© 1997 American Heart Association, Inc.
Articles |
Antithrombotic Efficacy of Inactivated Active Site Recombinant Factor VIIa Is Shear Dependent in Human Blood
From Nycomed Imaging AS, Oslo, Norway, and Novo Nordisk AS, Gentofte, Denmark (M.E., U.H.).
Correspondence to Kjell S. Sakariassen, Nycomed Imaging AS, Gaustadalléen 21, N-0371 Oslo, Norway. E-mail kjell.sakariassen{at}nycomed.telemax.no
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Abstract Several studies have indicated a profound role for factor VII(a) [FVII(a)] in venous and arterial thrombogenesis. In the present study, we quantified the inhibitory efficacy of dansyl-glutamyl-glycyl-arginyl-recombinant FVIIa (DEGR- rFVIIa) on acute thrombus formation. Thrombus formation was elicited by immobilized tissue factor (TF) in a parallel-plate perfusion chamber device at blood flow conditions characterized by wall shear rates of 100 s-1 (veins) and 650 s-1 (medium-sized healthy arteries). Native human blood was drawn directly from an antecubital vein by a pump into a heparin-coated mixing device in which DEGR-rFVIIa (0.09 to 880 nmol/L final plasma concentration) or buffer was mixed homogeneously with flowing blood. Subsequently, the blood was passed over a plastic coverslip coated with TF and phospholipids in the parallel-plate perfusion chamber. Fibrin deposition, platelet-fibrin adhesion, and platelet thrombus volume triggered by this surface were measured by morphometry. DEGR-rFVIIa inhibited thrombus formation in a dose-dependent manner, but the efficacy was shear rate dependent. At a wall shear rate of 100 s-1, the IC50 (50% inhibition) was 30 nmol/L, whereas at 650 s-1, the IC50 was 0.6 nmol/L. Binding studies to immobilized TF under flow conditions using surface plasmon resonance revealed a significantly higher on-rate for DEGR-rFVIIa and FVIIa than for FVII, 2.8x10,5 2.6x105, and 1.8x105 M-1 s-1, respectively. This indicates that a contributing factor to the shear-dependent efficacy may be a differential importance of on-rates at arterial and venous blood flow conditions.
Key Words: DEGR-rFVIIa • tissue factor • thrombus formation • blood flow
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Introduction |
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Blood coagulation is initiated when plasma factor VII (FVII) or activated factor VII (FVIIa) bind to tissue factor (TF) on a cell surface membrane or on a phospholipid-rich surface. FVII is rapidly activated to the two-chain serine protease FVIIa. The TF/FVIIa complex activates factor IX (FIX) and factor X (FX) to factor FIXa (FIXa) and factor Xa (FXa), respectively, resulting in generation of thrombin and fibrin formation at a vascular lesion.1
Few studies have been devoted to the possibility of interrupting arterial thrombus formation by interfering with the TF/FVIIa activity. In these studies, monoclonal antibodies (MAbs) directed against TF were used to inhibit the TF/FVIIa catalytic activity.2–7 Inclusion of such MAbs efficiently blocked thrombus formation in the various models. Disseminated intravascular coagulation induced by infusion of Escherichia coli in nonhuman primates was blocked by an anti-TF MAb as well.8 Other studies have focused on tissue factor pathway inhibitor (TFPI). In a canine femoral artery model, TFPI prevented reocclusion in arteries subjected to intimal injury following tPA-induced thrombolysis.9 Preincubation of the extracellular matrix of human endotoxin–stimulated endothelial cells with TFPI in the presence of FXa and FVIIa reduced fibrin deposition with more than 90% in a human ex vivo model of thrombus formation.10 Thus, there are a number of observations indicating that interference with the complex formation of TF/FVII(a) may prevent intravascular thrombosis.
