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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3294-3301
Activation of Clotting Factor XI Without Detectable Contact
Activation in Experimental Human Endotoxemia
By
M.C. Minnema,
D. Pajkrt,
W.A. Wuillemin,
D. Roem,
W.K. Bleeker,
M. Levi,
S.J.H. van Deventer,
C.E. Hack, and
H. ten Cate
From the Center for Hemostasis, Thrombosis, Atherosclerosis and
Inflammation Research; the Central Laboratory of the Netherlands Red
Cross Blood Transfusion Service, Laboratory for Clinical and
Experimental Immunology; the Laboratory of Experimental Internal
Medicine, University of Amsterdam, Academic Medical Center; the
Department of Internal Medicine, Slotervaart Hospital, Amsterdam, The
Netherlands; and the Central Haematology Laboratory, University
Hospital, Bern, Switzerland.
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ABSTRACT |
Evidence of factor XI (FXI) activation in vivo is scarce. In
addition, it remains uncertain whether thrombin, factor XIIa (FXIIa),
or perhaps another protease is responsible for FXI conversion. We
investigated the activation of FXI in eight healthy volunteers after
infusion of a low dose of endotoxin (4 ng/kg of body weight). Activation of prekallikrein FXII, FXI, and prothrombin was measured with sensitive enzyme-linked immunosorbent assays (ELISAs), and FXI
activation was measured with a novel enzyme capture assay that detects
noncomplexed FXIa. Activation of FXI was apparent with a significant
plasma peak level of noncomplexed FXIa of 10 to 11 pmol/L at 1 and 2 hours after endotoxin infusion, followed by a gradual increase in
FXIa-FXIa inhibitor complexes, measured in the ELISAs, with a summit of
11 to 15 pmol/L at 6 and 24 hours, respectively. In accordance with
previous studies, thrombin generation was detected 1 hour after
endotoxin infusion to become maximal after 3 to 4 hours. In contrast,
we did not find any evidence of contact activation, because markers of
activation of prekallikrein and FXII remained undetectable. From the
FXIa data a theoretical model was constructed which suggested that
inhibition of FXIa does not take place in the plasma compartment, but
is localized on a surface. These data provide the first evidence for
FXI activation in low-grade endotoxemia and suggest that FXI is
activated independently of FXII.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
IN 1991 IT WAS SHOWN that, besides factor
XII (FXII), thrombin is capable of activating factor XI (FXI) in
vitro.1,2 Thrombin-dependent activation of FXI constitutes
an amplification pathway because activation of FXI leads, via
activation of factors IX and X, to the formation of additional
thrombin.3 This additional thrombin may be important for
the activation of a carboxypeptidase B, called TAFI
(thrombin-activatable fibrinolysis inhibitor), resulting in the
attenuation of fibrinolysis.4-6 FXII-independent activation
of FXI would provide an explanation for the observed difference in
clinical phenotype between FXII-deficient patients who do not have an
increased bleeding risk, and FXI-deficient patients who do show a
bleeding tendency.7,8 Although FXII-independent activation
of FXI has been shown in plasma and in the presence of high molecular
weight kininogen (HK)9,10, the physiological significance
is questioned11,12 and there is no in vivo evidence for the
existence of this activation pathway.
The administration of endotoxin, the lipopolysaccharide (LPS) part of
the gram-negative bacterial cell membrane, to human volunteers under
controlled experimental conditions elicits a procoagulant response.
This model has been used to study the activation of the hemostatic
system in great detail.13 In animal studies, is was clearly
shown that initiation of the coagulation system in low-grade
endotoxemia as well as lethal Escherichia coli sepsis proceeded
via the tissue factor-factor VIIa (TF-FVIIa) complex, because
inhibition of FVIIa or TF attenuated activation of the coagulation
system.14-16 In contrast, no effect on coagulation was
observed when FXII was inhibited in a lethal sepsis
model.17 Therefore, although activation of the contact
system occurs in induced endotoxemia in humans and
animals18,19 as well as in patients with
sepsis,20,21 it does not contribute to activation of the
coagulation system.
We used the low-grade endotoxin model in humans to study the activation
of FXI. FXI activation was assessed with sensitive enzyme-linked
immunosorbent assays (ELISAs) for FXIa in complex with C1-inhibitor,
 1-antitrypsin, 2-antiplasmin, or antithrombin.22 Furthermore, we developed a sensitive enzyme capture assay (ECA) for
measurement of noncomplexed FXIa. FXI activation was studied in
relation to thrombin formation and contact activation, which was
analyzed by novel ELISAs that measure complexes of FXIIa or kallikrein
irreversibly bound to C1-inhibitor.
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MATERIALS AND METHODS |
Study group and design.
