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1995 86: 4134-4143
Monoclonal antibody F1 binds to the kringle domain of factor XII and
induces enhanced susceptibility for cleavage by kallikrein
DM Ravon, F Citarella, YT Lubbers, B Pascucci and CE Hack
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Monoclonal Antibody F1 Binds To the Kringle Domain of Factor XI1 and
Induces Enhanced Susceptibility for Cleavage by Kallikrein
By Dorothea M. Ravon, Franca Citarella, Yvonne T.P. Lubbers, Barbara Pascucci, and C. Erik Hack
In a previous study we have shown that monoclonal antibody F1 (MoAb Fl),directed against an epitope
on theheavy
chain of factor XI1 distinct from the bindingsite for anionic
surfaces, is able t o activate factor XI1 in plasma (Nuijens JH,
et al: JBio/Chem264;12941,1989).Here, we studiedin detail
the mechanism underlying the activation of factor XI1 by
MoAb F1 using purified proteins. Formation of factor Xlla
was assessed by measuring its amidolytic activity towards
in
the chromogenic substrateH-D-Pro-Phe-Arg-pNA(S-2302)
the presence of soybean trypsin inhibitor and by assessing
cleavage on sodiumdodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Upon incubation with MoAb F1
alone, factor XI1 was auto-activated in a time-dependent
fashion, activation being maximalafter 30 hours. Factor XI1
incubated in the absence of MoAb F1 was hardly activated
by kallikrein, whereas in the presence of MoAb F1, but not
in that of a control MoAb, the rate of factor XI1 activation
by kallikrein was promoted at
least 60-fold. Maximal activation offactor XI1 with kallikrein in thepresence of MoAb F1
was reached within 1 hour. This effect of kallikrein on the
cleavage of factor XI1 bound t o MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator couldnot substitute for
kallikrein. Also, trypsin could easily activate factor XII, but in
contrast t o kallikrein, this activation was independent of
MoAb F1. SDS-PAGE analysis showed that theappearance
of amidolytic activitycorrelated well with cleavage of factor
XU. MoAb F1-induced activation offactor XI1 in this purified
system wasnot dependent on the presence of high-molecular-weight kininogen (HK), in contrast to the activation of
Experiments with
the contact systemin plasma by MoAb Fl.
deletion mutants revealed that theepitopic regionfor MoAb
F1 on factor XI1 is located on the kringledomain. Thus, this
study shows that binding
of ligands t o t h ekringle domain,
which does not contributeto theproposed binding site for
negatively charged surfaces, may induce activation of factor
XII. Therefore, these findings pointto theexistence of multiple mechanisms of activation of factor XII.
0 1995 by The American Societyof Hematology.
T
upon binding to an activator to be the key event for initiating
activation of factor W and, hence, of the contact
These conformational changes render factor XI1 much more
susceptible to proteolytic activation by other plasma or cellular proteases, in particular plasma kallikrein. The site of
factor XI1 involved in binding to an activator has been localized between amino acids 134 and 153, the fibronectin type
I domain of factor XII," and between amino acids 2 and
12,1x.19
Preliminary experiments suggest an additional binding site in the epidermal growth factor-like domain and/or
kringle domain.20Factor XI1 bound to an activator also displays auto-activation, an intermolecular process in which
activator bound-single chain factor XI1 is activated by factor
XIIa.2'~Z4
Previously, we described that the contact system in plasma
can be activated by a monoclonal antibody (MoAb), MoAb
F1, which is not directed against the binding site of factor
XI1 for anionic surfaces.*' A detailed knowledge of the binding site for MoAb F1 on factor XI1 as well as that of the
mechanism of activation by MoAb F1 may, therefore, provide more insight into the molecular changes during the
activation process of factor XII. In this report we analyze in
detail the activation of purified factor XI1 by MoAb F1 and
localized its epitope on the heavy chain region. Our results
indicate that MoAb F1 is directed against the kringle domain
and that binding of MoAb F1 to this domain induces a conformational change in factor XI1 rendering it a better substrate for plasma kallikrein.
HE CONTACT SYSTEM of human plasma consists of
the zymogens factor XI1 (Hageman factor), prekallikrein (PK), factor XI, and the nonenzymatic cofactor highmolecular-weight kininogen (HK).' In vitro, factor XII, an
80-kD glycoprotein, readily binds to anionic surfaces such
as silicates, dextran sulfate or sulfatides, and thereby activates the contact systemwhich
inturn
in
may
initiate activation of the intrinsic pathway of blood coagulation, the fibrinolytic system, the complement cascade and
the production of kin ins.'^'
Protein sequencing as well as cloning of full-length cDNA
have shown that the heavy-chain region of factor XI1 contains a number of domains homologous to those recognized
previously in other proteins, ie, (from the aminoterminal to
the carboxyterminal region) a fibronectin type-I1 domain, an
epidermal growth factor-like domain, a fibronectin type-l
domain, a second epidermal growth factor-like domain, a
kringle, and a proline-rich region, the latter being unique
for factor XII. The light-chain region contains the catalytic
domain characteristic for serine proteases.'-" Several investigators have proposed conformational changes in factor XI1
From the Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service and Laboratory for Experimental and Clinical
Immunology, University of Amsterdam, Amsterdam, The Netherlands; andthe Dipartimento di Biopatologia Umana, Sezione di
Biologia Cellulare, Universita di Roma "La Sapienza, "Rome, Italy.
Submitted November 15, 1994; accepted June 8. 1995.
Supported by Grant No. 89.127 fromthe Dutch Heart Foundation.
Address reprint request to C. Erik Hack, MD, c/o Publication
Secretariat, Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service, PO Box 9406, 1006 AKAmsterdam, The Netherlands.
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.
0 1995 by The American Society of Hematology.
