 0.9) similar to that of native FXII
(Table 1). A similar activity was found
when rFXII-19 was optimally activated by kallikrein in the presence
of DS500 (data not shown).
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| Fig 1.
Electrophoretic analysis of recombinant FXII proteins.
Vaccinia virus-infected HepG2 cells were metabolically labeled and the
culture media were immunoprecipitated and electrophoresed as indicated
in Materials and Methods. Culture media from HepG2 cells infected with
vFXII (lane 1) or with vFXII.19 (lane 2) immunoprecipitated with
MoAb KOK5; culture media from HepG2 cells infected with vFXII (lane 3)
or with vFXII.19 (lane 4) immunoprecipitated with MoAb B7C9.
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Clotting activity of rFXII-19.
Because MoAb B7C9 was shown to inhibit kaolin-induced FXII clotting
activity, we asked whether rFXII-19, lacking the amino acid residues
recognized by MoAb B7C9, had an impaired clotting activity. Clotting
activity was determined using a modified kaolin-activated partial
thromboplastin time. As shown in Table 1, full-length recombinant FXII,
rFXII, had a specific clotting activity comparable to that of plasma
FXII, whereas rFXII-19 specific clotting activity was 44% of that
of native FXII. Furthermore, on preincubation with MoAb B7C9 (at a
protein:MoAb ratio 1:10), the clotting activity of plasma FXII and
rFXII was inhibited to 55%, whereas the clotting activity of
rFXII-19, although decreased itself, was not affected (data not
shown) consistent with the observation (see above) that this protein
lacks the epitope for MoAb B7C9.
Binding of rFXII-19 to kaolin.
As rFXII-19, lacking amino acid residues 3-19, possibly lacks a
proposed binding site for negatively charged surfaces, the impaired
clotting activity of rFXII-19 might be due to impaired binding to
kaolin. Therefore, we investigated the binding of radiolabeled rFXII-19 to kaolin. 125I-rFXII-19 did bind to
kaolin, which binding was inhibited in a similar dose-dependent manner
by either unlabeled FXII or rFXII-19 (Fig 2A), but not by bovine serum albumin
used as control (not shown). As well, rFXII-19 inhibited the binding
of 125I-FXII to kaolin (Fig 2B). The IC50 was
the same for FXII and rFXII-19 (IC50 = 7.2 ± 2.2 nmol/L), which indicated that both proteins bound to kaolin with the
same efficiency. These results suggested that amino acid residues 3-19 were not involved in the binding of FXII to kaolin. Further, a
synthetic peptide corresponding to residues 3-19 did not inhibit the
binding of labeled FXII to kaolin even at a 106-fold molar
excess (not shown).
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| Fig 2.
Binding of 125I-FXII and
125I-rFXII-19 to kaolin and inhibition by unlabeled
proteins. A volume of 50 µL of fivefold dilutions of nonlabeled
proteins in PBS 0.1 % (wt/vol) Tween-20 (PT) was added to 50 µL of
125I-rFXII-19 (A) or 125I-FXII (B) (0.025 pmol/mL) and then incubated with 50 µL of kaolin (0.032 mg/mL in PT)
for 10 minutes at room temperature. The tubes were centrifuged for 2 minutes at 10,000g and the pellets were counted for
radioactivity. Binding was expressed as the percentage of the total
counts added and are the means ± standard error (SE) of two
independent experiments in duplicate. Symbols: FXII ( ), rFXII-19
( ).
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Activation of rFXII-19 by kallikrein.
To elucidate further the cause of the diminished specific clotting
activity of rFXII-19, we studied the activation of the purified
recombinant protein by kallikrein, together with that of rFXII and
plasma FXII, in the presence or in the absence of a negatively charged
surface, such as DS500.
The activation rate of recombinant full-length FXII by kallikrein alone
was low and, similar to that of plasma FXII, was enhanced in the
presence of DS00 (Fig 3A). The activation
rate of rFXII-19 by kallikrein was about four times higher than that
of rFXII in the absence of DS500 and was enhanced in the presence of
DS500, reaching levels similar to that of rFXII (Fig 3B). These results indicated that rFXII-19 had a configuration more susceptible to
cleavage by kallikrein than full-length FXII and was efficiently activated by this enzyme in the presence of negatively charged surfaces.
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| Fig 3.