Eight volunteers (mean, 23 years; range, 19-32 years) were injected
with endotoxin. The E coli LPS (endotoxin) preparation, lot
EC-5 (D. Hochstein, Bureau of Biologics, Food and Drug Administration, Bethesda, MD) was administered as a 1-minute infusion in an antecubital vein at a dose of 4 ng/kg. All study subjects had a normal medical history, physical examination, and routine laboratory examination.
The study was approved by the Research and Ethical Committees of the
Academical Medical Center, Amsterdam, the Netherlands, and written
informed consent was obtained from all volunteers before the study
entry.
Blood sampling.
Venous blood was collected by separate punctures from antecubital veins
at t = 0 (before LPS infusion) and at 1, 2, 3, 4, 6, and 24 hours after
the LPS infusion in siliconized vacutainer tubes (sodium citrate, 0.105 mol/L or EDTA, 0.34 mol/L; Becton Dickinson, NJ). To the
EDTA tubes a solution of 0.4 mL containing Polybrene (Janssen Chimica,
Beerse, Belgium) to yield a final concentration of 0.05% wt/vol and
100 mmol/L benzamidine (Acros, NJ) was added to prevent
any ex vivo activation of clotting factors and subsequent complex
formation to inhibitors. Platelet-poor plasma was obtained
by centrifuging the blood samples at 1.600g for 20 minutes at
room temperature. Plasma samples were stored at  70°C until
assayed.
Proteins.
Lyophilized purified human FXIa (Kordia Laboratory Supplies, Leiden,
The Netherlands) was reconstituted with distilled water to the original
volume. This FXIa preparation had been made by incubating FXI with
FXIIa, after which FXIIa had been removed by absorption onto a corn
trypsin inhibitor column. Complete activation of FXI was observed on
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.
Human -thrombin was a generous gift from Dr P.J. Lentink (Department
of Coagulation, CLB, Amsterdam, The Netherlands). Activated human
protein C was a kind gift from Dr J.C.M. Meijers (Department of
Haematology, Academic Hospital Utrecht, The Netherlands).
FXIIa was made by incubating human FXII, purified via affinity
chromatography, by a monoclonal antibody (MoAb) F3 with dextran sulfate
(DXS; final concentration, 10 µg/mL) for 3 hours at
37°C.23 Plasmin and kallikrein were purified as
described.24
FXI-deficient plasma was obtained from a female patient with a
homozygous FXI deficiency (FXI: c < 1%; FXI: Ag < 1%). HK-, prekallikrein-, and FXII-deficient plasmas were obtained from George
King Biomedical (Overland Park, KS).
Amidolytic assay.
The amidolytic activity of FXIa was measured as follows: A mixture of
95 µL phosphate-buffered saline (PBS), pH 7.4, containing the
chromogenic substrate S-2366 (Chromogenix, Mölndal, Sweden) 1 mmol/L (final concentration) and 5 µL of FXIa at different final concentrations (range, 8-500 pmol/L), preincubated with or without benzamidine (100 mmol/L, final concentration), were added to wells of
microtiter plates (Dynatech, Plochingen, Germany). Microtiter plates
were read on a Multiskan plate reader (Labsystems, Helsinki, Finland)
after incubation for 72 hours at room temperature at 405 nm.
In vitro contact activation.
Varying concentrations of DXS (molecular weight, 500,000; Pharmacia
Fine Chemicals, Uppsala, Sweden), 0.05 and 0.2 mg/mL, or kaolin
(Brocacef BV, Maarsen, The Netherlands), 2.5 and 5 mg/mL, were
incubated with an equal volume of EDTA plasma for 20 minutes at
37°C. The activation was stopped by adding 3 vol of PBS containing 0.1 mg/mL soybean trypsin inhibitor (SBTI; Sigma Chemical Co, St Louis,
MO) and 0.05% (wt/vol) Polybrene. Kaolin was removed by centrifuging
the reaction mixture for 5 minutes at 13,000g.
Assays for FXI activation.
FXI antigen was measured in an ELISA as described,25 using
MoAb XI-5 as a capture antibody and biotinylated MoAb XI-3 for detection of FXI antigen.
FXIa-FXIa inhibitor complexes were assayed as described.22
The total amount of FXIa-FXIa inhibitor complexes was calculated as the
sum of the measured complexes in the four ELISAs.
A novel assay was developed for measuring noncomplexed FXIa, which was
based on a previously described enzyme capture assay for activated
protein C.26 Wells of microtiter plates (Dynatech) were
incubated with MoAb XI-5 at 3 µg/mL in carbonate buffer, 0.1 mol/L,
pH 9.5 (100 µL/well), overnight at 4°C. The plates were washed 5 times with wash buffer (PBS containing Tween 0.02% [wt/vol]; J.T.
