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RAPID COMMUNICATION
Measurement of Basal Levels of Factor VIIa in Hemophilia A and B Patients
By Peter Wildgoose, Yale Nemerson, Lisbeth Lyng Hansen, Finn E. Nielsen, Steven Glazer, and Ulla Hedner
Previous results, presented in abstract form, indicate that
replacement of thromboplastin with a mixture of phospholipid and truncated soluble tissue factor apoprotein results in
a coagulation assay that can directly measure plasma factor
Vlla levels without interference from zymogen factor VI1
(Atherosclerosis Thromb 11:1544a, 1991 [abstr]). We have
exploited the specificity and sensitivity of such a factor Vlla
specific coagulation assay to directly assess the in vivo
relationship of factor Vlll and factor IX on the production of
factor Vlla levels under nonthrombotic and nonstimulatory
conditions. Normal individuals (n = 20) were found to possess an average circulating factor Vlla level corresponding to
4.34 f 1.57 ng/mL, or approximately 1% of their total factor
VI1 antigen. Severe factor Vlll deficient patients (n = 13)
F
ACTOR VI1 is a trace vitamin K-dependent glycoprotein that participates in initiation of the extrinsic
pathway of blood coagulation. Under normal conditions
factor VI1 circulates in blood mainly as a precursor to its
activated form, factor VIIa. However, on vascular injury
factor VI1 forms a bimolecular complex with its high affinity
cell surface cofactor, tissue factor, which is presented on
many cell types. Once bound to tissue factor, factor VI1 is
rapidly converted to its activated two-chain form, factor
VIIa. Precisely which protease is responsible for the conversion of factor VI1 to VIIa under in vivo conditions is
unknown. However, it is known that factor IXa,’ factor Xa?
factor XIIa?g4 thrombin: and factor VIIa6 are all efficient
activators of factor VI1 in the test tube. Once activated, the
factor VI1a:tissue factor complex rapidly activates its protein substrates factors IX and X by limited proteolysis,
which eventually leads to thrombin formation and a fibrin
clot.
Despite major advances into the biochemistry and in
vitro behavior of the coagulation factors, little is known
about the coagulation mechanism as it functions in vivo
under basal (ie, in the absence of thrombosis or provocative
stimuli) activation levels. The development of immunoassays for the activation peptides of human factor IX,7 human
factor X,8 and human prothrombin9 have provided tools for
the indirect measurement of coagulation activation. Based
on these assay systems it has been suggested that the basal
activity of the hemostatic system is primarily caused by
factor VI1a:tissue factor function, which is responsible for
the continuous generation of factor IXa, factor Xa, and
thrombin. To investigate more closely the role of factor
VIIa under basal conditions, we have used a factor VIIa
specific coagulation assaylOJ1 to measure and directly compare the circulating factor VIIa levels in normal plasma as
well as plasma from severe hemophilia A and B patients. In
this report we provide direct evidence that basal factor VIIa
levels in factor IX deficient patients is greatly reduced in
comparison with normal individuals. These results are
consistent with the hypothesis that factor IXa is the
principal in vivo activator of factor VI1 under basal conditions.
Blood, Vol80, No 1 (July 1). 1992: pp 25-28
possesseda slightly lower but statisticallysignificant (P < .01)
decrease in their basal factor Vlla levels (2.69 f: 1.52 ng/mL),
corresponding to 360% of that observed in normal individuals. On the other hand, severe factor IX deficient patients
(n = 7) were found to possess even lower levels of factor Vlla
correspondingto 0.33 k 0.15 ng/mL, or less than 10% of that
observed in normal individuals. Measurement of total factor
VI1 antigen levels shows that the variation in basalfactor Vlla
levels stems from differences in the degree of factor VI1
activation as opposed to differences in factor VI1 antigen
levels. Our present data are consistent with the hypothesis
that factor IXa is the principal in vivo activator of factor VI1
under basal conditions.
0 1992 by The American Society of Hematology.
MATERIALS AND METHODS
Bovine serum albumin (BSA) (fatty-acid free) was obtained
from Sigma (St Louis MO). Bovine brain phospholipids (Thrombofax) were purchased from Ortho Diagnostics (Raritan, NJ). Factor
VI1 deficient plasma (< 1% factor VI1 antigen), obtained from a
hereditary factor VI1 deficient patient, was generously provided by
Dr Walter Kisiel (University of New Mexico, Albuquerque).
