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RAPIDCOMMUNICATION
Human Monocytes Support FactorX Activation by Factor VIIa, Independent
of Tissue Factor: Implicationsfor the Therapeutic Mechanismof High-Dose
Factor VIIa in Hemophilia
By Maureane Hoffman, Dougald M. Monroe, and Harold R. Roberts
High doses ofrecombinant factorVlla are useful in managing bleeding in hemophiliacswith inhibitors. Whether this
therapeutic effect of factor
Vlla is dependent on tissue factor (TF) is a matter of debate. W e examined the ability of
freshly isolated human monocytes(which lack TF) tosupport the activation of coagulation-factorX by factor Vlla.
The rate of factor-X activation by factor Vlla was accelerated in the presence of monocytes compared
with the rate
of X activation in solution. This activation of factor X on
monocytes was saturable with a K,,2 of about400 to 600
pmol/L factorVlla. The rate of activation was not inhibited
by an excess of inhibitory
anti-TF antibody ora Gla-containing fragment of prothrombin. In contrast to monocytes, an
endothelial cell line did
not support activation of factor
X by
factor Vlla. Our findings suggestthat at least one cell type
can accelerate activation for factor X by factor Vlla in the
absence of TF. This activity requires higher concentrations
of factor Vlla than does the TF mechanism. The concentrations ofVlla required are of
a similar order of magnitude
to
those required for a therapeutic effect of Vlla in bleeding
hemophiliacs with inhibitors.
0 1994by The American Society
of Hematology.
F
bosis to complicate its use. These complications are seen
when preparations of the activated vitamin-K-dependent
coagulation proteins, such as Autoplex, are used to treat patients with inhibitors. High-dose factor VIIa therapy has not
shown a propensity to initiate DIC orthrombosis in clinical
trial^.^ Factor VIIa can activate factor X invitro in the presence of phospholipid vesicles at a greater rate than in solution, although much slower than in the presence of TF.‘
Therefore, one group has postulated that the therapeutic
effect of high concentrations of factor VIIa is independent
of T F and may be the result of X activationon endogenous
phospholipid surfaces6 The current work was designed to
test the hypothesis that some cell membranes can support
activation of factor X by high concentrations of factor VIIa
in the absence of TF and factors IX and VIII.
We wanted to examine two types of cells that would be
in contact with plasma: endothelial cells and mononuclear
phagocytes. We used an immortalized human hybrid endothelial line, EA.hy 936 cells’ as our model endothelial cell.
Endothelial cells are generally viewed as having anticoagulant, rather than procoagulant properties. They can express
T F after exposure to inflammatory cytokines, but this process requires several hours.’ However, endothelial cells have
binding sites for factor VI1 and VIIa, which are not dependent onT F expression.’ The functional significance ofthese
binding sites is not known. It ispossible that such sites might
be part of a TF-independent mechanism for X activation.
Mononuclear phagocytes can express several procoagulant activities. These include tissue factor,” a Mac-l associated X-activating activity,’’ thefactor Vllla/lXa X-activating complex,12 prothrombinase activity.13 and HLA-DRassociated procoagulant activity.14TF is not normally present on circulating monocytes,15 but is expressed after
exposure to cytokines or endotoxin for several hours.16
The purpose of the current work wasto examine the ability of monocytes and a hybrid endothelial line, Ea.hy 926,
to support factor X activationby factor VIIa independent of
TF. and thuspossibly provide an explanation for the therapeutic effect ofhigh-dose VIIa in bleeding hemophiliacs.