Baker Chemical, Philipsburg, NJ). Residual binding sites for proteins
were blocked by incubating the wells for 30 minutes with 150 µL/well
2% (vol/vol) bovine milk in PBS. After washing 5 times with wash
buffer, the wells were incubated for 1 hour with the test samples or
standards diluted in dilution buffer, ie, PBS containing Tween 0.1%
(wt/vol), gelatine 0.2% (wt/vol), normal mouse serum 1% (vol/vol)
(CLB, Amsterdam, the Netherlands), Polybrene 0.05% (wt/vol), 1 mol/L
NaCl, 2 µg/mL aprotinine (Boehringer Mannheim GmBH, Mannheim,
Germany), and benzamidine 100 mmol/L (all final concentrations). Plates
were then washed 9 times with wash buffer, incubated for a period of 10 minutes, and washed again for another 9 times (total, 18) to ensure
complete removal of benzamidine as well as contaminating plasma
enzymes, nonspecifically bound to the plate. After the last washing
cycle the chromogenic substrate S-2366, 1 mmol/L in PBS, was added to
the wells (100 µL/well). Plates were sealed and kept at room
temperature. Hydrolysis of the substrate was monitored at 405 nm on a
Multiskan plate reader after 72 hours of incubation time. Dilutions of
purified FXIa were used as standard. Measured absorbance values of the
samples were compared with those of the standards, and expressed as
pmol/L. Molar concentrations of free FXIa activity were calculated
based on a molecular weight of FXIa of 80 kD, thus calculating the
active sites of FXIa.
The specificity of the assay was assessed by incubating FXI-deficient
plasma, containing Polybrene (0.05%, final concentration) and
benzamidine (100 mmol/L, final concentration), with thrombin, plasmin,
kallikrein, FXIIa, or activated protein C all plasma proteases known
to react with S-2366. The total amount of added protease corresponded
to 10% activation of the zymogen concentration present in normal
blood.
ELISAs for FXII and PK antigen.
Microtiter plates (Maxisorp, Nunc, Roshilde, Denmark) were
coated overnight at room temperature with 100 µL of 2 µg/mL
anti-human FXII MoAb B7C9 (kindly provided by Dr R.W. Colman,
Philadelphia, PA) or anti-human PK MoAb K1520 in 0.1 mmol/L
carbonate buffer, pH 9.6, and blocked for 30 minutes with 150 µL PBS,
pH 7.4, containing 2% (vol/vol) bovine milk. All subsequent
incubations were in 100-µL volumes at room temperature for 1 hour and
plates were washed after each incubation with PBS containing Tween
0.02% (wt/vol). The plates were then incubated with plasma samples
diluted in PBS-milk 2% (vol/vol) and bound FXII or PK were detected by
biotinylated MoAbs F323 or 13G11 (also kindly provided by
Dr R.W. Colman), diluted in PBS-milk 2% (vol/vol), respectively. FXII
and PK were detected by streptavidin-horseradish peroxidase (Amersham
International plc, Amersham, UK) in High Performance ELISA buffer
(CLB). The plates were developed with a solution of 100 µg/mL of
3,5,3 ,5-tetramethylbenzidin (TMB; Merck, Darmstadt,
Germany) with 0.003% (vol/vol) H2O2 in 0.11 mol/L sodium acetate, pH 5.5, for 30 minutes. The reaction was stopped
by the addition of 2 mol/L H2SO4 to the wells
and absorbance was read at 450 nm on a Multiskan plate reader.
Serial dilutions of normal pooled plasma from 30 volunteers was used as
a 100% standard. The interassay coefficient of variation of these
ELISAs was less than 10%. FXII or PK deficient plasma yielded no
response in the corresponding assay.
ELISAs for FXIIa- and kallikrein-C1-inhibitor complexes.
Microtiter plates (Maxisorp) were coated overnight at room temperature
with 100 µL of 10 µg/mL MoAb KOK-12, which binds complexed and
inactivated C1-inhibitor,20 in carbonate buffer, pH 9.6. Plates were blocked for 30 minutes with 150 µL PBS, pH 7.4, containing 2% (vol/vol) bovine milk. All subsequent incubations were
in 100-µL volumes at room temperature for 1 hour and plates were
washed after each incubation with PBS containing Tween 0.02% (wt/vol). The plates were then incubated with plasma samples diluted in PBS-milk
2% (vol/vol). FXIIa-C1-inhibitor complexes were detected by incubation
of the plates with biotinylated MoAb F3, and kallikrein-C1-inhibitor complexes were detected by incubation with biotinylated MoAb K15, each
diluted in PBS containing milk 3% (vol/vol) and normal mouse serum
1%. Next, the plates were incubated for 30 minutes with a 1:10,000
dilution of polymerized horseradish peroxidase bound to streptavidin
(poly-HRP; CLB) in PBS-milk 2% (vol/vol). The plates were developed
and read as described above. As a standard, we used pooled EDTA plasma
from normal volunteers, which was activated by incubation with 0.1 mg/mL (final concentration) DXS as described above. The plasma was
stored in small quantities at 70°C.