0006-4971/95/8611-0115$3.00/0
4134
MATERIALS AND METHODS
Reagents. Dextran sulfate (molecular weight 500.000; DXS
500), Protein G , CNBr-activated Sepharose 4B, and benzamidineSepharose were obtained from Pharmacia Fine Chemicals AB (Uppsala, Sweden); hexadimethrine bromide (Polybrene) from Janssen
Chimica (Beerse, Belgium); Tween-20 from J.T. Baker Chemical
CO (Phillipsburg, NJ); soybean trypsin inhibitor (SBTI) from BDH
Biochemicals Ltd (Poole, UK); nitrocellulose membranes from
Scheiler and Schull (Dassel, Germany); the chromogenic substrates
H-D-Pro-Phe-Arg-p-nitroanilide
(S-2302), H-D-Val-Leu-Lys-pNA
Blood, Vol 86, No 1 1 (December I), 1995:pp 4134-4143
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MoAb INDUCED ACTIVATION OFFACTOR
XI1
(S2251), and H-D-Ile-Pro-kg-pNA (S2288) were from Chromogenix AB (Mlilndal, Sweden); and restriction endonucleases, T4
DNA ligase and the Klenow fragment of DNA polymerase I were
purchased from New England Biolabs GnbH (SchwalbachlTaunus,
Germany).
Proreins. The MoAbs F1 and F3, against human factor XII,
and MoAb K15, against human (pre)kallikrein, have been described
previously.2s.26MoAb B7C9, directed against an epitope on the first
28 amino-terminal amino acids of the heavy-chain of factor XII,"
was a kind gift from Dr R.A. Pixley (Temple University, Philadelphia, PA). Human urokinase (uPA) was obtained from Laboratoire
Choay (Paris, France) and recombinant tissue-type plasminogen activator (r-tPA) from Boehringer Mannheim Biochemica (Mannheim,
Germany). a-Factor XIIa was obtained from Kordia (Leiden, The
Netherlands). The enzymatic activities were established usingthe
chromogenic substrates S2288 for tPA and uPA, and S2302 for afactor XIIa.
Plasminogen (PLG) prepared using MoAb APl, directed against
human plasmin(ogen), according to Levi et al." Plasminogen was
converted into plasmin using uPA (molar ratio of PLG to uPA was
1 0 0 to 1). Plasmin activity was established using the chromogenic
substrate S225 l.
Bovine trypsin was obtained from Sigma Chemical CO (St Louis,
MO) and was further purified by cation-exchange chromatography
on a Phast S column (Pharmacia).
Factor XI1 was purified as described previously.**The preparation
was found to contain approximately 0.7% of activated factor Xll as
determined with S-2302 using the low-molecular-weight form of
active factor X I , termed &factor XIIa as a reference (the term afactor XIIa is used herein to denote the 80-kD form of activated
factor XII, p-factor XIfa refers to the 28-kD activated factor XIl
fragment). 8-FactorXIla was obtained by incubating factor XIl
with trypsin at a 500 to 1 molar ratio, followed by anion-exchange
chromatography of the mixture on a Sephadex DEAE-A50 column
(Pharmacia). The resulting p-factor XIIa preparation contained no
detectable trypsin activity and migrated as a single protein band of
approximately Mr 30,000 on nonreduced as well as reduced sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Prekallikrein (PK) was purified according to a similar procedure
as described for factor XI1 with some exceptions. Briefly, fresh
citrated human plasma was filtered through a column of MoAb K15Sepharose. PK was eluted with 3 moVL KSCN and dialyzed against
phosphate-buffered saline, pH 7.4 (PBS). Then, the PK preparation
was sequentially applied onto columns of F3-Sepharose to remove
traces of factor Xi1 species, protein G-Sepharose to remove Igs,
and benzamidine-Sepharose to remove contaminating proteases. The
final preparation was adjusted to pH 5.2 as described for factor ?Qfs
and stored at -70°C. Purified PK (20 pg) migrated as a doublet of
Mr 86,000 and Mr 88,000 on nonreduced as well as reduced SDSPAGE. PK was converted into kallikrein by incubation with f3-factor
XIIa coupled to Sepharose 4B (molar of ratio p-factor XIIa to PK
1 to 100) for 1 hour at 37"C, after which the p-factorXIIa-Sepharose
was removed by centrifugation. SDS-PAGE analysis showed complete conversion of PK into kallikrein.
Protein concentrations were determined by radioimmunoassay
(kallikrein and factor XUz6)or by spectrophotometer using
= 16.1 for plasmin, 15.6 for trypsin and 14 for MoAb F1.All
proteins were stored in aliquots at -70°C.
Localization of the epitope for MoAb F1 on factor X l l . The
epitopic region of MoAb F1 on factor XII was localized using the
following factor XI1 variants: full-length recombinant (r) factor XI1
[rFXIIl; r-factor XI1 lacking the N-terminal fibronectin type II domain, the first epidermal growth factor-like domain, the fibronectin
type I domain, and the second epidermal growth factor-like domain
IrFXII-kringlel; and c-factor XI1 containing only the entire serineprotease region and part of the proline-rich hinge [rFXII-lpc]. Con-
4135
struction of cDNAs, vaccinia vectors, and expression of recombinant
proteins by HepG2 cells have been described for rFXLI and for
rFxU-1~c.Z~
The other variant was obtained in an analogous way.
Briefly, to obtain a plasmid carrying the sequences coding for rFXIIkringle (PBFXILKringle), plasmids carrying the sequences coding
for full-length FXII cDNA (pBFXII) were digested with HincIl and
AvrII restriction endonucleases. The cDNA fragment comprising
nucleotides 48 through 925 (numbering according toref 29) was
removed and digested with Sty I restriction endonuclease to isolate
the cDNA fragment comprising nucleotides 639 through 925. This
latter fragment was ligated into pBFXII digested with HincII-AvrII,
together with a synthetic deoxyribonucleotide obtained by annealing
two chemically synthesized deoxyribonucleotides: 5'-AACACTTKGATTGACAC-3' and 3'"lTGTGAAAGCTAACTGTGGTTCS. The synthetic nucleotide was used to reconstitute the proper cleavage site after the leader peptide and the correct reading frame of the
cDNA. As confirmed by sequence analysis according to Sanger et
al?' the FXlI cDNA inserted in pBFXII.Kringle was 1,739 bp in
length and codes for the complete leader peptide (amino acid residues - 19 to +l), the kringle domain, the proline-rich region, and
the complete catalytic domain (amino acid residues +l93 to +596).