Active site–directed inactivation of enzymes is a powerful tool for studies of enzyme mechanisms. Peptide chloromethyl ketone (Peptide-ck) is one class of such irreversible enzyme inactivators. Dansyl-Glu-Gly-Arg-ck (DEGRck) reacts with rFVIIa, forming a covalent bond between the peptide (DEGR) chloromethyl group and the active site Ser and His in rFVIIa. The serine protease is thus inactivated by the physical blockage of the active site region. DEGR-rFVIIa will compete with FVII(a) for the binding to TF and consequently impede TF/FVIIa catalytic activity.11 Kirchhofer et al12 showed recently that inactivated active site rFVIIa (Phe-Phe-Arg-rFVIIa) efficiently interrupted TF/FVIIa-induced thrombus formation in human native blood in the parallel-plate perfusion device at a venous shear condition. Efficient interruption of in vivo arterial thrombosis by DEGR-rFVIIa in nonhuman primate and rabbit has been reported as well.13,14
In the present study, we have evaluated the antithrombotic efficacy of DEGR-rFVIIa in human blood in an ex vivo perfusion model of thrombus formation at venous and arterial shear. A dose-dependent and shear-dependent inhibition was observed. At arterial shear, 50% inhibition (IC50) was in the subnanomolar range (0.6 nmol/L) and about 50 times lower than the IC50 value at venous blood flow (30 nmol/L). Apparently, the inhibitory capacity of DEGR-rFVIIa is potentiated by increasing shear and is a very efficient inhibitor of TF/FVIIa-induced thrombus formation at arterial blood flow.
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Materials
DEGRck was from Calbiochem. The chromogenic plasmin and streptokinase-activated plasminogen substrate S-2251 (H-D-Val-Leu-Lys-pNA) and the chromogenic FXa substrates S-2765 (N-
-Z-D-Arg-Gly-Arg-pNA) and S-2288
(H-D-Ile-Pro-Arg-pNA) were from Chromogenix. Recombinant
human apo tissue factor and lipidated tissue factor were from American
Diagnostica, Inc., and human FX was from Enzyme Research
Laboratories, Inc. Transformed human umbilical vein
endothelial cells, ECV304, were a kind gift of Dr Jordi
Felez, Research Institute for Oncology, Barcelona, Spain. Collagen
coated dextran microcarrier beads (Cytodex-3) were from Pharmacia.
ThromborelS, a preparation rich in TF and phospholipids, and the assay
for measurement of plasma levels of thrombin-antithrombin III complexes
(Enzygnost TAT micro) were from Behringwerke AS.
Preparation of Dansyl-Glutamyl-Glycyl-Arginyl rFVIIa
(DEGR-rFVIIa)
Recombinant human factor VIIa (rFVIIa) was purified from culture
media of a transfected baby hamster kidney cell line as previously
described.15 rFVIIa was inactivated
by addition of a fivefold molar excess of DEGRck. The mixture was
incubated overnight at 4°C. Inactivation of FVIIa was considered
complete when residual FVIIa activity was <0.5% as measured in a
FVIIa-specific assay.16,17 Unreacted DEGRck was
separated from inactivated FVIIa by ion exchange
chromatography. No residual DEGRck was present as
measured in a chromogenic substrate assay system of
12.5 µmol/L of DEGR-rFVIIa, 20 nmol/L of trypsin,
and 400 µmol/L of S-2251.
All in vitro and ex vivo experiments were performed with a single lot of DEGR-rFVIIa. The stock solution of 3.12 mg/mL (62.4 µmol/L) DEGR-rFVIIa in buffer (in mmol/L: glycyl-glycin 10, NaCl 150, CaCl2 10, pH 8) was further diluted as described in each experiment.
In Vitro Assays
One-Stage Amidolytic Assay
Reactions were performed directly in microtiter wells, using a
final incubation volume of 200 µL. DEGR-rFVIIa (0.1 to 200
nmol/L final concentration), FVIIa (2 to 20 nmol/L),
apoTF (1 to 5 nmol/L), and CaCl2 (5
mmol/L) in MOPS buffer (100 mmol/L), pH 7.4,
containing NaCl (100 mmol/L), and BSA (1 mg/mL) were
preincubated at ambient temperature for 15 minutes.
Chromogenic substrate S-2288 was then added, and the
enzymatic activity of the apoTF/FVIIa complex was monitored by
measurement of absorbance increase at 405 nm using a Labsystems
Multiscan Multisoft plate reader (Labsystems).