The amount of complexes in DXS-activated plasma was quantitated by
comparison with purified FXIIa- or kallikrein-C1-inhibitor complexes.
FXIIa- or kallikrein-C1-inhibitor complexes were prepared by incubating
10 µmol/L C1-inhibitor (Behringwerke AG, Marburg, Germany) overnight
at 37°C with 1.4 µmol/L FXIIa or 2.3 µmol/L kallikrein,
respectively. None of the complex mixtures showed amidolytic activity
against the chromogenic substrate S-2302 (Chromogenix). For the
quantification of the complexes it was therefore assumed that in these
mixtures FXIIa or kallikrein was completely bound to C1-inhibitor and
that the amount of the complexes in the mixtures was equal to the
initial molar concentration of FXIIa or kallikrein.
The specificity of the assays was assessed by incubating purified
FXIIa-C1-inhibitor and kallikrein-C1-inhibitor complexes in FXII-and
PK-deficient plasma, respectively, and by DXS activation of FXII- and
PK-deficient plasma for 20 minutes at 37°C.
Thrombin formation.
Prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin (TAT)
complexes were assayed using commercially available kits according to
the manufacturers' instructions (Enzygnost F1 + 2 and Enzygnost TAT,
respectively; Behringwerke).
Data analysis.
Several theoretical models were constructed to simulate the in vivo
generation and inhibition of FXIa. The calculated time course of the
plasma concentrations of FXIa and FXIa-FXIa inhibitor complexes was
determined by stepwise calculation with 15-second time intervals using
Microsoft Excel (Microsoft Corporation). First-order
kinetics were assumed for association, dissociation, and elimination of
the different substances.
Statistical analysis.
Results are presented as mean ± standard error of the mean (SEM).
Changes of variables over time were analyzed using Friedman's nonparametric repeated measured test and Dunn's test. FXI, FXII, and
PK antigen levels were analyzed by ANOVA and Newman-Keuls test. A
two-sided P value (P value over time) < .05 was
considered to reflect a significant difference.
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RESULTS |
ECA for activated FXI.
To protect FXIa from irreversible ex vivo inactivation by plasma
protease inhibitors in the plasma sample to be tested, addition of a
sufficient amount of a reversible inhibitor in the dilution buffer as
well as in the plasma samples was necessary. In the presence of
benzamidine (100 mmol/L, final concentration) only 3.4% ± 0.8% (n = 5) of added FXIa was complexed to its inhibitors within 1 hour,
whereas in the presence of 50 mmol/L benzamidine the corresponding
value was 9% ± 2.2% (n = 5) (results not shown). We therefore
added 100 mmol/L benzamidine to all samples.
The amidolytic activity of FXIa in the ECA was similar to that of FXIa
directly incubated with the chromogenic substrate S-2366, indicating
almost complete binding of FXIa to the MoAb-coated plates used in the
ECA (Fig 1A). As expected, no activity was found in the amidolytic assay in the presence of benzamidine. However,
in the ECA, preincubation of FXIa with buffer containing benzamidine
(100 mmol/L) yielded the same response as FXIa in buffer without
benzamidine. This indicates the efficiency of the extensive washing
cycle to remove benzamidine from the active sites of FXIa, allowing
conversion of the added chromogenic substrate by FXIa in the final
incubation step of the ECA.
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| Fig 1.
Enzyme capture assay for FXIa. (A) Effect of benzamidine
on the activity of purified FXIa in the amidolytic assay and in the
ECA. Microtiter plates coated with MoAb XI-5 were incubated with
various concentrations of FXIa in the presence or absence of
benzamidine (100 mmol/L, final concentration). The plates were washed
extensively and incubated for 72 hours with S-2366. Results obtained
were expressed as the change in absorbance at 405 nm and compared with
those obtained with an amidolytic assay in which FXIa was incubated in
the presence or absence of benzamidine (100 mmol/L, final
concentration) with S-2366. The means of four experiments ± SD are
given. FXIa in the ECA incubated in the absence ( ) or in the
presence ( ) of benzamidine. FXIa directly tested in the amidolytic
assay in the absence ( ) or in the presence ( ) of benzamidine. (B)
Recovery in the ECA of purified FXIa added to plasma. Dilutions of
purified FXIa were added to FXI-deficient plasma or EDTA plasma
containing 100 mmol/L benzamidine. Samples were tested in the ECA as
described in Materials and Methods. The means of four experiments ± SD are given. FXIa in dilution buffer (), in FXI-deficient plasma
(), or in EDTA plasma (). Control EDTA plasma without FXIa
( ).