Activation of purified recombinant FXII proteins by
kallikrein. FXII recombinant proteins (125 nmol/L) were activated with
kallikrein (12.5 nmol/L) as described in Materials and Methods in the
presence (filled symbols) or in the absence (open symbols) of DS500. At
several time intervals, the reaction was stopped by adding 50 µL of
assay mixture containing SBTI and S-2302, and the increase in
absorbance at 405 nm was immediately recorded. Results are expressed as
the percentage of the maximum amount of FXIIa present in the wells
after full activation and represent the mean ± SE of three
experiments. The absence of standard error bars indicates that the
variation was too little to portray visually. Symbols: plasma FXII
(), rFXII ( ), rFXII-19 ().
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Autoactivation of rFXII-19.
Because, in vitro, proteolytic cleavage of single-chain FXII may be
mediated not only by kallikrein, but also by autoactivation, we studied
the capability of the recombinant FXII protein to undergo autoactivation in the presence of DS500. As shown in
Fig 4, FXII and rFXII-19 did undergo
autoactivation in the presence of DS500. The rate of autoactivation and
optimum DS500 concentration able to induce autoactivation (1 to 2 µg/mL) were similar for FXII and rFXII-19 and were consistent with
the proposed template model.14,36
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| Fig 4.
Autoactivation of FXII proteins. A total of 25 µL of
FXII proteins (400 nmol/L), 25 µL of twofold serial dilutions of DS
500 and 50 µL of buffer yielding final concentrations of 50 mmol/L
Tris-HCl, 50 mmol/L NaCl, 0.1% (wt/vol) Tween-20, pH 7.8, were
incubated for 60 minutes at 37°C. Thereafter, 50 µL of assay
mixture containing S2302 were added and the increase in absorbance at
405 nm was immediately recorded. Results are expressed as percentage of
total FXII activity and represent the mean of two experiments. Symbols:
FXII (), rFXII-19 ().
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Contact system activation in plasma by rFXII-19.
The results of the above experiments indicated that rFXII-19 was
able to bind to negatively charged surfaces, to undergo autoactivation,
and to be activated by kallikrein. All together, these results
indicated that the amino acid residues 3-19 do not harbor the binding
site for negatively charged surfaces and the impaired clotting activity
of rFXII-19 was not due to reduced binding to kaolin. Therefore, we
hypothesized that the impaired clotting activity of this protein might
be due to an impaired interaction with FXII substrates, ie, factor XI
or prekallikrein.
To answer this question, we investigated the activation of
prekallikrein and factor XI in FXII-deficient plasma, reconstituted with rFXII-19. The activation of the contact system proteases was
evaluated by measuring the generation of FXIIa-, factor XIa-, and
kallikrein-C1-inhibitor complexes. We found that equal amounts of FXII
and rFXII-19, added to FXII-deficient plasma, in the presence of
kaolin induced the generation of a similar amount of
FXIIa-C1-inhibitor complexes, indicating that the two proteins were
equally well activated in plasma. However, whereas the amount of
kallikrein-C1-inhibitor complexes generated also was the same, the
generation of factor XIa-C1-inhibitor complexes was 50% lower in
FXII-deficient plasma reconstituted with rFXII-19 than in that
reconstituted with FXII, thus indicating that rFXII-19 efficiently activated prekallikrein, but was not able to efficiently activate factor XI (Fig 5).
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| Fig 5.
Activation of contact system in plasma by FXII proteins.
A volume of 20 µL of FXII proteins (40 nmol/L) in PT was added to 40 µL of FXII-deficient plasma and then incubated with 20 µL of kaolin
(5 mg/mL in PT) for 20 minutes at 37°C. The reaction was stopped by
addition of 120 µL of stop solution (PBS, 0.1 mg/mL SBTI, 0.05%
[wt/vol] Polybrene), followed by centrifugation for 2 minutes at
10,000g to discard the kaolin pellet. The amount of
FXIIa-C1-inhibitor ( ), kallikrein-C1-inhibitor ( ), and factor
XIa-C1-inhibitor () complexes generated in EDTA-plasma was
determined as described in Materials and Methods. Results are expressed
as nmol/L and represent the means ± SD of three independent
experiments each performed in duplicate (n = 6). * P < .05 as compared with the amount of factor XIa-C1-inhibitor complexes
generated by FXII.