ACTORS VI11 AND IX are required for normal hemostasis, as evidenced by the severe bleeding diathesis
seen in patients lacking either factor. Recently, hemorrhage
in hemophiliacs with high titer inhibitors has been successfully treated with infusion of large amounts of recombinant
factor VIIa.’ High doses of factor VIIa also tend to normalize the hemostatic defect in an animal model of hemo~ h i l i a .Thus,
~ , ~ high concentrations of VIIa can at least partially overcome the requirement for a functionalXase (IXa/
VIIIa) complex. The mechanism behind the success of this
therapy is currently unclear. Factor VIIa requires its membrane-bound cofactor, tissue factor (TF), for optimal activation of factor X.4 The kd for binding of factor VIIa to
T F is around 100 pmol/L. Effective therapy of hemophilic
bleeding requires doses of factor VIIa that should bring
plasma levels into the low nanomolar range.’ Veryhigh
plasma doses of factor VIIa might be required to get the factor into areas of tissue injury. However, if factor VIIa acts
through a TF-dependent mechanism,it seems unlikely that
levels more than 10-fold above the K,,, should be required
for effective therapy. Itis equally unlikely that high-dose factor VIIa is effective by
virtue of its limited ability to activate
factor X in solution. If factor VIIa were activating significant
amounts of factor X in solution,one would expect disseminated intravascular coagulation (DIC) andpossibly thromFrom the Department of’ Pathology, Durham Veterans Affair5
Medical Center and Duke University, Durham. NC; and the Depurtment ofA4edicine. Division of Hematology, Universily ofNorth Carolina at Chapel Hill.
Submitted August2. 1993; accepted October 11, 1993.
Supported in part by Grant No. R01 HL48320fiom the National
Institutes of Health and by theUS Department of Veterans Affairs.
Address reprint requests to Maureane R. Hoffman, MD, PhD.
Laboratory Service (113) Durham VA Medical Center, Durham.
NC27705.
The publication costs of this article were defiayed 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.
01994 by TheAmerican Society of’Hematology.
0006-4971/94/8301-0038$3.00/0
38
MATERIALS AND METHODS
Materials. Dulbecco’s modified Eagle medium (DMEM)and
Eagle’s minimum essential medium (EMEM) were purchased from
Blood, Vol83, No 1 (January l ) , 1994: pp 38-42
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FACTOR X ACTIVATION BY HIGH-DOSE VllA
the Tissue Culture Facility ofthe Lineberger Cancer Research Center at The University of North Carolina-Chapel Hill. Mono-Poly
Resolving Medium (MPRM) was purchased from Flow Laboratories, Inc. Lipopolysaccharide (LPS) from Escherichia coli was
purchased from Sigma
Chemical CO (St Louis, MO).
A monoclonal
antibody (MoAb) to Mac-l (CD1 lb/CDl8) was purchased from
Boehringer Mannheim Biochemicals(Indianapolis, IN). The antibody producedby this clone (M 1/70)inhibits 98%of the binding of
factor X to monocytesvia the Mac-l antigen.” An inhibitory
MoAb againsthuman TF was purchased from American Diagnostics, Inc (Greenwich, CT). Recombinant human factor VIIa and
TF pathway inhibitor (TFPI) were the kind gifts of Novo Nordisk
(Gentofte, Denmark). Recombinant hiruden was purchased from
Accurate Chemical CO (Westbury, NY). Immunoaffinity-purified
factor X was purchasedfromEnzymeResearch
Laboratories
(South Bend, IN). The factor-X preparation was treated with an
inhibitor mixture (1 pmol/L tosyl-Lysyl-chloromethylketone, tosyl-Phenyl-chloromethyl ketone, Glu-Gly-Arg-chloromethyl ketone, Phe-Pro-Arg-chloromethyl ketone, and Phenyl-methyl-sulfonyl fluoride)followed by exhaustivedialysis to removeactive
proteases. Afterthis treatment, factor X contained less than 0.0 1%
Xa. Spectrozyme FXa chromogenic substrate was purchased from
American Diagnostica. A purified chymotryptic fragment of prothrombin from amino acids 1-42’* wasthe kind gift ofDr Richard
Hiskey (Department Chemistry, University of North Carolina). All
other chemicals were of high
commercial grade.
