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Influence of endothelial cell protein C receptor on plasma
clearance of factor VIIa
R. GOPALAKRISHNAN,* U. R. PENDURTHI,* U. HEDNER,? H. AGERSØ,? C. T. ESMON§
and L. V. M. RAO*
*Center for Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, TX, USA; ?Department of Medicine, Malmo
University Hospital, University of Lund, Malmo, Sweden; ?Pharmacology, Biopharmaceuticals Research Unit, Novo Nordisk A/S, Maaloev,
Denmark; and §Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, Howard Hughes Medical Institute, Oklahoma City,
To cite this article: Gopalakrishnan R, Pendurthi UR, Hedner U, Agersø H, Esmon CT, Rao LVM. Influence of endothelial cell protein C receptor on
plasma clearance of factor VIIa. J Thromb Haemost 2012; 10: 971–3.
Both plasma factor (F)VII andFVIIa arecleared relatively fast
from circulation with a half-life of 3–6 h in humans [1,2]. The
mechanism(s) responsible for the clearance of FVII/FVIIa
from circulation are unknown. Pharmacokinetic studies in
mice have shown that FVII, FVIIa, active-site blocked FVIIa
and pre-formed FVIIa–antithrombin complexes are cleared
with similar rates, indicating that plasma elimination kinetics
for FVII were independent of its activation and subsequent
inactivation by plasma inhibitors . Nonetheless, recent
studies have implicated that a substantial fraction of pharma-
cologically administered FVIIa activity is inactivated by
antithrombin (AT) in humans and dogs, which could explain
differences observed in FVIIa activity and antigen clearance
curves in these previous studies [4,5]. Recent studies from our
laboratory and others showed that endothelial cell protein C
receptor (EPCR) acts as a true cellular receptor for FVII or
FVIIa [6–8] and promotes FVIIa endocytosis in cell model
systems [7,9]. Administration of murine EPCR blocking
antibodies was shown to reduce FVIIa clearance from
circulation, particularly in the initial phase (a-phase) of
clearance, indicating that EPCR may play a role in FVII
clearance in vivo . However, EPCR blocking antibodies that
could fully block FVIIa binding to murine EPCR prolonged
Correspondence: L. V. M. Rao, Center for Biomedical Research,
The University of Texas Health Science Center at Tyler, Tyler, TX,
Tel.: +1 903 877 7332; Fax: +1 903 877 7426.
Received 8 February 2012, accepted 15 February 2012
? 2012 International Society on Thrombosis and Haemostasis
Letters to the Editor 971
the circulatory half-time of FVIIa only modestly and did not
block the clearance of FVIIa from circulation . Together
these data suggest EPCR may play a role in the initial, rapid
phase of FVIIa clearance but other mechanism(s) may be
responsible for its clearance in the terminal phase. To further
investigate the potential role of EPCR in FVIIa clearance in a
more stringent model system, in the present study we evaluated
plasma elimination kinetics of FVIIa in wild-type, EPCR-
deficient and EPCR-over expressing mice. Given that human
binds only negligibly [10,11], we have used human FVIIa in the
Wild-type littermate controls, EPCR-deficient mice  or
EPCR-over expressing mice  were injected with125I-labeled
human FVIIa (5 lg kg)1) as a single intravenous bolus via the
tail vein. The low concentration of FVIIa dosing was chosen to
reflect elimination kinetics of FVII at its plasma concentration.
All mice were bled retro-orbitally at 3 min after FVIIa
administration and thereafter at one or two pre-set time
points. Mice were anesthetized by isoflurane gas for tail vein
injection and blood sampling, and experiments wereconducted
Guide for the Care and Use of Laboratory Animals and
approved by the Institutional Animal Use and Care Commit-
tee. Except in a rare instance, three or more mice were used per
time point. Blood (45 lL) was collected into citrate anticoag-
ulant (5 lL of 0.13 M tri-sodium citrate) and plasma was
obtained by centrifugation at 4000 · g for 5 min using a table-
top Eppendorf centrifuge. FVIIa concentration in plasma was
quantified by measuring radioactivity. Pharmacokinetics were
evaluated by a standard non-compartmental method or fitting
the data to a two-compartmental model using the NONMEM
modeling program (GloboMax/ICON, Ellicott City, MD,
As shown in Fig. 1A, relatively large differences were
observed among the genotypes at the first sampling time point
(3 min). At this point, approximately 20% more FVIIa was
recovered in the circulation of EPCR-deficient mice compared
with the wild-type littermates. In contrast, FVIIa recovery in
plasma of EPCR-over expressing mice was reduced by 30%
compared with wild-type mice. These data indicate that a
fraction of FVIIa administered to wild-type and more so to
EPCR-over expressing mice was removed from circulation
almost instantaneously after dosing. This indicates that FVIIa
administered to mice readily associates with EPCR on the
vascular endothelium. These data also suggest that a
substantial fraction of EPCR on the vascular endothelium is
plasma concentrations and exogenously administered FVIIa is
capable of binding to unoccupied EPCR in vivo. Interestingly,
when FVIIa elimination kinetics in plasma were normalized to
the mean maximum concentration of FVIIa in plasma
measured at 3 min, the pharmacokinetic curves were almost
identical among all three genotypes (Fig. 1B). Analysis of the
data using the standard non-compartmental method showed a
very similar half-life of FVIIa, between 2.2 and 2.4 h, in all
type, 60 ng mL)1; EPCR deficient, 78 ng mL)1; EPCR over-
expressors, 45 ng mL)1).