Two-Stage Amidolytic Assay With Lipidated TF
DEGR-rFVIIa (0.01 to 1000 pmol/L final concentration),
FVIIa (5 to 100 pmol/L), lipidated TF (5 pmol/L), and FX
(50 nmol/L) were mixed in 175 µL of MOPS buffer (100
mmol/L), pH 7.4, containing NaCl (100 mmol/L) and
BSA (1 mg/mL), and were preincubated for 15 minutes at ambient
temperature. The reaction was initiated by addition of 25 µL
CaCl2 (5 mmol/L). Incubations were
continued for 30 minutes and quenched with EDTA (50
nmol/L). FXa formation was quantified by monitoring hydrolysis
of the chromogenic FXa substrate S-2765.
Two-Stage Amidolytic Assay With TF-Expressing Cells
ECV304 cells constitutively express TF.18
The cells were suspended in 15 mL (5 to 7x106
cells/mL) of fetal calf serum–supplemented medium and mixed with
collagen-coated dextran microcarriers beads in a ratio of 50 to 100
cells per microcarrier. The suspension was gently mixed on a roller for
2 hours at 37°C, after which the cells were adherent and uniformly
distributed on the microcarriers. The microcarriers bearing ECV304
cells was used as a TF source.19
ECV304-microcarriers (300 to 400 beads/mL final concentration),
DEGR-rFVIIa (0.001 to 10 nmol/L), FVIIa (0.25 nmol/L) in
MOPS buffer (100 mmol/L), pH 7.4, containing NaCl (100
mmol/L), CaCl2 (5 mmol/L), and
BSA (1 mg/mL) were preincubated at ambient temperature for 30
minutes before addition of FX. FXa formation was quantified as
described for the two-stage amidolytic assay with lipidated TF (section
II).
One-Stage Clotting Assay With TF-Expressing Cells
Experiments were performed with microcarriers bearing ECV304
cells (section III) at a concentration of 7000 to 8000 beads/mL in
10 mmol/L HEPES (pH 7.0) containing NaCl (150
mmol/L), CaCl2 (12.5 mmol/L),
and BSA (1 mg/mL). To assay cell surface procoagulant, an
aliquot of microcarrier suspension (200 µL) was allowed to
preincubate for 2 minutes at 37°C in the coagulometer before addition
of an aliquot (200 µL) of DEGR-rFVIIa (0.1 to 100 nmol/L) in
citrated, platelet-free plasma pool (37°C). The clotting times
for four replicate incubations were simultaneously
determined automatically in an electromagnetic coagulometer
(Thrombotrack 4, Nycomed Pharma AS).
Surface Plasmon Resonance
Surface plasmon resonance (SPR) studies were performed on
BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden). Full length
TF was immobilized on the sensor chip surface at a
concentration of 50 µg/mL in 50 mmol/L acetate
buffer, pH 4.0. The carboxylated surface of the sensor chip was first
activated with N-hydroxysuccinimide
(NHS)/N-ethyl-N'-[3-(dimethylamino)propyl]carbodiimide hydrochloride
(EDC) using a protocol provided by the manufacturer. TF was
immobilized on the chip until a SPR signal of 1500 to 2000
resonance units over baseline. Unreacted NHS was blocked by injection
of an aliquot of 1 mol/L ethanolamine. The flow cell was
equilibrated with 50 mmol/L MOPS, pH 7.4, 100
mmol/L NaCl, 5 mmol/L CaCl2,
and 0.005% P20 at a flow rate of 5 µL/min. The ligand, FVII, rFVIIa,
or DEGR-rFVIIa, at 50 nM was injected in buffer at a flow rate of 5
µL/min, corresponding to a shear rate of 400
s-1. Between each run, the sensor chip surface
was regenerated by eluting bound ligand with an injection of 50
mmol/L EDTA. The chip with the covalently
immobilized TF showed good stability, and no decrease in
ligand binding was observed during the course of the experiment. Data
points were collected continously during the binding and dissociation
processes, and the association rate constant
(kon) and the dissociation rate constant
(koff) were estimated by using the software
(BIA Evaluation) supplied by the manufacturer. The dissociation
equilibrium constant (KD) was calculated as
the ratio of
kon/koff.