The recombinant insert from pBFXII.Ktingle plasmid was subcloned
into the HindIII-Sac I sites of vector p1 lkd6-131." Construction
and selection of a recombinant vaccinia virus, v-rFXII.&ingle, containing the FXEKringle cDNA sequences, were performed as described for rFXII and r F X I l - l ~ c . ~ ~
The epitopic region of MoAb F1 on XI1 was localized as follows:
human hepatoma cells (HepG2), grown to subconfluency in Dulbecco's modified Eagle's medium containing 1Wo(voYvol) fetal
calf serum, were infected with wild-type vaccinia viruses or with
recombinant vaccinia viruses carrying cDNA sequences coding for
the different factor XI1 variants. Twenty-four hours after infection,
cells were washed with minimum essential medium (MEM) without
L-cysteine (GIBCO-BRL, Gaithersburg, MD) and incubated for 2
hours with MEM without L-cysteine. Then, themediumwas replaced with MEM without L-cysteine containing 50 pCimL of Promi~:L-[~'s]in vitro cell labeling mix (Amersham, Bucks, UK),
which contains approximately 70% L-t3*S]methionine and 30% L["Slcysteine, and the cells were grown for an additional 6 hours.
Then, approximately S X IO6 cells were washed with PBS and lysed
with 1 mL of cold NP-40 lysis buffer (ie, 150 mmolfL NaCI, 1%
(wt/vol) Nonidet P-40 (NP-40; ICN Biomedicals. Aurora, OH), and
50 mmoVL Tris, pH 8.0). The cell lysates were centrifuged for 30
minutes at 4°C at 10,OOOg and the supernatant collected. Labeled
recombinant factor XI1 mutants were absorbed from the supernatant
using MoAb Fl.OT2, and B7C9 coupled to CNBr-activated Sepharose 4B in the presence of bovine serum albumin (BSA) (0.3%,wt/
vol). After 4 hours of incubation, the samples were Centrifuged at
1,5OOg, the supernatant discarded and the Sepharose washed four
times with PBS containing Tween-20 (0.1% wt/vol), and thereafter
once with PBS containing 1 moVL NaCl and once with PBS containing SDS (0.196, wt/vd). The Sepharose beads were dried and
sample buffer for gel electrophoresis was added. SDS-PAGE was
performed in the presence of dithiotreitol (DTT) using a 5% (wt/
vol) polyacrylamide stacking gel and 12% (wt/vol) polyacrylamide
separating gel. Thereafter, the gels were incubated for 30 minutes
with Enlightening (Dupont, Boston, MA), dried, and protein bands
were visualized by autoradiography using x-ray films. A detailed
description of the construction and characterization of these recombinant factor XI1 variants is submitted (F. Citarella et al, manuscript
submitted).
Chromogenic assays. Assays were performed at37°Cin Tris
buffer (ie, 100mmovL Tris containing 150 mmoW NaCl and 0.1 %
[wt/vol] Tween-20, pH 8.0). H-D-Pro-Phe-kg-p-nitroanilide ( S 2302) was used to quantitate factor XIIa generated. The substrate
was dissolved in distilled water and the concentrations of stock
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4136
solutions were determined by absorbance at 316 nm (molar extinctioncoefficient cman= 1.27 X IO4 [mol/L]".cm").Thesubstrate
was usually dissolved at a concentration of approximately 4 mmol/
L and was stored at -20°C. Before
use S-2302 was diluted to 0.5
mmol/L with Tris buffer containing 0.1% (wt/vol) Polybrene to prevent binding of factorXI1to surfaces, andSBTI (230 pg/mL) to
inhibit kallikrein activity (substrate solution). The substances added
to the Tris buffer had no influence on the hydrolysis of S-2302 by
factor XIIa. Control experiments established that kallikrein trypand
sin up to a concentration of 25 nmol/L (final concentration per well)
was not able to convert S2302 in this substrate solution.
Activation of MoAb Fl-bound factor XI1 in the presence of kallikrein orjbrinolytic proteases. Ten microliters of factor XI1 (final
concentration 1,475 n m o m in Tris buffer) was preincubated for 30
minutesat37°Cwith
S pL ofMoAbF1(finalconcentration
15
pmolL) in 1.5 mL polypropylene tubes (Eppendorf). Then, 5 pL
of kallikrein,plasmin,uPA,r-tPA,
or trypsin(finalconcentration
125 nmol/L), prewarmed at37T, were added and the mixtures were
incubatedfor25minutesat37°C.Then,10
pL of samplewere
transferred to a 96-well microtiter plate (Nunc, Roskilde. Denmark)
containing 195 p L substrate solution per well, prewarmed at 37°C.
Thereactionwasfollowedbyassessingthechangeinabsorbance
at405nm,recordedevery
3 minutesusing a Titertektwinreader
(Flow Laboratories, Irvine,UK), equipped with a thermostat (37°C).
The amount of factor XlIa present
in the mixtures was calculated
from the rate of conversion of S-2302 as estimated from the changes
in absorbance at 405 nm (AA405/min),by reference to a calibration
curve of factor XIIa versus AA40Wmin. This calibration curve consisted of serial dilutions of factor XI1 maximally activated by kallikrein in the presence of MoAb F1. In a parallel experiment, MoAb
F3,directedagainstthelightchainoffactorwasusedas
a
control antibody. Under the experimental conditions used,
no hydrolysis of S-2302 byuPA, r-tPA, plasmin, kallikrein, or trypsin was
observed in the absence of factor XII.