Monocyte isolation. Blood was collected from medication-free
healthy volunteersinto citrate anticoagulant.A “mononuclear cell”
band was purified on density gradients (MPRM) as described.19
Platelets were removed fromthe monocytes by washing twice with
cold Versene buffer. The resulting mononuclear cell preparation
was analyzed by flowcytometryon a FACScan flowcytometer (Becton Dickinson, Mountainview, CA) after staining with CD45-fluorescein isothiocyanate/CD14-PE Dual Color Reagent (Olympus
Immunochemicals, Lake Success, NY). All leukocytes stain with
the anti-CD45 antibody, but not erythrocytes or platelets. Monocytes are stained strongly with
the anti-CD14 antibody, neutrophils
are stained weakly, and lymphocytes are not stained. Thus, the proportion of monocytes in the preparation can be accurately determined. Cell preparations consisted of 10%to 20% monocytes,80%
to 90% lymphocytes,
and less than 1% neutrophils. Total cell counts
were determined by hematocytometer or automated counting on a
Sysmex KlOOO cell counter (Baxter Diagnostics, McGaw Park, IL).
Sufficient mononuclear cells were plated
to give 100,000 monocytes
per microtiter well. Monocytes were separated from lymphocytes
by plating for 1 hour at 37°C in DMEM with 5% neonatal bovine
serum (NBS; Biocell,Inc, Carson, CA). Nonadherent cells were removed by washing with phosphate-buffered saline, and all subsequent incubations were performed in DMEM without NBS. All
buffers, sera,and media contained less than 0.06 ng/mL endotoxin.
TF expression by monocytes was induced by culturing them for 18
hours in the presence of500 ng/mL LPS.
Hybrid endothelial (Ea.hy 926) cell culture. The Ea.hy 926 cell
line is a continuous human-derived cell linethat displays many features characteristicof vascularendothelial cell^.'^-*^ Ea.hy cells were
cultured in EMEM with 10%fetal calfserum. For the X-activation
assays, Ea.hy cells were plated
in 96-well platesand grown to confluence.The cell monolayerswere washed thoroughly with HEPESbuffered saline (HBS)
(pH = 7.4) containing 1% bovine serum albumin beforeuse.
Activity assays. Activation of factor X was measured using a
specific chromogenic substrate. TF-dependent and independent
factor-)< activation was assayed as follows. Monocyte monolayers
were incubated for 1 hour in 20 mmol/L HEPES, 150 mmol/L
39
0.0
2.0
4.0
6.0
8.0
.J
10.0
antl-TF Antibody (uglml)
Fig 1. Inhibition of factorX activation on LPS-treated monocytes
by anti-TF antibody. Monocytes were plated and cultured for l 8
hours in the presence of 500 ng/mL LPS and assayed for factor-)<
activation in the presence of 500 pmol/L factor Vlla as described
in Materials and Methods. The indicated amount of antibody was
added 5 minutes before the incubation of monocyte monolayers
ofthe
with factors Vlla and X. After the incubation period, aliquots
monocyte supernatants were transferred to wells containing the
chromogenic substrate solution, and
the rate of substrate cleavage
monitored for the succeeding 20 minutes. The data points represent the results ofthree separate experiments.
NaCI, pH 7.4 (HBS) containing 3.5 mmol/L CaClz, 170 nmol/L
purified factor X (plasma concentration) and different concentrations of added factor VIIa. Activationof factor X was verifiedto be
linear for incubation periods with the monocytes ofup to 90 minutes. At the end of the incubation period, a 1 0 0 pL aliquot of the
supernatant was removed and added to an equal volume of HBS
containing 2.2 mmol/L EDTA, l mmol/L Spectrozyme m a , and
I mg/mL albumin. The absorbance at 405 nm wasmonitored continuously in a plate reading spectrophotometer(Vmax, Molecular
Devices Corp, Menlo Park, CA). Any baseline-Xa activity (cleavage of the chromogenic substrate in the absence of added factor
VIIa) was subtracted from the values measuredfor all of the other
wells.