Blood Donors
Blood for ex vivo perfusion experiments was donated by healthy
nonsmoking individuals who denied ingestion of aspirin or other
nonsteroidal anti-inflammatory drugs for at least 10 days before the
blood donations. All donors gave informed consent to participate in the
study. Hemoglobin, hematocrit, and platelet count were within the
normal ranges for all volunteers (Auto Counter AC 920, Swelab
Instruments).
Preparation of TF Surface for Ex Vivo Perfusion
Experiments
One vial of Thromborel (Thromborel®S) was
dispersed in 2 mL of deionized water, incubated for 15 minutes at
37°C and diluted 1:50 in coating buffer (0.1 mol/L sodium
carbonate, pH 9.5). Thermanox plastic coverslips (Miles Laboratories)
were stored in 70% ethanol and rinsed in deionized water before being
incubated at 4°C for about 17 hours in 2 mL of the Thromborel
dilution. The Thromborel-coated coverslips were rinsed six times with
sterile phosphate buffered saline (PBS) and stored in PBS for a maximum
of 7 hours before use in perfusion experiments.20
A full characterization of Thromborel in the one-stage clotting assay
and as a surface in the ex vivo perfusion model has been previously
reported.20 Thrombus formation triggered by this
surface is in the perfusion model almost completely dependent on
TF.
Ex Vivo Perfusions With DEGR-rFVIIa, Fixation, and
Embedding
Perfusion experiments with human nonanticoagulated
blood21 and the parallel-plate perfusion chamber
device22,23 connected with a mixing
device24 were performed at 37°C. A
heparin-coated mixing device for homogenous mixing of DEGR-rFVIIa or
buffer (control) in the flowing blood was placed vertically between a
No. 19 Butterfly Infusion Set (Abbot Ireland Ltd., Sligo, Republic of
Ireland) transferring blood from an antecubital vein of the blood donor
and the parallel-plate perfusion chamber.24 Both
devices were prefilled with Buffer C (in mmol/L: NaCl 130,
KCl 2, NaHCO3 12, CaCl2
2.5, MgCl2 0.9; pH 7.4, 37°C), as recently
described.21 Blood was drawn from an antecubital
vein of a healthy donor with the Butterfly Infusion Set and mixed with
different concentrations of DEGR-rFVIIa or buffer (control; in
mmol/L: glycyl-glycin 10, NaCl 150, CaCl2
10, pH 8) in the mixing device. Both DEGR-rFVIIa and buffer were
diluted in saline and infused into the bloodstream with a syringe pump
(Alitea, Wetek AS) at a flow rate of 0.2 mL/min. The blood entered the
parallel-plate perfusion chamber and were subsequently exposed to the
coverslip coated with TF/phospholipids, which triggered thrombus
formation. The blood flow rate was maintained at 10 mL/min for 4
minutes by a nonocclusive roller pump (Gilson) placed distal to the
perfusion chamber. However, the reactive surface was exposed to blood
for 3.5 minutes owing to the 0.5-minute transit time of blood from the
inlet of the mixing device to the perfusion chamber. The shear rates at
the thrombogenic surface corresponded to venous blood flow of 100
s-1 or to arterial blood flow of 650
s-1, whereas the shear and the blood transit
time from the vein, through the mixing device, and to the flow inlet of
the perfusion chambers were similar, irrespective the shear at the
thrombogenic surface in the chamber.
The blood flow direction and the flow rate were continuously measured immediately downstream from the Butterfly Infusion Set and the mixing device. The parameters were monitored and recorded by a laser Doppler flowmeter (Transonic Systems Inc., BBS Medical Electronic AB, Kista, Sweden) to ensure that the blood donor was not exposed to DEGR-rFVIIa or buffer. The flow rate was maintained at 10 mL/min in all experiments, except for the 30 seconds when blood samples were collected for determination of plasma levels of TAT. During this period, there was an increase in the flow rate of about 25%, corresponding to a transient increase in the wall shear rate from 100 s-1 to about 125 s-1 and from 650 s-1 to about 800 s-1, respectively.