Kinetic analysis of the activation of MoAb FI-bound factor XI1
by kallikrein. Six differentconcentrations of factor XI1 (125 to
1,250 nmol/L)in Tris buffer, were preincubated for 1 hour at 37°C
with MoAb F1 (15 prnollL) in polypropylene tubes. Kallikrein (140
of 10
nmolL) was added to start the activation reaction. Samples
pL, diluted 1/20 in substrate solution, were assayed for factor XI1
activityat10-secondintervalsduringthe
first 60 seconds.Factor
XIIa generation was linear with time during this period. The amount
of factor XIIa generated was determined as described above. The
initial ratesof activation were obtained from least-squares regression
lines obtained by plotting generated factor XIIa versus incubation
time. A double-reciprocal plot of these rates versus the initial factor
XI1 concentration was used to determine Km, Vmax, and K,,of the
activation reaction.
Kinetics of auto-activation of factor XI1 in the presence of MoAb
F l . Different amounts of factor XI1 (125 to 1,250 nmolk) in Tris
buffer,wereincubatedfor30to
300 minutes with MoAb FI (15
MmolL) at 37°C in polypropylene tubes. At intervals (varying from
20 to 60 minutes)duringtheincubation,smallaliquots(7.5
&)
were withdrawn from the mixtures, diluted 1/20 in substrate solution
and assayed for generated active factorXI1 as described above. The
change in absorbance at 405 nm were recorded every 10 minutes
for up to 2 hours.
RAVON ET AL
of DTT using a 5% (wt/vol) polyacrylamide stacking gel and 10%'
(wt/vol)polyacrylamideseparatinggel.Afterelectrophoresisproteins were transferred electrophoretically onto nitrocellulose membranes. Electroblotting was performed for 1 hour at 200 V in 0.192
m o l L glycine, 19.28 (vol/vol)ethanol and 0.025 mol/L Tris. pH
8.3. Subsequently, the nitrocellulose sheets
were incubated overnight
with 'Z'I-radiolabeled anti-factorXI1 antibodie? (5 X 10"cpm, ie,
approximately150 ngof antibodies) in PETbuffer (ie, PBS-10
mmolL EDTA and 0.1% [wt/vol] Tween-20) containing
S'% (wt/vol)
nonfat drymilk to block remaining bindingsites on the nitrocellulose
sheets. Blots were washed, air dried, and protein bands were visualized by autoradiography using preflashed Kodak X-Omat AR films.
The extent of factor XI1 cleavage was judged from the intensity of
the uncleaved factor XI1 band, as measured by densitometry.
RESULTS
Localization of the epitope for MoAb FI on factor XII.
Previous experiments indicated that MoAb F1 reacts with
an epitope on the heavy chain of
factor XII.*' To further
map the epitopic region of MoAb F1 on factor XII, we
assessed binding of MoAb F1 to severaldeletionmutants
lacking the various N-terminal heavy
chain domains. As a
control,bindingof
MoAb OT2, whichisdirected
against
the catalytic domain of factor XII?' and B7C9, which is
directed against an epitope on the aminoterminal 28 amino
acids of factor XII," to the r-factor XI1 variants was also
assessed. Figure 1 shows that MoAbF1 was able to immunoprecipitate rFXII of Mr 77,500 and 67,000 (lane 9;
the band
of Mr 67,000 representsincompletelyglycosylated
factor
XII, as was established in experiments with endoglycosidase
F), and the mutant containing the kringle domain, the proline-richregion
andthecatalyticdomain
of factor XI1
(rFXII-kringle) (lane 10) with doublet of Mr of 46,500 and
46,000, which represent differential glycosylation,
as confirmed by endoglycosidase F treatment. However, MoAb F1
did not bind to the r-factor XI1 variant containing only part
of the proline-rich hinge in addition to the entire catalytic
domain (rFXJI-lpc) (lane 11). As expected,MoAb OT2
boundall
threefactor XI1 variants(lanes
5 through 7 )
whereas B7C9 bound only full-length factor XII (lane I),
confirming that the other variants lacked the aminoterminal
28 amino acids. Thus, the epitope for MoAb F1 appeared to
be located on the kringle domain of factor XII.
Kinetics of activation of factor XII by kallikrein in the
presence or absence of MoAb FI. To assess activation of
factor XI1 by kallikrein in the presence or absenceof MoAb
F1, we measured the conversion of the chromogenic substrate S2302 by factor XIIa. Toprevent conversion by kallikrein, SBTI was added to the substrate solution and in control experiments it was established that under the conditions
usedkallikrein upto afinalconcentrationof
25 nmollL
did not hydrolyze S2302. Purified factor XII, absorbed with
benzamidine-Sepharose, had negligible amidolytic activity
Assessment of cleavage of factor XI1 during auto-activation or
against S-2302. Upon addition of kallikrein a slight increase
activation by kallikrein inthe presence of MoAb FI. Factor XI1
of factor XI1 activity was observed (Fig 2) corresponding to
was incubatedfor various times with
MoAb F1 aloneor preincubated
2% activation of factor XII, and in agreement with observafor 30 minutes with MoAb F1 and then incubated for various times
tions by others showing lessthan 5% activation of fluidwith kallikrein at 37°C in polypropylene tubes.At intervals, samples
were withdrawn and assayed for factor XlIa activity using the S2302phase factor XI1 at a kallikrein-factor XI1 ratio of IO%.'' In
contrast, in the presence of MoAb F1, kallikreinrapidly
substrate solution. Simultaneously, samples were transferred
to tubes
cleaved factor XI1 yielding, for example, 7 1 % factor XIIa
containing 1% (wt/vol) SBTI. Thereafter, sample buffer
for gel elecunder the experimental conditions shown in Fig 2. The actitrophoresis was added. SDS-PAGE was performed in the presence
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MoAb INDUCED ACTIVATION OF FACTOR XI1
41 37
40.5-
Fig 1. Localization of the epitope for MoAb F1 on factor XII. Human hepatoma cells (HepG2) were infected with recombinant vaccinia
viruses carring cDNA sequences coding for factorXI1 variants. After infection, cells were labeled with PRO-MIX:L-[35SIin vitro celllabeling mix
and lysed with NP-40 lysis buffer. Cell lysates were then immunoprecipitated with MoAb B7C9 (lanes 1 through 41,OTZ (lanes 5 through 81,
and F1 (lanes 9 through 121.SDS-PAGE was performed under reducing conditions, the gels were incubated for 30 min with Enlightening,
dried, and protein bands were visualized by autoradiographyas described in Materials and Methods. Lanes 1,5, and 9 full-llength r-factor XII;
lanes 2, 6, and 10: r-factor XII-kringle; lanes 3,7, and 11: r-factor XII-lpc; lanes 4, 8, and 12: wild-type vaccinia virus. The positions of molecular
weight markers in kilodaltons are indicated at theleft.