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The supposed role of residues 3-19 in the activation of factor XI by
FXIIa was further substantiated by experiments in which purified factor
XI was preincubated with a synthetic peptide corresponding to this
sequence of FXII. After preincubation with the synthetic peptide, FXI
was added to factor XI-deficient plasma and activation of contact
system was induced by adding kaolin. The synthetic peptide 3-19 (net
charge 0.82 at pH 7) inhibited the generation of
FXIa-C1-inhibitor complexes in FXI-deficient plasma in a
dose-dependent manner, the IC50 being 3.1 ± 0.7 mmol/L
(Fig 6). On the contrary, the synthetic
peptide 3-19 did not have any effect on the generation of
FXIIa-C1-inhibitor and kallikrein-C1-inhibitor, further suggesting that residues 3-19 are involved in the activation of factor XI by
FXIIa. The synthetic peptide BVEAAAASAISVA (net charge 1.01 at
pH 7), used as an irrelevant control, showed no effects on FXIa-C1-inhibitor complexes, as well as on FXIIa- and
kallikrein-C1-inhibitor complexes (Fig 6).
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| Fig 6.
Inhibition of factor XI activation in plasma by synthetic
peptides. A total of 10 µL of serial dilutions (16 mmol/L to 0.2 mmol/L) of peptides in PBS 0.1% (wt/vol) Tween-20 (PT) were incubated
with 10 µL of purified factor XI (160 nmol/L) overnight at 4°C.
Thereafter, 40 µL of factor XI-deficient plasma were added and
contact activation was started by adding 20 µL of kaolin (5 mg/mL).
After a 20-minute incubation at 37°C, the reaction was stopped by
adding 120 µL of stop solution followed by centrifugation to discard
kaolin pellet. The amount of FXIIa-C1-inhibitor ( ),
kallikrein-C1-inhibitor ( ), and factor XIa-C1-inhibitor
(+--+) complexes generated in EDTA-plasma was determined as
described in Materials and Methods. Results are expressed as percentage
of the amount of complexes generated in the absence of peptides and
represent the means ± SE of two different experiments in duplicate.
In the inset, the results obtained with an irrelevant peptide, tested
as control at 8 mmol/L, are shown. Symbols: FXIIa-C1-inhibitor (),
kallikrein-C1-inhibitor (), and factor XIa-C1-inhibitor ()
complexes.
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Activation of factor XI by FXII proteins.
Factor XI circulates in plasma noncovalently complexed with HK and
forms a ternary complex with FXIIa in the presence of negatively charged surfaces.37-40 The results of the experiment
performed in plasma (see Fig 5) indicated that the recombinant protein
rFXII-19 had an impaired clotting activity due to the fact that it
had an impaired capability to activate factor XI, but did not give any
indication about the role of FXII residues 3-19 in the formation of the
ternary complex. Therefore, we studied the influence of HK on the
activation of purified factor XI by FXIIa and rFXII-19.
As shown in Fig 7, preincubation with HK
increased the rate of activation of factor XI by both full-length FXII
(either recombinant FXII or plasma FXII) and rFXII-19. The FXIa
generated by rFXII-19a was significantly lower than that generated
by an equal amount of FXIIa. However, the relative amount of factor XI
activated by rFXII-19 compared with full-length FXII was very
similar either when HK was present or absent (33.2% and 37.7%,
respectively), thus indicating that the impaired capability of
rFXII-19 to activate factor XI was independent on the presence of
HK.
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| Fig 7.
Factor XI activation by FXII proteins. FXII and
rFXII-19 (50 nmol/L) were first activated by incubation with DS500
(1 µg/mL) as described in Materials and Methods. Thereafter, 25 µL
of the mixture 1 to 20 diluted were added to 25 µL of purified human
factor XI (40 to 80 nmol/L) preincubated 5 minutes at room temperature
with 25 µL of HK (16 to 33 nmol/L, respectively) (A) or with 25 µL
of buffer (B). After a 15-minute incubation at 37°C, the reaction
was stopped by adding 25 µL of MoAb OT2 (100 nmol/L) and the rate of
factor XI activation was measured by adding 50 µL of the chromogenic
substrate S-2366 (1 mmol/L) and recording the increase in absorbance at
405 nm. Results, expressed as the nmol/L of factor XIa generated,
represent the means ± SD of four different experiments. * P
< .005 as compared with the amount of factor XI activated by FXII
and rFXII.
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Furthermore, in the purified system we observed that the synthetic
peptide 3-19 inhibited in a dose-dependent manner the activation of
factor XI by FXIIa (IC50 = 1.66 ± 0.8 mmol/L), whereas
the activation of factor XI by rFXII-19 was not inhibited at all (Fig 8).