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Besides benzamidine, NaCl (1 mmol/L) and aprotinine (2 µg/mL) were
also added to the dilution buffer to minimize any nonspecific conversion of the chromogenic substrate S-2366 by proteases other than
FXIa aspecifically sticking to the solid phase. Using these conditions,
parallel curves were obtained when FXIa was diluted in the dilution
buffer or in FXI-deficient EDTA plasma or normal EDTA plasma (1:10
diluted), both containing benzamidine (Fig 1B).
Hydrolysis of the chromogenic substrate was linear over time with
different concentrations of FXIa, also showing the stability of FXIa
during the 3 days of incubation.
Because other plasma proteases can also convert the chromogenic
substrate S-2366,27,28 the specificity of the ECA was
assessed by reconstituting FXI-deficient plasma with thrombin, plasmin, kallikrein, FXIIa, or activated protein C. The plasma samples, diluted
1:10, yielded a similar response in the ECA as FXI-deficient plasma or
dilution buffer (results not shown).
MoAb XI-5 coated on the microtiter plates binds native as well as
activated FXIa. Therefore, we investigated whether FXI competed with
FXIa for binding to the limited amount of MoAb XI-5 bound to the
microtiter plate. FXI-deficient plasma was reconstituted with 5 µg/mL
FXI and diluted in the dilution buffer. Recovery of 100 pmol/L FXIa,
added to the diluted, reconstituted plasma, was 106.6% ± 4.8% (n = 4) when tested in the ECA at a plasma dilution of 1:10, and was
63.8% ± 9.5% when diluted 1:5 (results not shown). Thus, at
plasma sample dilutions of 1:10 or more, native FXI did not interfere
with the measurement of FXIa.
The interassay coefficient of variation, calculated from the results of
30 samples measured on six different occasions in a 3-month period, was
6.1% ± 1.6%.
ELISAs for FXIIa and kallikrein-C1-inhibitor complexes.
MoAb KOK-12, directed against complexed C1-inhibitor, was used to
quantify FXIIa- and kallikrein-C1-inhibitor complexes. In initial
experiments the optimal conditions for the ELISAs to detect the FXIIa-
and kallikrein-C1-inhibitor complexes were established (see Materials
and Methods). Parallel dose-response curves were obtained from the
prepared FXIIa- and kallikrein-C1-inhibitor complexes of known
concentrations and those obtained with DXS-activated plasma, whereas
background absorbance values were observed with dilutions of fresh
plasma (Fig 2).
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| Fig 2.
ELISAs for FXIIa-C1-inhibitor (A) and
kallikrein-C1-inhibitor (B) complexes. Absorbance values of dilutions
of preformed complexes of FXIIa-C1-inhibitor (A) or
kallikreinC1-inhibitor (B) in PBS (), and absorbance values of
DXS-activated reference plasma () and of fresh plasma () are
shown.
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The specificity of the assays was assessed by incubating the preformed
complexes in FXII- and PK-deficient plasmas and dilutions of these
mixtures were tested in both the FXIIa- and kallikrein-C1-inhibitor complex assays. Only in the corresponding ELISA was a significant signal detected. Activation of plasma deficient in FXII or PK with DXS
yielded only a response in the FXIIa-C1-inhibitor ELISA for
PK-deficient plasma, probably due to auto-activation of FXII (results
not shown).29
Because the capture antibody KOK-12 not only binds complexed
C1-inhibitor but also inactivated C1-inhibitor, which is present in
normal plasma,30 we established the minimal dilution of the plasma samples to be tested at which no competition of inactivated C1-inhibitor was observed. Dilutions of normal plasma were added to the
preformed complexes and tested in the respective ELISAs. At a plasma
dilution of 1:20 no effect on the recovery of the preformed complexes
was observed (results not shown). Therefore, plasma samples were
diluted at least 1:20 in the ELISAs.
The lower detection limits of the ELISAs, set as the values that
correspond to twice the absorbance of the background, were 80 pmol/L
for each ELISA.
The interassay coefficient of variation, calculated from the results of
four standard samples in a 2-month period, were 3.4% and 16.1% for
the FXIIa- and kallikrein-C1-inhibitor complexes, respectively.
In vitro contact activation.