After 4 minutes of blood perfusion, the mixing device and the blood donor were disconnected from the parallel-plate perfusion chamber device, which was immediately perfused with Buffer C (37°C) for 20 s at 10 mL/min, followed by a perfusion with fixation solution consisting of 2.5% glutaraldehyde in 0.1 mol/L cacodylate, pH 7.4 (21°C) for 40 seconds. The flow was not interrupted during the 5-minute period of perfusion with blood, buffer, and fixation solution. The coverslip was removed from the chamber and kept in a freshly prepared fixation solution for 1 hour, and then stored in 0.1 mol/L cacodylate/7% sucrose at 4°C until being embedded in epoxy resin.25
Plasma Levels of TAT
Plasma TAT levels were measured proximal to the mixing
device before perfusion and distal to the perfusion chamber after 3
minutes of perfusion. Further processing of the blood samples for
quantitation of plasma TAT levels was performed according to the
manufacturer of the assay kit (Behringwerke AS).
Thrombus Morphometry
Microscopic evaluation of thrombotic deposits was performed on
1-µm-thick epoxy resin–embedded sections prepared perpendicularly to
the direction of the blood flow 1 mm downstream from the upstream
edge of the coverslip.26 The sections were
stained with toludine blue and basic
fuchsin.27
Percent surface coverage with fibrin (percent fibrin deposition) and platelet-fibrin adhesion (percent platelet-fibrin adhesion) were assessed by standard morphometry.27 The evaluations were carried out at 1000xmagnification using a Zeiss Standard 25 light microscope (Carl Zeiss).
Computer-assisted morphometry was used to quantify platelet thrombus area (µm2/µm sectional length). Platelet thrombus volume (µm3/µm2) was derived from the sectional thrombus area as previously described.26 The evaluations were performed by a Kontron Vidas Image analyzing unit (Zeiss) at magnification of 2000x.
Representative light micrographs of sections prepared perpendicular to the direction of the blood flow were taken with a Zeiss Axiophot (Carl Zeiss).
Statistical Analysis
Results are expressed as mean±SEM. Significance for paired data
were calculated with two-tailed Student's t-test. Values of
P< 0.05 were considered significant
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In Vitro Assays
The inhibitory capacity of DEGR-rFVIIa was assessed by examining the ability of increasing concentrations of DEGR-rFVIIa to compete with FVII(a) in TF/FVII(a) complex formation in in vitro assays. TF was presented as an apo protein, inserted into lipid vesicles, or expressed on the surface of living cells. A dose-dependent inhibition was found in all three types of assays with complete blockage of coagulation at higher concentrations of inhibitor. Representative dose-response curves are shown in Figs 1
and 2. Kinetic analyses in the form
of double reciprocal plots (1/v as a function of
1/S) indicated that DEGR-FVIIa was competitive with rFVIIa
for binding to TF and noncompetitive with respect to FX (data not
shown), as has been reported previously.11,28
Irrespective of the assay system used, DEGR-rFVIIa was similar to
FVII(a) in its affinity for TF. The IC50 values (Table 1) that are determined depend on the
concentrations of FVII(a) and TF used in relation to the dissociation
constant for the FVII(a)/TF complex in the particular assay, which is
about 0.2 to 0.3 nmol/L and 3 to 34 pmol/L in the absence
and the presence of the substrate, FX,
respectively.11,29 Although the
IC50 values in the two-stage amidolytic assay are
higher than the FVII(a) concentrations used, the dissociation constant
is similar and in accordance with the Cheng-Prusoff equation:
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Assuming a dissociation constant of 3 to 34 pmol/L for the FVII(a)/TF complex in the presence of FX, the constant calculated for the DEGR-rFVIIa becomes 5 to 22 pmol/L. In the two assay types using cell-expressed TF, the dissociation constant becomes 6 to 60 pmol/L in the two-stage amidolytic assay and 5 to 54 pmol/L in the one-stage clotting assay.