In some assays, TFPI was added to the monocyte monolayer.
TFPI binds to factor Xa, then the bimolecularcomplex inhibits TF/
VIIa. When TFPI concentrations near that in plasma (100 ng/mL)
were added to LPS-treated or fresh monocytes,no significantfactorXa activity couldbe measured. We selected the lowest concentration of TFPI ( I O ng/mL) that gave significantinhibition of factor-X
activation by LPS-treated monocytes.This concentration of TFPI
only minimallyinhibited the amount of factor Xa activityin wells
containing fresh monocytes. To verify that the chromogenic substrate cleavage wasnot actually causedby a small amount of thrombin being generated, we assayed the factor-X-activating activityof
fresh monocytes inthe presence of the thrombin inhibitor hiruden.
At a concentration of 2 U/mL, hiruden had no effect on chromogenic substrate cleave in assays
of freshmonocytes.
The inhibitory activity of the anti-TF antibody against human
monocyte TF was verified as follows. Monocyte monolayers were
cultured for 4 hours with I pg/mL LPS to induce TF expression,
then assayed foractivation of factor X in the presence of200 pmol/
L factor VIIa and varying concentrations of the anti-TF antibody.
An antibody concentration of 10 pg/mL was found to completely
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HOFFMAN,MONROE,ANDROBERTS
ing all the abovecomponents was compared with thrombin generation in the presence of 250 nmol/L prothrombin fragment 1-42.
The cleavage of the chromogenic substrate (absorbance
at 405 nm)
was monitored continuously in a V,
plate reading spectrophotometer. As a control, cleavage of the chromogenic substrate in
wells lacking factor Va was also measured. This represents direct
cleavage of the chromogenic substrate by factor Xa, and by the
small amount of thrombin generated by free factor Xa. Addition of
the Gla-containing peptide to the prothrombinase assay reduced
the rate of color development to the same level seen in the absence
of added factor Va. Thus, the Gla-containing prothrombin fragment inhibited prothrombinase complex activity in a purely phospholipid-dependent system.
Data analysis. The datawere fitted to a right hyperbola defined
by the equation:v = Vmax X [VIIa]/(K1,2+ [VIIa]) using the curvefitting function of DeltaGraph Professional (Delta Point, Inc, Monterey, CA).
L
I
1000l
500
I
2000
1
I
1500
pM VIIa
Fig 2. The amount of factor X activated by fresh and LPS-cultured monocytes as a function offactor Vlla concentration. Monocytes were plated at 100,000 per well and cultured for 18 hours in
the presence of 500 ng/mLof LPS (LPS-Monos [Ell), or isolated and
plated at 100,000 per well for 1 hour (FreshMonos [G]). Both LPS
and fresh monocytes were from thesame donorand were assayed
for factor X activation at the same time. Results shown are for a
typical experiment assayed in triplicate. Factor X activation was assayed as describedin Materials and Methods. The inset shows an
expansion of the curve for fresh monocytes and representsthe values from nine separate experiments performed on separate days.
inhibit the factor-VIIa-dependent activation of factor X (Fig l).
This concentration of anti-TF antibody was used to eliminate any
contribution of T F in our studies of factor-X activation on fresh
monocytes. TF-dependent Xase activity was defined as factorVIla-dependent factor-X activation that could be inhibited by the
anti-TF antibody. Non-TF-dependent activity was factor Xactivation that occurred in the presence of 10 pg/mL of the anti-TF antibody. As reported by others, freshly isolated unactivated monocytes
contained no TF-dependent Xase activity (at the limit of detection
of the assay). There was no color development in the assay when
factor X was omitted from the assay, thus eliminating the possibility
that the monocytes secrete esterase activity that cleaves the chromogenic substrate in theabsence of factor Xa.