vation of factor XI1 by kallikrein in the presence of MoAb
F1 was not inhibited by the presence of Polybrene (data
not shown). Moreover. a similar activation of factor XI1 by
kallikrein occurred in the presence of F(&')? and F(&')
fragments of MoAb F1 (data not shown).
To determine the initial rate of activation of factor XI1 by
kallikrein in the presence or absence of MoAb FI, different
amounts of factor XI1 were preincubated with MoAb FI or
Tris buffer alone, andthereafterincubated
with kallikrein
for 10 to 60 seconds. During this time period up to 10%
activation was observed for each concentration of factor XII.
Figure 3 shows thatat increasing concentrations of factor
2.00
1.20
1.50
1.00
0.50
1
./-
h
v)
\
3
c
I ;/
0
0.00
v
-a
5
0.40
10
20
30
40
Time (min)
Fig 2. Time-course of substrate hydrolysis by factor Xlla generated by kallikrein in the presence or absence of MoAb Fl.Factor XI1
11,475 nmollL in Tris buffer) was preincubated with MoAb F1 (15
pmollL1 or buffer alone for 30 minutes at 37°C. Then kallikrein (125
nmol/LI or buffer was added, and the mixtures were incubated for
25 minutes at 37°C. Finally, lO-@L samples were withdrawn and
added to the S2302 in substrate solution and the
change in absorbance at 405 nm wasmeasured as outlined in Materials and Methods. The mixtures contained factor XI1 and buffer ,(l.
factor XII,
MoAb F1 and kallikrein (01,factor XI1 and kallikrein ( l, and factor
XI1 and MoAb F1 (A).The experiment shown was repeated three
times with identicalresults.
0.00
Fig 3. Michaelis-Menten plot for the activation
of factor XI1 in the
presence or absence of MoAb F1. Six different concentrations of factor X11 (125 t o 1,250 nmollL in Tris buffer), preincubated with MoAb
F1 for l hour at 37°C. were incubated for 10 t o 60 seconds with
kallikrein (140 nmol/L). Generated factor Xlla, and therefrom the initial rates of activation, were determined as described in Materials
and Methods.The symbols represent incubation mixtures containing
factor XII, MoAb F1, and kallikrein (01;factor XI1 and kallikrein (01;
and factor XI1 and MoAb F1 (Al.
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4138
XII, the rate at which factor XIIa was formed increased. Not
surprisingly, the rate of activation was also dependent on
the concentration of MoAb F1. However, in separate experiments we established that the rate of activation of factor XI1
reached a plateau at 4 p m o K of MoAb F1. Therefore, in
further experiments we added MoAb F1 at a concentration
of 15 pmol/L to rule out an influence of the concentration
of MoAb F1 on the observed activation. It is to be noted
that in separate experiments we established that the affinity
(Kassoc,ation)
of MoAb F1 for native factor XI1was 6 X IO6
(moK)". This low affinity is probably because of the fact
that MoAb F1 has to induce a conformational change in
factor XI1 upon binding, which explained the necessity for
a molar excess of MoAb F1 in the experiments (we reported
previously that MoAb F1 has an affinity of about 200 times
higher for activated factor XI1 than native factor XII). Furthermore, there was no significant contribution of auto-activation to the total activation rate under the experimental
conditions used: Samples containing the highest concentration of factor XI1 (1,250 nmoVL) and to which SBTI was
added after a l-minute incubation with kallikrein, were assayed immediately and after 5 or 10 minutes for factor XIIa
activity. The rate of substrate hydrolysis was the same in all
cases (data not shown).
From a Lineweaver-Burk plot, a double-reciprocal plot of
the rates of activation versus the initial factor XI1 concentration, we calculated values of 10 pmol/L for the Km (Michaelis constant), 10 nmol/L for the Vmax (maximal rate of
catalysis), and 0.07 S" for the L,, for the activation of factor
XI1by kallikrein in the presence of MoAb F1. However,
these values could not be determined very accurately using
higher concentrations of factor XII, because the amount of
factor XIIa present in the preparation was too high, resulting
in high blank values in the absence of kallikrein. In addition,
it was also impossible to determine the kinetic constants for
the activation of factor XI1 incubated with MoAb F1 or
kallikrein alone, because the rate at which factor XIIa was
formed, was too low.