Surface Plasmon Resonance
Full-length TF, covalently immobilized on the BIAcore
sensor chip, bound FVII, rFVIIa, and DEGR-rFVIIa from the flow. As
expected, the interaction was calcium dependent, and the bound ligand
could be eluted by injection of 50 nmol/L of EDTA. Ligands
were run at 50 nmol/L in triplicate and in random order. Binding curves
were collected, and association and dissociation rate constants and the
dissociation equilibrium constants were estimated from these data
(Table 2). On-rates for rFVIIa and
DEGR-rFVIIa were significantly higher (P<.01) than the
on-rate for FVII. The KD value for
DEGR-rFVIIa was clearly lower in comparison to FVII
(P<.001) and rFVIIa (P<.01), mainly owing to a
low koff. The difference between rFVIIa and
FVII was smaller but significant (P<.05) here, mainly owing
to a higher kon. Results are similar to
previously reported values for FVII and FVIIa using the BIAcore
technique.30–32 Under the conditions of our
experiments (flow rate of 5 µL/min), the wall shear rate over the
TF-coated sensor chip was 400 s-1, corresponding
to arterial flow conditions. It is noteworthy that the
clear difference in KD values between
rFVIIa and DEGR-rFVIIa observed in the BIAcore experiments differs from
those in the in vitro experiments described above, performed under
nonflow conditions, in which no significant difference in
KD was observed.
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Effect of DEGR-rFVIIa on TF-Induced Thrombus Formation
The TF surface was exposed for 3.5 minutes to nonanticoagulated
human blood, containing different concentrations of DEGR-rFVIIa or
buffer diluted in saline (control). The wall shear rate at the TF
surface was 100 s-1 or 650
s-1.The DEGR-rFVIIa plasma levels were
calculated from the hematocrit values. At the wall shear rate of 100
s-1, they were as follows (mean±SEM, in
nmol/L): 1.98 (n=1), 2.16 (n=1), 8.36±0.07 (n=3), 41.9±1.3
(n=3), and 82.8±2.4 (n=3). At the wall shear rate of 650
s-1, the DEGR-rFVIIa plasma levels were
calculated to be 0.09±0.001 (n=6), 0.90±0.02 (n=6), 4.46±0.04 (n=6),
8.82±0.26 (n=6), 84.08±0.34 (n=3), 417.10±3.90 (n=4), and
880.80±15.10 (n=3). Individual values for fibrin deposition,
platelet-fibrin adhesion and thrombus volume in the presence of
DEGR-rFVIIa at the concentrations indicated above were calculated as
percent of control. The results are given in Figs 3 and 4.
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Thrombus formation was inhibited in a dose-dependent manner by DEGR-rFVIIa, but the efficacy was shear rate dependent. At 100 s-1, the IC50 value for fibrin deposition was about 30 nmol/L. Platelet-fibrin adhesion and platelet thrombus volume in the absence of DEGR-rFVIIa were less than 5% and 0.6 µm3/µm,2 respectively. At 650 s-1, fibrin adhesion was significantly reduced by 77% at a plasma concentration of 4.5 nmol/L DEGR-rFVIIa. Platelet-fibrin adhesion was significantly reduced by 66% at a plasma concentration of 0.9 nmol/L DEGR-rFVIIa. The thrombus volume was inhibited >95% at concentrations of inhibitor >8.8 nmol/L. IC50 values for these thrombus parameters were between 0.5 nmol/L and 0.7 nmol/L DEGR-rFVIIa.
Light micrographs from sections prepared perpendicular to the direction
of blood flow from perfusion experiments with different concentrations
of DEGR-rFVIIa at a wall shear rate of 650 s-1
show the pronounced reduction in thrombus formation at increasing
concentration of DEGR-rFVIIa (Fig 5).
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Effect of DEGR-rFVIIa on TAT Plasma Levels
Mean values of TAT plasma levels, measured distal to the perfusion
chamber at 3 minutes perfusion time at a wall shear rate of 650
s-1 are given as a function of increasing
concentration of DEGR-rFVIIa in Fig 6.