When the data were fitted to a two-compartmental model
using Nonmem modeling, it confirmed the variation in
bioavailability of FVIIa among the wild-type, EPCR-deficient
and EPCR-over expressing mice. The bioavailability of FVIIa
compared with wild-type mice, suggesting that less FVIIa was
sequestered in these mice. In EPCR-over expressing mice, the
bioavailability of FVIIa in plasma was decreased by 30%,
indicating that a larger fraction of FVIIa was bound to EPCR
immediately after its administration. Only minor differences
were found in the CL (clearance) values among the wild-type
(0.018 mL min)1), EPCR-deficient (0.017 mL min)1) and
EPCR-over expressing mice (0.014 mL min)1), indicating that
FVIIa is cleared in a similar profile in these mice. After the
initial disparate sequestration trends in EPCR transgenic mice,
FVIIa was eliminated from circulation thereafter in all three
FVIIa in circulation
FVIIa in circulation
(% of initial value)
0 30 6090120
150 180 210 240
0 3060 90120150180210 240
Fig. 1. In vivo elimination of factor (F)VIIa in wild-type littermates,
endothelial cell protein C receptor (EPCR)-deficient and EPCR-over
expressing mice.125I-labeled human FVIIa (5 lg kg)1body weight,
approximately 125 ng per mice) was administered to mice as a single dose
intravenously via the tail vein. FVIIa levels in circulation were determined
240 min. The data were shown as ng FVIIa present in circulation (A) or
normalized to the percent concentration of125I-FVIIa present in circula-
tion at 3 min (B). The symbols denote: (d), wild-type; (h), EPCR-defi-
cient; ( ), EPCR-over expressing mice.
? 2012 International Society on Thrombosis and Haemostasis
972 Letters to the Editor
2.2–2.8 h. Other pharmacokinetics values, such as Q, V1 and Download full-text
V2, were identical among the three genotypes. Taken as a
whole these data indicate that EPCR may play a role in
modulation of FVII(a) levels in the circulation by sequestering
it on the vascular endothelium but it is unlikely to influence the
rate of FVII(a) clearance.
Overall, FVIIa pharmacokinetics observed in the present
study were similar to that reported earlier . Any minor
variation in CL, V1 and V2 values reported in the present
study and the previous study could reflect variation in the
methods for measuring FVIIa concentration in plasma
(radioactivity vs. clotting activity) and a difference in the
dosage levels (5 vs. 10 mg kg)1). In an earlier study, we found
that blockade of EPCR with EPCR-blocking antibody pro-
longed the t1/2aof FVIIa clearance from 19 to 31 min .
These data were interpreted as EPCR serving a role in FVIIa
clearance. However, based on the present study that showed
no significant differences in FVIIa clearance rates among wild-
type, EPCR-deficient and EPCR-over expressing mice, it is
unlikely that EPCR-mediated FVIIa internalization plays a
significant role in FVII(a) clearance in vivo, at least at
concentrations close to the endogenous plasma concentration
of FVII. In our earlier study , the reversible nature of
antibody binding to the receptor which could allow the
exchange between antibody and ligand binding to EPCR
coupled with potential differences in the bioavailability of
FVIIa in control and EPCR blocking antibody-treated mice,
might have given the impression that blockade of EPCR
prolonged the initial phase of FVIIa clearance modestly but
statistically significantly. Although the present data suggest
that EPCR does not appear to play a significant role in the rate
of FVIIa clearance from plasma, we can not completely rule
out the possibility of EPCR influencing FVII clearance in
humans as human EPCR may behave differently than murine
EPCR. Irrespective of its role (or lack there of) in FVII
clearance from plasma, EPCR still could play an important
role in the continual, prolonged transport of a small but
physiologically meaningful amount of FVIIa from circulation
into extravascular compartments. Studies investigating this
possibility are in progress within the authors? laboratories.
This work has been partly supported by a grant from Novo
Nordisk and NHLBI grants (HL 58869 and 107483).
Disclosure of Conflict of Interests
This work is partly supported by a grant from Novo Nordisk.
One of the authors (H. Agersø) is an employee of Novo
Nordisk, Denmark. U. Hedner is a consultant to Novo
Nordisk A/S, Zurich, Switzerland.
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