Specijicity of the increased susceptibility of cleavage of
factor XII bound to MoAb F1 for kallikrein. It has been
suggested that factor XI1may be involved in fibrinolysis.8,27.3Z-3STherefore, we compared the rate of cleavage of
MoAb Fl-bound factor XI1 by kallikrein with those by trypsin, a-factor XIIa and the fibrinolytic proteases plasmin, uPA
and tPA. As shown in Fig 4, the fibrinolytic enzymes were
hardly able to activate MoAb F1-bound factor XII. Activation of MoAb F1-bound factor XI1by these proteases resulted in less than 1% activation of factor XI1 during an
incubation period of 25 minutes. Conversely, kallikrein and
trypsin activated 70% and 100% of MoAb F1-bound factor
XII, respectively. However, the activation by trypsinwas
not dependent on MoAb F1 in contrast to that by kallikrein
(Fig 4). We also examined the susceptibility of MoAb F1bound factor XI1 for a-factor XIIa. However, because a factor XIIa is able to hydrolyze the substrate S2302, we used
a 10-fold lower concentration of a-factor XIIa, ie, 12.5 nmoV
L (and also of kallikrein for comparison). a-Factor XIIa
activated MoAb F1-bound factor XI1 at a much lower rate
than kallikrein. About 0.4% activation was observed (in addition to that induced by MoAb F1) during an incubation
RAVON ET AL
n
80
S
v
PI
(0
X
Y
40
0
buffer
pli
uPA
tPA
kal
tryp
Fig 4. Specificity of activation MoAb F1-bound factor Xli. Factor
XI1 (1,475 nmol/L in Tris buffer), preincubated for30 minutes at 37°C
with MoAb F1 (15 pmol/L) orbufferalone, was incubated for 25
minutes with 125 nmol/L of plasmin (pli), uPA, r-tPA, kallikrein (kall.
and trypsin (tryp). Tan-microliter samples were assayed for factor
Xlla activity (fordetails, see Materials and Methods). Resultsare
expressed as percentage of maximal activation. (a],Activation of
factor XI1 in the absence of MoAb F1; (Dl, activation of factor XI1 in
the presence of MoAb F1.
periodof 20 minutes, whereas a similar concentration of
kallikrein'yielded 12%activation under these conditions. DFactor XIIa at a similar concentration did not activate MoAb
FI-bound factor XII.
In all the experiments detailed above, MoAb F3, an antibody against the light chain of factor XI1 was studied as a
control. In the presence of this MoAb, no significant changes
in the rate of activation of factor XI1 by kallikrein or the
other enzymes, except for trypsin, was observed (data not
shown).
Auto-activation offactor XII induced by MoAb F l , Purified factor XI1 in the presence of negatively charged surfaces
may undergo autoactivation. Therefore, we studied the effect
of various concentrations of factor XI1 on the rate of autoactivation induced by MoAb F1. It is to be noted that to study
autoactivation, different conditions had to be used than those
used to study susceptibility for kallikrein cleavage. For the
latter an incubation of up to 1 hour was used, whereas for
autoactivation at least 5 hours incubation was necessary.
Figure 5A shows that in the presence of MoAb F1 concave
upward progress curves were obtained with the highest factor
XI1 concentrations, consistent with an continuously increasing rate of formation of activated factor XII. The purified
factor XI1 was found to contain about 0.7% of activated
enzyme, which could catalyze the autocatalytic process. Incubation of MoAb F1-bound factor XI1 with @factor XIIa
did not support activation of factor XII. However, this autoactivation process was very slow compared with the activation of MoAb F1-bound factor XII by kallikrein. For example, with the highest concentration of factor XI1 (1,250 nmoV
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4139
MoAb INDUCED ACTIVATION OF FACTOR XI1
I
150
0
a0
160
240
320
160
200
Time (min)
100
B
75
h
I
-C
v
l3
50
II
25
0
0
60
100
Time (min)
Fig 5. Progress curves for autoactivation of factor XI1 in the presence of MoAb F1. (A) Various concentrations of factor XI1 were incubated at 37°C with MoAb F1 for the times indicated. Aliquots (10
pL) of the incubation mixtures were assayed for factor XI1 activity.
Experimental procedures are described in Materials and Methods.
The initial concentrations of factor XI1 were 125 W, 250 IO), 500
(D), 750 (A),1,000 (0)and
. 1250 (A)nmol/L. (Inset) Percentage of
activated factor XI1 formed after 5 hours of incubation with buffer
(B) Factor XI1 1 5 0 0 nmol/L) was incubated
alone (a)or MoAb F1 (0).
a t 37°Cwith buffer alone (01,DXS 500 (2 pgfmL final concentration;
0).
MoAb F1 ( l 5 pmol/L final concentration; D), or together with
MoAb F1 and DXS 500 (A) for the timesindicated. Factor XI1 activity
was assayed as described above.
L) used, only10%of
the native factor XI1was activated
after 5 hours of incubation (inset, Fig 5A), whereas 70% of
factor XI1 was activated in the presence of kallikrein within
1 hour (Fig 6B). Autoactivation in the absence of MoAb F1
was even slower, yielding for example, 1.5% activation of
the native factor XI1 after 5 hours at 37°C (inset, Fig 5.4).
TO test the effects of negatively charged surfaces in the
presence of MoAb F1, factor XI1 (500 nmol/L) was incubated with buffer alone, MoAb F1 (15 pmolk), DXS 500
(2 pg/mL), or together with MoAb F1 and DXS 500 at
37°C. As shown inFig 5B, under the conditions used no
autoactivation was observed in the presence ofDXS 500
alone. In the presence of MoAb F1 autoactivation was observed, and this was potentiated in the presence of DXS 500:
for example, after 3 hours of incubation, about 9% of factor
XI1 was activated in the presence of MoAb F1 alone, whereas
in the presence of both MoAb F1 and DXS, about 21% of
factor XI1 was activated.
The molar ratio of MoAb F1 to factor XI1 during the
autoactivation experiments was atleast 10 to 1 (Fig 5).
Therefore, most, if not all, of factor XII-MoAb F1 complexes
consisted of one molecule factor XI1 and one molecule
MoAb F1, andthis apparently was sufficient to allow autoactivation. Consistent herewith, a similar autoactivation was
observed when F(ab')-fragments of MoAb F1 wereused
(data not shown), definitely ruling out the possibility that
the observed autoactivation was due to a spatial positioning
of two factor XI1 molecules by one MoAb F1 molecule.