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| Fig 8.
Inhibition of FXI activation by synthetic peptides. A
total of 15 µL of serial dilutions (4.5 mmol/L to 0.2 mmol/L) of
peptide in PT were incubated with 15 µL of purified factor XI (80 nmol/L) for 6 hours at 4°C. Thereafter, 15 µL of HK (33 nmol/L)
were added and after 5 minutes at room temperature, factor XI
activation was started by adding 25 µL of FXII (2.5 nmol/L) activated
as in Materials and Methods. After a 15-minute incubation at 37°C,
the reaction was stopped by adding MoAb OT2 and the rate of factor XI
activation was determined by adding 50 µL of the chromogenic
substrate S-2366 (1 mmol/L) and recording the increase in absorbance at
405 nm. Results are expressed as the percentage of factor XIa formed by
either protein, FXII () or rFXII-19 () in the absence of
peptide and represent the means ± SD of three different
experiments.
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| |
DISCUSSION |
Since FXII protein is coded by 14 exons corresponding, in part, to the
structural domains at the amino acid level, it is possible to construct
recombinant deletion mutants of FXII that comprise the serine protease
domain and one or more of the other structural domains of the intact
molecule.24,25 In this report, we describe the production
and characterization of a recombinant FXII protein, rFXII-19,
deleted of the amino acid residues 3-19, ie, the residues that are
encoded by the second exon of FXII gene and that contain a putative
binding site for negatively charged surfaces. Our results, however,
indicate that this sequence is not involved in the binding of FXII to
negatively charged surfaces, but comprises a site for the interaction
of FXII with its substrate factor XI.
rFXII-19, produced by using vaccinia virus as expression system, was
first characterized by two MoAbs directed against the heavy chain
region of FXII, MoAbs KOK5 and B7C9. The recombinant protein,
consisting of one polypeptide chain with the expected electrophoretic
mobility, was immunoprecipitated by MoAb KOK5 from the culture media of
HepG2 cells infected with the recombinant virus, which indicated that
the recombinant protein was correctly folded and secreted. On the
contrary, MoAb B7C9 failed to immunoprecipitate rFXI-19.
A putative surface-binding site of FXII has been localized within amino
acid residues 134-153 and/or 1-28 by mapping the epitope for
MoAb B7C9, which inhibits kaolin-induced FXII clotting activity to
60%.22,23 Because the clotting activity of kaolin-bound FXII was not inhibited by MoAb B7C9, it was suggested that MoAb B7C9
recognized the binding site of FXII for negatively charged surfaces, as
this binding site may not be accessible on kaolin-bound FXII.22 Our results indicated that the recombinant FXII
protein deleted of amino acid residues 3-19 did not bind MoAb B7C9:
rFXII-19, indeed, was not immunoprecipitated by immobilized MoAb
B7C9, its clotting activity was not affected by preincubation with this MoAb, and it was not detected in an ELISA with MoAb B7C9 as a capturing
antibody and MoAb F3 (against the light chain region) as a detecting
antibody. Hence, the epitope for MoAb B7C9 resides within the amino
acid residues 3-19 of FXII molecule. Yet, rFXII-19, lacking this
region, normally bound to kaolin and to dextran sulfate, as it was
evident from the following observations: 125I-rFXII-19
bound to kaolin equally well as 125I-FXII; unlabeled
rFXII-19 inhibited the binding of 125I-FXII to kaolin
with the same efficiency of unlabeled FXII; the activation rate of
rFXII-19 by kallikrein was increased in the presence of dextran
sulfate; dextran sulfate supported the autoactivation of rFXII-19 at
the same concentration and at the same rate as that of plasma FXII.
These results do not support a role of amino acid residues 3-19 in the
binding of FXII to negatively charged surfaces.
Consistent with the observation that MoAb B7C9 inhibits the clotting
activity of FXII, rFXII-19, lacking the epitope for MoAb B7C9, had a
lower clotting activity (44%) than full-length FXII but, as discussed
above, this impaired clotting activity cannot be explained by lack of
binding to kaolin.
It is well accepted that the proteolytic enzymes of the cascade systems
in plasma have a catalytic region as well as binding site(s) for their
substrates necessary to efficiently catalyze the conversion of the
substrate to the product. Our results indicating that rFXII-19 did
bind to negatively charged surfaces (as well as FXII) and was
efficiently activated by kallikrein, led us to predict that the
impaired clotting activity of this recombinant protein was due to
either (1) an impaired enzymatic activity or (2) an impaired
interaction with one of FXII substrates: prekallikrein or factor XI.