Because the detection limits of the assays for FXI activation were
eightfold lower than those of the FXII- and kallikrein-C1-inhibitor complex ELISAs, we performed a series of experiments using DXS and
kaolin as activators of the contact system to investigate the ratio
between FXIIa- or kallikrein-C1-inhibitor and FXIa-C1-inhibitor complexes. Activation of EDTA plasma with 0.05 or 0.2 mg/mL DXS generated 2.6 and 3.9 nmol/L FXIa-C1-inhibitor complexes, 32 and 51 nmol/L FXIIa-C1-inhibitor complexes, and 61 and 60 nmol/L
kallikrein-C1-inhibitor complexes, respectively. Kaolin activation,
either 2.5 or 5 mg/mL, resulted at both concentrations in formation of
15 nmol/L FXIa-C1-inhibitor complexes, 163 nmol/L FXIIa-C1-inhibitor
complexes, and 79 and 90 nmol/L kallikrein-C1-inhibitor complexes,
respectively (Fig 3).
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| Fig 3.
Generation of FXIa-, FXIIa- and kallikrein-C1-inhibitor
complexes on in vitro contact activation in plasma. EDTA plasma was
activated with DXS (0.05 or 0.2 mg/mL, final concentration) or with
kaolin (2.5 or 5 mg/mL), respectively. FXIa-, FXIIa-, and
kallikrein-C1-inhibitor complexes were measured using specific ELISAs
(see Materials and Methods). White bars () represent
FXIa-C1-inhibitor complexes, shaded bars ( ) represent
FXIIa-C1-inhibitor complexes, and black bars () represent
kallikrein-C1-inhibitor complexes.
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LPS infusion in volunteers.
In all eight volunteers, flu-like symptoms such as headache, chills,
nausea, vomiting, myalgia, and fever were observed after LPS
administration. After 24 hours all volunteers were symptom free.
Infusion of LPS induced increases of the plasma levels of the
prothrombin fragment F1 + 2 and TAT complex plasma concentrations with
peak levels at 4 hours of 7.1 ± 2 nmol/L and 26.8 ± 3.3 µg/L, respectively (Fig 4). Both increases were
significant over time (P < .0001). Thrombin generation was
first evident after 1 hour by significant increase of TAT complexes of
8.5 ± 3.4 µg/L. Thrombin production was still detectable at 24 hours, 0.16 nmol/L and 3.6 µg/L for F1 + 2 and TAT complexes,
respectively.
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| Fig 4.
F1 + 2 and TAT complexes after LPS infusion. Mean ± SEM of F1+2 () and TAT complexes () after LPS administration
(4 ng/kg). The left y-axis indicates the absolute difference
from the mean value at t = 0 for F1 + 2, and the right y-axis
indicates the absolute difference from the mean value at t = 0 of the
TAT complexes. The P value indicates difference over time of
both F1 + 2 and TAT complexes.
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FXI activation was assessed using the ECA, which measures noncomplexed
FXIa, and the FXIa-FXIa inhibitor ELISAs, which detect FXIa complexed
to its inhibitors in plasma. As shown in
Fig 5, the administration of LPS induced a
significant increase in FXIa activity as measured in the ECA with a
peak concentration at 2 hours of 11.2 ± 3.8 pmol/L, followed by a
subsequent decline to baseline values at 6 and 24 hours. After a delay
the FXIa-FXIa inhibitor complex concentration also increased at 3 hours
after LPS infusion and reached maximal levels at 6 and 24 hours of 11.1 ± 6.1 pmol/L and 15.4 ± 6.7 pmol/L, respectively (P
value over time, P < .001 for the ECA and P < .0001 for the FXIa-FXIa inhibitor complexes).
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| Fig 5.
Activation of FXI after LPS infusion. Mean ± SEM of FXIa levels measured in the ECA () and the total amount of
the FXIa-FXIa inhibitor complexes () after LPS administration (4 ng/kg). The y-axis presents the absolute difference from the mean value
at t = 0 of both the ECA and the FXIa-FXIa inhibitor complexes. The
P value indicates difference over time (P < .001 for
the ECA, P < .0001 for the FXIa-FXIa inhibitor complexes).
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As shown in Table 1, the activation of FXI
was accompanied by a small but significant decline in FXI antigen
levels after 2 hours as measured by ELISA; the levels returned to
baseline values at 24 hours.
No changes over time were measured in the ELISAs for PK antigen and
FXII antigen (Table 1) and also no increases of FXIIa- or
kallikrein-C1-inhibitor complexes were detected (results not shown).
Data analysis.
The relationship between the observed time course of FXIa generation,
measured by ECA, versus FXIa-FXIa inhibitor complex formation was
studied by computer simulations.