Taken together, these findings indicated that autoactivation
of factor XI1 is accelerated in the presence of MoAb F1.
Cleavage of factor XII because of autoactivation or by
kallikrein inthe presence of MoAb F1. The experiments
shown above indicated that in the presence of MoAb F1 ,
factor XI1 obtained amidolytic activity, which process was
enhanced by kallikrein. To assess whether this increase in
amidolytic activity was due to generation of two-chain factor
XIIa, we studied the coincidence of cleavage of factor XI1
and the generation of amidolytic activity during incubation
of factor XI1 with MoAb F1 in the presence or absence of
kallikrein. Cleavage of factor XI1 was assessed by SDSPAGE under reducing conditions. The electrophoresed factor
XI1 species were visualized by immunoblotting with '"1polyclonal anti-factor XI1 antibodies followed by autoradiography. The extent of cleavage was determined by densitometry. Figure 6 shows the time course of the appearance
of amidolytic activity of MoAb F1-bound factor XI1 in the
absence (A) or presence of kallikrein (B) and the time course
of cleavage of factor XI1 under these conditions (C). During
the course of activation, factor XI1 was cleaved into a-factor
XIIa as evidenced by the presence of the heavy chain with
Mr 50,000 (Fig 6C). After 1 hour, about 70% of MoAb Flbound factor XI1 was cleaved in the presence of kallikrein
(Fig 6C, lane 13), which correlated well with the generation
of factor XIIa amidolytic activity (inset, Fig 6B). In contrast,
only about 17% of the maximal amidolytic activity was observed when factor XI1 was incubated for 5 hours with MoAb
F1 alone (Fig 6A), which was accompanied by a comparable
extent of cleavage of factor XII, as was judged from the
intensities of the uncleaved factor XI1 bands (Fig 6C; inset,
Fig 6A). Upon prolonged incubation of MoAb F1-bound
factor XI1 in the presence (not shown) and absence of kallikrein, cleavage products of Mr =50,000 and Mr =40,000
and minor products of Mr 30,000 and Mr 18,OOO were observed (Fig 6C, lane 5). In none of the experimental condi-
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RAVON ET AL
41 40
B75
n
50
S
v
-m
X
Y
25
l
0 1
0
100
200
7 5 300
Time (min)
0
50 0
25
Time (min)
n
- 106
- 80
- 49.5
- 32.5
- 27.5
Fig 6. Comparison of cleavage of factor XI1 and appearance of amidolytic activity. (A)Factor XI1 (1,400 nmol/L) wasincubated with MoAbF1
(15 pmollL) alone for various times at37°C and generation of amidolytic activity wasassessed. The extent of cleavage was analyzed by SDSPAGE (under reducing conditions) followed by immunoblotting
with '251-anti-factorX11 antibodies as described in Materials and Methods.
Generation of factor XI1 amidolytic activity is expressed as percentage of maximal amidolytic activity. (B) In a parallel experiment, factor XI1
(1,400 nmollL) was incubated for
30 minutes at37°C with MoAbF1 (15 pmol/L) and activated by kallikrein(140 nmol/L) forvarious times. Factor
XI1 amidolytic activity ( 0 )and the extent ofcleavage (inset A andB; CI) were assessed as described above. (Cl Autoradiographs of the cleavage
of factor XI1 in the presence of MoAb F1 alone (lanes 1 through 5) and MoAb Fl-boundfactor XI1 in the presence of kallikrein (lanes 7 through
13) corresponding to the experiments shown in (A) and (B), respectively. Incubation time with MoAbF1 alone: 2 hours (lane l ) , 3 hours (lane
21, 4 hours (lane 31, 5 hours (lane 4). and 27 hours (lane5). Incubation time in thepresence of kallikrein: 1 minute (lane7). 3 minutes (lane 8 ) . 6
minutes (lane9). 10 minutes (lane10). 20minutes (lane 11). 30 minutes (lane121, and 60 minutes (lane 13). Lane 6, factor XI1 alone. The positions
of the molecular weights markers (in kilodaltons) are indicated on the right.
tions studied did we observe significant amidolytic activity
accompanied by the presence of only the 80.000 daltons
form of factor XII.
DISCUSSION
Previously wehave shown that MoAb F1 activates the
contact system in plasma."As this MoAb is not directed
against the surface-binding site,'< unraveling ofhow this
MoAb induces activation may show an alternative mechanism of activation of factor XII. Therefore, in this study we
performed a detailed analysis of the changes of factor XI1
that occurred after binding to MoAb FI. We found that in
a purified system, factor XI1 was rapidly cleaved and activated by kallikrein in the presence of MoAb FI. in contrast
to the slow activation that occurred in the absence of MoAb
F t . The latter is consistent with the concept that factor XI1
in the fluid phase is a poor substrate for kallikrein."..'"
Optimal activation of the contact system in plasma by
MoAb F1 was dependent on the presence of both factor XII.
PK.and HK,'5 whichreflect the importance of reciprocal
activation of factor XI1 and PK in theMoAb FI-induced
activation of plasma. We observed no significant effect of
the presence of HK (up to concentrations of 80 nmol/L. data
not shown). Thus, activation of MoAb FI-bound factor XI1
in this purified system by kallikrein was independent of HK.
The explanation for these seemingly discrepant results is
presumably that in this study we focused on the changes that
occurred in factor XI1 after binding toMoAb Ft. and for
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MoAb INDUCEDACTIVATION OF FACTOR XI1
this reason we added kallikrein rather than PK. Experiments
are now underway to establish whether HK is required for
contact activation by MoAb F1 when using factor XI1 and
PK (rather than kallikrein) or in the presence of plasma
inhibitors such as Cl esterase inhibitor.