Recombinant human factor VIIa was purified from BHK cell
culture medium as described previously.12 Recombinant soluble
tissue factor apoprotein (residues 1through 218) was purified from
the culture media of transfected BL-21 (DE3) Escherichia coli cells
by a combination of ammonium sulphate precipitation and
S-Sepharose and Q-Sepharose ion exchange ~hromatography.’~
Total factor VI1 antigen levelswere determined using a solid-phase
double-antibody enzyme-linked immunoassay (ELISA) kit obtained from Novo Nordisk (Bagsvaerd, Denmark).14This assay is a
two-site monoclonal antibody assay in which a sandwich is formed
between a solid-phase catching antibody, the factor VIIIVIIa
sample, and a peroxidase-conjugated anti-factor VI1 detecting
antibody. Total factor VIIIVIIa levels are determined by comparison to a standard curve constructed with known amounts of
recombinant factor VIIa.
Coagulation assays. All plasma factor VIIa levels were measured in a one-stage clotting assay using an ACL300R automated
coagulation instrument purchased from Instrumentation Laboratories (Ascoli Piceno, Italy). Test samples were diluted fivefold in 0.1
molIL NaC110.05 molIL Tris-HC1/0.1% BSA pH 7.4 (TBSIBSA)
and mixed with an equal volume of hereditary factor VI1 deficient
plasma to yield a total volume of 100 pL. Each aliquot was
From Novo Nordisk AIS, Biophaimaceuticals Division, Gentofte,
Denmark; and the Division of Thrombosis Research, the Deparhent
of Medicine, Mount Sinai School of Medicine of the City University of
New York, New York
Submitted March 16,1992; accepted April 18, 1992.
Address reprint requests to Peter wildgoose, PhD, Novo Nordisk
AIS, Niek Steensens Vej 1, DK-2820 Gentofte, Denmark.
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 1992 by TheAmerican Society of Hematology.
0006-4971f 9218001-0046$3.00/0
25
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WILDGOOSE ET AL
26
subsequently incubated for 5 minutes at 37°C with 50 KLof bovine
phospholipids (Thrombofax). Coagulation was then initiated by
the addition of a 1OO-kL aliquot of 10 nmol/L TF1.218 diluted in
12.5 mmol/L CaC12/0.1 mol/L NaCl/O.O5 mol/L Tris/l% BSA pH
7.4. Coagulation times were subsequently converted to factor VIIa
concentration (nanograms per milliliter) by comparison to a
standard curve constructed with varying concentrations (0.05 to 50
ng/mL) of purified recombinant factor VIIa diluted in TBS/BSA.
Collectwn and processing of blood samples. Venipunctures were
performed autraumatically and blood samples drawn into citrated
vacutainers. The citrated samples were centrifuged for 15 minutes
at 1,200g after which time the plasma was removed with a plastic
pipette and stored at -80°C. Normal plasma samples were collected from 10 fasting and 10 nonfasting individuals who had a
negative history for bleeding as well as thrombosis and were not
taking any medications at the time of sample collection. Patient
samples were obtained from 13 severe hemophilia A and seven
hemophilia B patients ( < 1% FV1II:C and < 1%FIXC). Patients
were excluded from the study if they had received factor concentrates, cryoprecipitate, and/or antifibrinolytics within the previous
48 hours.
Informed consent Informed consent was obtained from all
patients before drawing of blood samples.
RESULTS
Results by Macik and Morrissey indicate that replacement of thromboplastin with a mixture of phospholipid and
truncated soluble tissue factor apoprotein results in a
coagulation assay that can directly measure plasma factor
VIIa levels without interference from zymogen factor
VII.lo,ll Use of a similar assay system has enabled us to
compare the circulating factor VIIa levels of normal individuals to that of individuals afflicted with hemophilia A or B.
In preliminary experiments we tested the effect of adding
increasing amounts of purified recombinant factor VIIa on
the coagulation time of factor VI1 deficient plasma in the
presence of phospholipid and a soluble truncated form of
recombinant tissue factor apoprotein (residues 1 through
218). By performing a log-log transformation of the coagulation data followed by a linear regression it was possible to
construct a standard curve from which one could readily
compare coagulation time with factor VIIa content (Fig 1).