First, a theoretical model was constructed in which all processes
related to FXIa generation and inhibition take place in the fluid phase
of the plasma compartment. The parameter values chosen were based on
observations in previous experiments, ie, a half-life for FXIa
inhibition in the order of minutes31,32 and a plasma
half-life of FXIa-FXIa inhibitor complexes between 100 and 350 minutes.32 However, in this model there was virtually no
time delay between the start of FXIa generation and the raise in
FXIa-FXIa inhibitor complexes. Furthermore, the modeled FXIa plasma
concentrations as measured in the ECA remained a factor 10 to 100 times
lower than the concentrations of FXIa-FXIa inhibitor complexes. Thus,
this model could not provide a satisfactory fit to the experimental
data assuming realistic parameter values. It was therefore concluded
that inhibition of FXIa generated after in vivo LPS infusion differs
from inhibition of FXIa infused intravenously. Other models were
constructed to fit the observed time course of noncomplexed FXIa and
FXIa-FXIa inhibitor complexes. A satisfactory fit was obtained with the
scheme shown in the inset of Fig 6, where
it is assumed that FXIa is rapidly removed from the plasma compartment
by reversibly binding to a surface or inhibitor (t1/2 = 1.5 minutes) and that the FXIa-FXIa inhibitor complex are formed or are
liberated into the plasma compartment very slowly (t1/2 = 72 h)
and cleared at a normal rate (t1/2 = 100 minutes). FXIa generation in this constructed model is maximal at 2 hours with 10 pmol/minute, but continues at a low rate until 23 hours, undetected by the ECA (Fig
6). When assuming a plasma volume of 3 L, the total amount of generated
FXIa is ± 670 pmol/L, which corresponds to 2% to 3% of the plasma
concentration of FXI.
View larger version (22K):
[in this window]
[in a new window]
| Fig 6.
Computer simulation for FXIa activation and inhibition in
LPS volunteers. A mathematical model was constructed to explain the
course of FXIa concentrations as measured in the ECA and ELISAs. The
inset shows a scheme of the assumed transfer of substances, in which
the formation of FXIa-FXIa inhibitor complexes occurs outside the fluid
phase plasma compartment. The graph shows the calculated time course
for the concentrations of noncomplexed FXIa ( - ) and FXIa-FXIa
inhibitor complexes () in the plasma compartment assuming inhibition
of FXIa outside the plasma compartment, a slow dissociation of bound
FXIa-FXIa inhibitor complexes into the plasma compartment, and a plasma
half-life of FXIa-FXIa inhibitor complexes of 100 minutes. The left
y-axis represents the absolute increase in FXIa and FXIa-FXIa inhibitor
complexes in pmol/L, the right y-axis represents the formation of
endogenous FXIa (----) in pmol/minute. The
measured FXIa in the ECA ( ) and FXIa-FXIa inhibitor complexes ()
are given for comparison.
|
|
| |
DISCUSSION |
According to current views, FXI can be activated by thrombin and is
primarily involved in sustaining thrombin formation.2,33,34 However, there is no evidence for such a mechanism occurring in vivo.
In this study we have used the well-characterized model of
endotoxin-induced coagulation activation in humans to investigate FXI
activation. For this purpose we developed a set of novel and sensitive
immunoassays, including an ECA for measuring free, noncomplexed FXIa.
This assay was developed because in a previous study it was shown that
intravenously infused human FXIa in chimpanzees has a half-life time of
inhibition of several minutes,32 indicating that also free,
noncomplexed FXIa could be detectable in the circulation. For the ECA
it was necessary to prevent any complexation of FXIa to its inhibitors
ex vivo, which was achieved by collecting blood samples at high
concentrations of the reversible inhibitor benzamidine.26 With this assay we detected an absolute rise in plasma FXIa of 10 to 11 pmol/L at 1 and 2 hours, respectively, indicating that FXI was
activated early after infusion of LPS (Fig 5). Thereafter, FXIa was
inhibited, reflected by a significant increment in FXIa-FXIa inhibitor
complexes after 2 hours, which was still detectable at 24 hours.
Consistent with activation of FXI was the decrease in antigen levels
after 2 hours, which remained low at least until 6 hours after the
endotoxin infusion, indicative for continuous activation of FXI (Table
1).