In vitro, factor XI1 can be activated by trypsin, plasmin,
factor XIa, kallikrein,12s36-38
factor XIIa,21-24and microbial
pro tease^.^^.^ In this study, the rate of activation of factor
XI1 by a-factor XIIa and the fibrinolytic proteases plasmins,
uPA and tPA, was at least 100-fold slower than that by
kallikrein. As shown in Fig 4, MoAb F1-bound factor XI1
was specifically cleaved by kallikrein, consistent with the
concept that kallikrein is the enzyme mainly responsible for
the generation of active factor XII.'2,366,37,41
p-Factor XIIa had
no effect on the activity of MoAb F1-bound factor XII, in
agreement with the results of Silverberg et al?' who observed
no cleavage of glass-bound factor XI1 upon incubation with
p-factor XIIa, whereas cleavage occurred with a-factor XIIa.
We observed that a-factor XIIa also is able to activate factor
XI1 bound to MoAb F1, although less efficiently than kallikrein. Thus, these data together show that a-factor XIIa is
the factor XI1 species that is able to propagate autoactivation
induced by MoAb F1, whereas p-factor XI1 has no role in
this process.
One hypothesis to explain the initial generation of active
factor XI1 during contact activation implies that factor XI1
gains intrinsic enzymatic activity without proteolytic cleavage, upon binding to a surface, because of a conformational
change in the molecule.'",42We studied this concept by incubating benzamidine-Sepharose absorbed factor XI1 with
MoAb F1, also absorbed with benzamidine-Sepharose. Under our experimental conditions, the generation of amidolytic
activity correlated well with cleavage of factor XI1 as was
assessed by SDS-PAGE, immunoblotting with 12'I-anti-factor XI1 antibodies and densitometry, upon incubation with
MoAb F1 alone (Fig 6). Thus, we found no experimental
evidence for the existence of a single-chain 80-kD enzymatically active species. In similar experiments with sulfatides>22 g l a ~ s , ~ ' .or
~ ' DXS," comparable findings have
been reported.
The surface-binding site of factor XI1 has been proposed
to consist of residues 134 through 153 (belonging to the
fibronectin type I region of factor XII), as proposed by Pixley
et al," and residues 1-28.'8,19Using deletion mutants, we
localized the epitope on factor XI1 for MoAb F1 on the
kringle domain of factor XII, corresponding to the residues
193 through 276:" ie, a region not considered to contribute
to the surface-binding site. Recent studies in our laboratory
indicate that factor XI1 may have another or a complementary surface-binding site located in the second epidermal
growth factor-like domain and/or kringle domain."However, MoAb F1 is not directed against any of these binding
sites, because it easily recognizes factor XI1 bound to glass2'
and is not able to inhibit binding of labeled factor XI1 to
glass or other activators (F.C., unpublished observations,
1994). In agreement with the concept that the binding sites
for MoAb F1 and negatively charged surfaces on factor XI1
are different, was the observation that MoAb F1 and DXS
500 together enhanced the observed increase in susceptibility
for proteolytic cleavage (Fig 5B).
4141
Generally, kringle domains are thought to function as autonomously folding modules specialized in protein-protein
interactions and to play regulatory roles in the functions of
the proteins in which they have been identified. The kringle
module has been implicated in the binding of fibrinolytic
proteins to fibrin, which binding seems to be essential for
effective activation of the p r ~ t e i n s ? ~Kringle
-~l
domains also
mediate the interaction of plasminogen with a2-antiplasmin
and tPA with plasminogen activator inhibitor type 1 (PAI1).52-5h
Plasminogen binds specificallyand in a functional
manner tohumanumbilicalvein
endothelial cells (HUVECs), which binding is mediated by the lysine-binding sites
present in its kringle
Factor XI1 has also been
shown to bindspecifically to H U V E C S , ~ ,although
~'
the
binding site on factor XI1 mediating this effect has not been
identified. Schmeidler-Sapiro et a16' have reported mitogenic
effects of factor XI1 on HepG 2 cells, although also the
binding site necessary for this effect is not known. The most
potent mitogen for mature hepatocytes is hepatocyte growth
factor, a protein homologous to pla~minogen.'~Binding of
hepatocyte growth factor to its receptor on hepatocytes is
mediated by its first and second kringle domain.ma Therefore, it is tempting to speculate that some of the effects of
factor XI1 on cells may occur via its kringle domain. Our
studies with MoAb F1 predict that such an interaction of
factor XI1 with cellular receptors may allow the stabilization
of a conformation resulting in an enhanced susceptibility for
proteolytic cleavage and an acceleration of autoactivation.
Such an enhanced susceptibility of proteins for other proteases, after binding via their kringles, is known. For example,
the enzymatic activity of single-chain uPAis potentiated
upon binding to its receptor on monocytes.67Furthermore,
plasminogen bound to its receptor, is more easily activated
by tPAs7,h8 oru P A . ~ ~ . ~ '
The classical kringle structure contains specific lysinebinding sites. Recently, however, Stephens et a17' described
a class of kringles with affinityfor polyanions. They reported
that the uPA kringle domain contains a heparin binding site,
which mediates the stimulation of activation of pro-uPA by
plasmin and the activation of plasminogen by uPA in cellfree system^.^"^^ Because factor XI1 is also activated when
bound to negatively charged surfaces, interactions of factor
XI1 with proteoglycans in vivo via its kringle domain may
also serve as the mechanism of factor XI1 activation.
Whether these mechanisms may contribute to activation of
factor XI1 under physiologic conditions remains to be established in further studies.
In conclusion, we have shown that MoAb F1 binds to the
kringle domain of factor XII, which domain does not contain
the proposed binding sites for negatively charged surfaces,
andthat this MoAb enhances susceptibility of factor XI1
for cleavage by kallikrein. These findings may point to the
existence of alternative mechanisms of activation of factor
XII.
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RAVON ET AL
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XI1
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