We found that this assay system could reproducibly measure factor VIIa levels over a wide range covering several
orders of magnitude between 100 pg/mL to 1 mg/mL (data
not shown).
In our initial experiments we measured the circulating
factor VIIa levels of normal individuals under fasting and
nonfasting conditions (n = 10 per group). The basal factor
VIIa levels ranged from 2.21 to 7.85 ng/mL with an average
of 4.34 c 1.57 ng/mL (Fig 2). No statistical significancewas
observed between the plasma of fasting and nonfasting
individuals. To determine the role of factor VI11 or factor
IX deficiency on basal factor VIIa levels, we also tested the
functional factor VIIa level of 13 severe hemophilia A
patients and seven severe hemophilia B patients. The
hemophilia A patients possessed a basal factor VIIa level
that varied between 1.32 and 6.24 ng/mL with an average of
2.69 5 1.52 ng/mL (Fig 2). Thus, factor VI11 deficiency
significantly decreases (P < .01) basal factor VIIa levels to
approximately 60% of that observed in normal individuals.
0.1
1
10
Factor VIIa Concentration (ndml)
Fig 1. Factor Vlla standard coagulation curve. Varying amounts of
recombinant factor Vlla were added to hereditaryfactor VI1 deficient
plasma in the presence of phospholipid and soluble tissue factor
apoprotein(residues 1 through 218). Coagulation was initiated by the
addition of CaCI, and the decrease in coagulation time measured on
an automated coagulation apparatus as described in Materials and
Methods.
Repetitive measurement of basal factor VIIa levels in
several hemophilia A patients over a time period of months
indicates that basal factor VIIa levels remain essentially
constant (data not shown).
Measurement of factor VIIa levels in hemophilia B
patients shows a drastically lower factor VIIa level in
comparison with both normal and hemophilia A patients
(P < .01). The seven hemophilia B patients tested in this
study were found to possess a circulating factor VIIa level
that varied between 0.19 and 0.59 ng/mL with an average of
A
A
E
8
8
U
n=7
0
Nmalt
Factor Vlll
Deficient
$-
Factor IX
Dendent
Fig 2. Factor Vlla levels in the plasma of patients with severe
factor Vlll deficiency, severe factor IX deficiency, and normalcontrols.
The horizontalbar representsthe mean factor Vlla level in each group.
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BASAL FACTOR Vlla LEVELS
0.33 2 0.15 ng/mL. Because the functional factor VIIa level
in normal and hemophilic plasma was not influenced by
prolonged incubation at room temperature or by such harsh
treatment as repetitive freeze thawing, it is highly unlikely
that any of the observed differences in factor VIIa levels are
caused by ex vivo events.
In control experiments all plasma samples were subjected to factor VI1 antigen testing. As expected there was
no significant (P > .1) difference in factor VI1 antigen
levels between the hemophilia A (445 2 93 ng/mL) and B
patients (430 2 89 ng/mL) and that of the normal control
group (455 2 72 ng/mL). Control experiments were also
conducted to assess the recovery of factor VIIa activity after
the addition of trace amounts of rFVIIa (5 to 10 ng/mL) to
normal, factor VI11 deficient, and factor IX deficient
plasmas. As expected, there was virtually a 100% recovery
of factor VIIa activity in each of the plasmas tested.
DISCUSSION
Measurement of the ratio of factor VI1 coagulant activity
to amidolytic activity has previously been used as a crude
measure of factor VIIa levels in hemophi1ia~s.l~
Unfortunately this technique is rather imprecise because zymogen
factor VI1 undergoes activation during the coagulation
assay and thus contributes to the observed coagulant
activity.16 However, this problem has recently been circumvented by the observation that a soluble form of tissue
factor apoprotein, lacking the cytoplasmic and transmembrane domains, retains cofactor activity toward factor VIIa
but is incapable of supporting the activation of zymogen
factor VII.” A study by Morrissey et a1 indicates that this
soluble form of tissue factor can be used in a modified
factor VI1 coagulation assay to specifically measure circulating factor VIIa levels.lOJ In the present study we have
developed a similar assay system based on the use of a
truncated tissue factor molecule spanning residues 1through
218. We find that this assay system is exquisitely sensitive to
trace amounts of factor VIIa and can reproducibly measure
recombinant factor VIIa levels between 0.01 ng/mL to 1
mg/mL.