Binding of vitamin-K-dependent coagulation factors to cell
membranes is at least partially mediated by calcium and Gla-dependent binding to anionic phospholipid.The Gla-containing fragment of prothrombin from residues 1-42 binds to phosphatidylserine-containing vesicles in a calcium dependent manner,18 and
should competitively inhibitGla-dependent coagulation factor
binding to phospholipid. Association of factor Xa with its cofactor,
factor Va, on a phospholipid surface dramatically accelerates prothrombin activation by factor Xa.24The ability of the 1-42 peptide
to interfere with a process dependent on anionic phospholipid was
verified by measuring its ability to inhibit the prothrombinase activity of purified factors Xa and Va on large unilamellar phospholipid vesicles. The vesicles were prepared by reverse-phase evaporation from 30%bovine brain phosphatidyl serine, and 70% synthetic
l-palmitoyl-2-oleoyl-phosphatidylcholine (both fromAvanti Polar
Lipids, Pelham, AL).25 The basic assay mixturecontained 40
mmol/L phospholipid vesicles, 500 nmol/L prothrombin, 1 nmol/
L factor Va, 25 pmol/L factor Xa, 2.5 mmol/L CaClz, and 0.5
mmol/L Chromozyme TH. Thrombingeneration in wells contain-
RESULTS
The rate of factor-X activation in the presence of LPStreated and fresh monocytes as a functionof increasing factor VIIa concentration is shown in Fig 2. The rate of factorX activationon monocytes was at least IO-fold greater after
18 hours in culture with LPS. The titration curve with respect to factor-VIIa concentration was saturable, implying
that a limited number of sites on themonocyte surface were
responsible for factor-X activation. The concentration of
added factor VIIa required for half maximal factor-X activation (K,,2) was 145 2 50 pmol/L VIIa (with the factor-X
concentration held constant at I70 nmol/L) for LPS-treated
*0°
1
.
I
T
anti-TF
baseline
Fresh Monos
TFPl
Fig 3. The effect of an anti-TF antibody and TFPl onthe amount
of factor X activation by fresh and LPS-treatedmonocytes. Monocytes were isolated and cultured as for Fig 2.The anti-TF antibody
(10 pg/mL) and TFPl (10 ng/mL) were added 5 minutes before the
addition of factor Vlla to the factor-X activation assays. After the
incubation period, aliquots were removed and added to separate
wells containing the chromogenic substrate solution. The rate of
chromogenic substrate cleavage was monitored over then succeeding 20 minutes. The figure representsthe results of four separate experiments. The amount of factor X activated by LPS Monos
was significantly reduced by anti-TF antibodies and by TFPl (P <
.05, Student's paired t-test). The amount of factor X activated by
fresh monocytes was also slightly lower in the presence of TFPI.
but thedifference was notstatistically significant.
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41
FACTOR X ACTIVATION BY HIGH-DOSE VllA
lo
l
No Gla Peptide
TT
H + Gla Peptide
0
500
2000
pM Factor Vlla
Fig 4. The effect of prothrombinfragment 1-42 on TF-independent activation of factor X on fresh monocytes. Monocytes were
isolated, plated, and assayed for factor-X activation as describedin
Materials and Methods in the presence and absence of a 100-fold
molar excess ofthe prothrombinfragment over factor Vlla.
monocytes, and 580 f 153 pmol/L for fresh monocytes.
When assayed in the presence of 10 pg/mL inhibitory antiT F antibody, the K1,2for fresh monocytes was 406 78
pmol/L factor VIIa. In the absence of monocytes, there was
no detectable increase in X activationat increasing concentrations of factor VIIa. The X-activating activity of LPStreated monocyteswas inhibited by 85%to 90% in the presence of anti-TF antibody or TFPI, whereas the factor-Xactivating activity of fresh monocytes was not inhibitedsignificant (Fig 3).
In contrast to theresults obtained with monocytes, Ea.hy
cells had little ability to support factor-X activation by VIIa.