The former possibility could be ruled out by the fact that the specific
amidolytic activity of activated rFXII-19 was comparable to that of
activated plasma FXII and that rFXII-19 did undergo autoactivation.
On the contrary, the finding that in the presence of kaolin, equal
amounts of kallikrein-C1-inhibitor complexes were generated in
FXII-deficient plasma by either FXII or rFXII-19, whereas only 50%
of FXIa-C1-inhibitor complexes were formed in the presence of
rFXII-19 compared with FXII, indicated that the impaired clotting
activity of rFXII-19 was due to an impaired interaction with factor
XI. This conclusion was supported by the observation that in a purified
system rFXII-19 also activated factor XI twice less efficiently than
full-length FXII independent on the presence of HK (Fig 7) and that a
synthetic peptide corresponding to amino acid residues 3-19 inhibited
in a dose-dependent fashion the activation of factor XI in plasma (Fig
6), as well as in the purified system (Fig 8).
Baglia et al35,41-44 have demonstrated the presence of
various binding sites for enzymes, substrate, and cofactor in the
different domains of the factor XI molecule such as a HK-binding site
in the A1 domain, a factor XIa substrate-binding site for factor IX in
the A2 domain, an enzyme-binding site for thrombin in the A1 domain and
a binding site for FXIIa in the A4 domain. Studying the effects of the
factor XI A4 domain peptide Gly326-Lys357 on the activation of factor
XI by FXIIa and on the amidolytic activity of FXIIa through the
chromogenic substrate S-2302, the investigators found that the
A4-derived peptide was a noncompetitive inhibitor of macromolecular
substrate (factor XI) conversion, whereas it is a competitive inhibitor
of small peptide (S-2302) hydrolysis. They postulated that the factor
XI A4-derived peptide binds to a small peptide substrate-binding site
near the catalytic site of FXIIa that does not appear to function as a
macromolecular substrate-binding site. They proposed that the putative
small peptide substrate-binding site near to the active site of FXIIa (recognized by the chromogenic substrate S-2302 and by the factor XI
A4-derived peptide) represents a secondary substrate-binding site. This
site may serve to positioning the factor XI cleavage site
(Arg369-Ile370) near the catalytic site of FXIIa once factor XI is
already anchored to FXIIa via a macromolecular substrate-binding site,
which may well exist elsewhere in the FXIIa molecule. Our results
support this model and provide indirect evidence for the existence of
two different substrate-binding sites. We speculate that the FXII amino
acid residues 3-19 may, indeed, represent the macromolecular
substrate-binding site for factor XI as the presence of these residues
increases the efficiency of factor XI cleavage by FXIIa. Cleavage of
factor XI apparently may occur without this site, which may explain
that the deletion of amino acid residues 3-19 did not completely
abolish the clotting activity of FXII, but reduced it to 44% of that
of plasma FXII. This reduction by 56% is in agreement with the finding
that maximum inhibition of FXII clotting activity by MoAb B7C9 is 60%,
only. Accordingly, the generation of factor XI-C1-inhibitor complexes
was only partially (50%) inhibited in FXII-deficient plasma
reconstituted with rFXII-19 recombinant protein.
In conclusion, we have produced and characterized a recombinant FXII
protein, which lacks the amino acid residues 3-19, encoded by the
second exon of FXII gene. The binding capacity of this recombinant
protein to negatively charged surfaces, its capacity to undergo
autoactivation, and its susceptibility for activation by kallikrein
were similar to those of full-length FXII. However, the recombinant
FXII deletion mutant displayed a reduced clotting activity, which
correlated with an impaired capability to activate factor XI.
Therefore, we suggest that residues 3-19 at the amino terminus of FXII
molecule do not contribute to the surface-binding site of FXII, but
play a role in the activation of factor XI.
| |
FOOTNOTES |
Submitted January 13, 1998;
accepted August 4, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Prof C. Erik Hack, c/o
Publication Secretariat, Central Laboratory of The Netherlands Red
Cross Blood Transfusion Service, Sanquin Blood Supply Foundation,
Department Pathophysiology of Plasma Proteins, Plesmanlaan 125, 1066 CX
Amsterdam, The Netherlands.
| |
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Abstract
Introduction