In this communication we have exploited the specificity
and sensitivity of this factor VIIa specific coagulation assay
to directly assess the in vivo levels of factor VIIa under
nonthrombotic and nonstimulatory conditions. Previous
attempts to assess the role of factor VIIa under basal
conditions have centered on measuring the activation
peptide that is released from factor IX on activation by
either factor VI1a:tissue factor or factor XIa.7 In this regard
it has recently been shown that factor VI1 deficient patients
have reduced plasma levels of factor IX activation peptide,
whereas FXI deficient individuals possess normal levels of
factor IX activation ~ e p t i d eIn
. ~ related experiments it has
been observed that factor VI1 deficient patients also possess lower levels of factor X activation peptide as compared
with factor VI11 and factor IX deficient patients who
possess essentially normal levels of factor X activation
peptide.8 Although these data indicate that the generation
of factor IXa and factor Xa under basal conditions is
primarily caused by the action of factor VI1a:tissue factor,
27
nothing is known about the role that these coagulation
factors play on the activation of factor VI1 under basal
conditions.
To investigate this question we initially measured the
basal factor VIIa levels of normal individuals under fasting
and nonfasting conditions. We found that normal individuals, regardless of fasting condition, possessed an average
functional factor VIIa level corresponding to 4.3 ng/mL or
approximately 1%of their total factor VI1 antigen (assuming an average factor VI1 antigen level of 400 ng/mLls). To
assess the in vivo role of factor VI11 and factor IX on basal
factor VIIa levels we also measured the factor VIIa levels of
severe ( < 1%activity) hemophilia A and B patients. Factor
VI11 deficiency was found to have a small but statistically
significant effect on circulating factor VIIa levels, with
patients on the average possessing a functional factor VIIa
level ~ 6 0 %
of that observed in normals. Factor IX deficiency, on the other hand, was found to have a far greater
effect with factor IX deficient patients on the average
possessing a basal factor VIIa level corresponding to 10%
of that observed in normal individuals.
Although previous in vitro studies indicate that factor Xa
is a far more efficient activator of factor VI1 than factor
IXa,*9J0our present data are consistent with the notion that
factor IXa is responsible for basal activation of factor VI1
under in vivo conditions. Of course, it may be argued that
the decrease in basal factor VIIa levels that we have
observed in the hemophilia B patients is caused by a
concomitant decrease in the functional levels of other
serine proteases capable of activating factor VII, such as
thrombin and factor Xa. However, this explanation is quite
unlikely in view of previous observations that factor IX
deficient and factor VI11 deficient individuals possess normal levels of factor X activation peptide and prothrombin
fragmentl+z. Precisely what role factor VI11 plays in the
activation of factor VI1 is less certain. However, one can
readily envision that the reduced factor VIIa levels observed in the factor VI11 deficient patients results from a
concomitant decrease in basal factor IXa levels. Whether
this hypothesis holds true or not will require measurement
of basal factor IXa levels in factor VI11 deficient patients.
In conclusion, we have developed a simple and highly
specific coagulation assay that can reproducibly measure
functional levels of factor VIIa in plasma. Using this assay
system we have been able to assess the in vivo role of factor
VI11 and factor IX on the production of circulating factor
VIIa levels under nonthrombotic and nonstimulatory conditions. Our results are consistent with the hypothesis that
factor IXa is the principal in vivo activator of factor VI1
under basal conditions.
ACKNOWLEDGMENT
The authors thank Dr Lilian Tengbom, Sahlgren’s Hospital,
Gothenburg, Sweden, and Dr Sam Schulman of Karolinska Sjukhuset, Stockholm, Sweden, for providing us with samples of factor IX
deficient plasma. We also thank Dr Walter Kisiel, University of
New Mexico, Albuquerque, for providing us with the hereditaly
factor VI1 deficient plasma and Carsten Christofferson for excellent technical assistance.
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WILDGOOSE ET AL
28
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1992 80: 25-28
Measurement of basal levels of factor VIIa in hemophilia A and B
patients [see comments]
P Wildgoose, Y Nemerson, LL Hansen, FE Nielsen, S Glazer and U Hedner
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