Low levels of factor-X activation were inhibited by the antiT F antibody, indicating that this activity was caused by
small amounts of T F expression (data not shown).
Factor Xcan be activated by factor VIIa in acell-free mixture containing calciumand phospholipid.6326
Interaction of
factors VIIa and X with the phospholipid surface is calciumdependent and mediated by Gla residues. We wanted to determine whether the activationof factor X by factor VIIa on
monocytes was primarily caused by the expression of anionic phospholipid on monocytes. Therefore, we examined
the effect o f a prothrombin fragment, from amino acids 142 of prothrombin, on monocyte-mediated factor-X activation. This peptide contains 10 y-carboxy Gla residues. As
described in Materials and Methods, this peptide inhibited
prothrombinase activity on phospholipid vesicles. It would
be expected to inhibit factor-X activation on monocytes if
activation is caused by only Gla-mediated binding of factor
VIIa to monocyte membrane^.^' We found that a 100-fold
molar excess ofthe prothrombinfragment (up to200 nmol/
L) over factor VIIa did not decrease factor-X activation over
the range ofconcentrations of factor VIIa tested (Fig 4). This
suggests that a specific binding site, rather than only Glamediated binding to anionic phospholipid plays a role in
*
activation of factor X on monocytes by high concentrations
of VIIa.
We hypothesized that TF-independent factor-X activation by highconcentrations of VIIa might be related to MacI-dependent factor-X binding and activation on monocytes." Therefore, we examined the effect ofan anti-Mac-l
antibody on factor X activation on monocytes. Not all antiMac-l antibodiesinhibit factor Xbinding and activation.""' We purchased a MoAb that inhibited Mac-l-dependent binding of factor X to monocytes by 98%
(M 1/70).17 We verified, by indirect immunofluorescence
and flow cytometry, that theantibody had bound tofreshly
isolated monocytes (data not shown). However, this antibody had no effect on factor-X activation on fresh monocytes in the presence of 2000 pmol/L factor VIIa (data not
shown).
DISCUSSION
Fresh monocytes, but not an endothelial cell line, supportedTF-independent activation of factor X by factor
VIIa. The rate of factor Xa generation was less than 10%of
the rate achieved after induction of T F expression on the
monocytes. The TF-independent factor X activation was
not simply caused by the provision of anionic phospholipid
by monocyte membranes, nor was it caused by a Mac-ldependentmechanism.This
factor-X-activating activity
was detected at concentrations of factor VIIa greater than
those required for TF-mediated activity. Thus, this TF-independent factor-X-activating activity could play a role in
the ability of highdoses of factor VIIa to overcome bleeding
in hemophiliapatients.
In this report,we only tested two cell types. The ability to
support factor Xactivation by factor VIIa may not be
unique to monocytes. However, the fact that endothelial
cells do not have this property is consistent with the finding
that administration of high doses of factor VIIa rarely (if
ever) leads to the development of DIC.' Monocytes comprise less than 5% of the total circulating leukocytes. Because the rate of factor Xa generation on monocytes is quite
low in the absence of TF, significant amounts of factor Xa
might only accumulate at sites of injury where leukocytes
and platelets adhere. At sites of injury tissue macrophages
or stromal cells might also be able to support factor Xa production in the presence of high doses of factor VIIa. TFindependent Xa generation might be an important source
of factor Xa in a hemophiliac, because it would not be inhibited by TFPI. Such an activity could continue to provide
factor Xa after the more potent TF-dependent activity has
been inhibited.
ACKNOWLEDGMENT
The authors acknowledge the excellent technical assistance of T.
Susan Wortham.
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1994 83: 38-42
Human monocytes support factor X activation by factor VIIa,
independent of tissue factor: implications for the therapeutic
mechanism of high- dose factor VIIa in hemophilia [see comments]
M Hoffman, DM Monroe and HR Roberts
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