These data together with results from previous studies in chimpanzees
and in vitro data were used to construct a model for FXI activation and
inhibition (Fig 6). This model predicts that inhibition of endogenously
formed FXIa does not take place in the plasma compartment, ie, is not a
fluid phase inhibition. The late rise in FXIa-FXIa inhibitor
concentrations as compared with the early detection of noncomplexed
FXIa can be explained by assuming that the inhibition of FXIa occurs
locally and that the FXI-FXIa inhibitor complexes are released very
slowly from their site of formation into the plasma compartment. This
site for FXIa inhibition is not necessarily the same as the site for
activation because some free, noncomplexed FXIa was also measured in
the plasma compartment. Possible locations for FXIa inhibition could be
activated platelets or endothelial cells that are both capable of
reversible binding of FXIa in vitro.35,36 Another
possibility is that FXIa is complexed to inhibitors linked to the
endothelial surface such as C1-inhibitor37 and antithrombin
via glycoaminoglycans.38 Also, reversible inhibition of
FXIa by protease nexin-2, an Kunitz-type inhibitor released by the
-granules of activated platelets,39,40 could explain the
observed results. However, the exact inhibitory mechanism of FXIa in
vivo is beyond the scope of this study and requires further
investigation.
The revised model of coagulation assumes that activation of FXI is
mediated by thrombin, generated via the TF-FVIIa pathway.2 Thrombin generation was already detected at 1 hour after the endotoxin infusion and the levels of both TAT complexes and F1 + 2 reached peak
levels at 3 and 4 hours (Fig 5). We speculate that the
initial formation of small amounts of thrombin directly activated FXI, whereafter activation of factors IX and X contributed to the generation of the larger amounts of thrombin measured at 3 and 4 hours, which maintained a prolonged generation of FXIa.
Previous studies from our group and others have suggested that
endotoxin-induced activation was solely dependent on the TF-FVIIa complex.14-16 To further establish this mechanism, we
developed novel ELISAs for monitoring contact activation, together with measurements of FXII and PK antigen concentrations. The ELISAs for the
detection of FXIIa- and kallikrein-C1-inhibitor were modified from
previously published radioimmunoassays29 and detected as low as 80 pmol/L activation of FXII or PK. Assuming a plasma
concentration of FXII and PK of 0.4 µmol/L and 0.47 µmol/L,
respectively (as measured in pooled standard plasma from 30 normal
volunteers), this corresponds to activation of 0.02% of total FXII or
PK, which is 50- to 100-fold more sensitive compared with previously
described assays.41,42
In this study we did not detect activation of the contact system,
neither by an increase in FXIIa- or kallikrein-C1-inhibitor complexes
nor by decreasing antigen levels of FXII or PK. Assuming similar ratios of FXIIa-, kallikrein-, and FXIa-C1-inhibitor complexes to be generated in vivo as observed in the in vitro experiments (Fig
3), and in view of the amount of FXIa generated after LPS infusion,
FXIIa- and kallikrein-C1-inhibitor complexes should have been detected
to explain FXI activation through the contact system. This finding
corroborates an earlier study in which a lower dose of endotoxin also
did not elicit detectable contact activation.43 However, it
should be noted that activation of the contact system is notoriously
difficult to measure in vivo, and only in some occasional patients with
severe septic shock was clear evidence for activation
found.20,21,25,44 Using a specific assay for
kallikrein-2-macroglobulin complexes, enhanced levels of this
contact marker have been detected in a lethal animal model of
sepsis,17,18 but also in a similar human endotoxemia model.19 An explanation for this discrepancy with our study may be a longer half-life of kallikrein-2-macroglobulin complexes as
compared with kallikrein-C1-inhibitor complexes.45 Thus, activation of the contact route cannot be excluded with certainty under
these experimental conditions.
We conclude that activation of FXI occurs early after endotoxin
infusion to humans. Given the established role of the TF-FVIIa complex
in coagulation activation in the human endotoxemia model, and
considering the lack of conclusive evidence for contact activation, our
results are consistent with the notion that FXI is activated via a
thrombin-dependent pathway in vivo.
| |
FOOTNOTES |
Submitted November 24, 1997;
accepted July 6, 1998.
M.L. is a fellow of the Royal Dutch Academy of Arts and Science.
Supported by Grant No. 94.024 from the Netherlands Heart Foundation
(M.C.M.), and by Grant No. 32-47016.96 from the Swiss National
Foundation for Scientific Research (W.A.W.).
Presented in part at the American Heart Association Meeting, held in
New Orleans, LA, November 7-11, 1996.
Address reprint request to M.C. Minnema, MD, Academic Medical Center,
University of Amsterdam, Center for Hemostasis, Thrombosis, Atherosclerosis and Inflammation Research, F4-277, PO Box 22660, 1100 DD, Amsterdam, The Netherlands.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank the members of the Laboratory of Special Hemostasis (AMC, G1)
and Y.P.T. Lubbers from the Laboratory of Clinical and Experimental
Immunology (CLB, U110) for their technical assistance.
| |
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Abstract
Introduction
Abstract