The TAT values were reduced to the normal range at 4.5 nmol/L
DEGR-rFVIIa. The IC50 was about 0.6 nmol/L
DEGR-rFVIIa, thus paralleling the IC50 value of 0.6
nmol/L of the fibrin deposition.
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TAT plasma levels of perfusions at 100 s-1 were not measured.
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Discussion |
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TF/FVIIa is the major initiator of coagulation in vivo.33–36 A number of studies point toward a profound impact of this binary complex on arterial thrombus formation as well.2,3,5,6 To characterize the role of TF/FVIIa in thrombus formation, we have selectively interfered with the TF/FVII(a) by using DEGR-rFVIIa in a human ex vivo model of venous and arterial thrombus formation. The primary goal was to measure and compare the antithrombotic efficacy of DEGR-rFVIIa at venous and arterial blood flow conditions.
The antithrombotic efficacy of DEGR-rFVIIa was significant. At venous blood flow (100 s-1), the IC50 of fibrin deposition was 30 nmol/L and thus in the range of the normal FVII plasma concentration of 10 nmol/L. However, at arterial blood flow (650 s-1), the IC50 of fibrin deposition was 50-fold lower, 0.6 nmol/L. Platelet-fibrin adhesion, platelet thrombus volume on the top of the fibrin mesh, and the TAT plasma levels were reduced concomitantly with the fibrin deposition. It is apparent that DEGR-rFVIIa is a very efficient antithrombotic with a shear dependent efficacy that increases by increasing shear.
There are a number of possible explanations for the shear-dependent
antithrombotic efficacy of DEGR- rFVIIa. For instance, it is known
that the conversion rate at the catalytic surface and the removal rate
of formed products are dependent on local flow conditions, ie,
shear rate.37,38 Recently, it was suggested that
at low shear rate, the FXa desorption rate from the reaction surface
may be rate limiting, causing underestimation of calculated kinetic
parameters.39,40 Our binding studies
using BIAcore were performed with a wall shear rate at the TF-coated
sensor chip corresponding to arterial flow conditions (400
s-1), which can be compared with experiments
performed in the parallel-plate perfusion chamber at 650
s-1. The results in Table 2
clearly show two
things: that rFVIIa and DEGR-rFVIIa have significantly higher on-rates
(kon) than FVII and that the off-rates
(koff) are so low that under the conditions
of the experiment, the binding of FVII, rFVIIa, or DEGR-rFVIIa to TF is
in practice irreversible (<0.1% of ligand is released from the
receptor). Since the increased shear rate at arterial flow
reduces contact time and thus puts more emphasis on the on-rates, it
may be speculated that at venous shear DEGR-rFVIIa competes with the
bulk of FVII (ie, ca. 10 nM FVII/FVIIa), whereas at
arterial shear, it competes with FVIIa only (ie, 
0.1
nmol/L). Thus, IC50 values are 30 nmol/L and 0.5 to
0.7 nmol/L at venous and arterial shear, respectively. In
addition, the enzyme reaction kinetics are enhanced by the fluid shear
forces generated by the flow,38 and the flow may
also induce conformational changes of the different enzymatic
complexes, altering their kinetic
behavior.38,41,42
The efficacy of inactivated active site rFVIIa has recently been evaluated in an ex vivo model similar to ours, using stimulated endothelial cells at a venous shear condition of 65 s-1.12 In these experiments, the IC50 of fibrin deposition was 3 nmol/L in whole blood and about 10 times lower as obtained in the present study at venous blood flow (100 s-1). However, a direct comparison of results should be performed with caution, because wall shear rates, mixing devices, and immobilized TF-pre- senting surfaces were different. Stimulated endothelium possesses a surface that modulate many thrombotic mechanisms,4,43 whereas the surface used in our study essentially consists of two components, namely, TF and phospholipids, which are entirely thrombus promoting in the model system.20 Thus, the thrombus-modulating properties of the two surfaces were quite different. A more trivial explanation for this discrepancy can be ruled out, since DEGR-rFVIIa was quantified by determination of levels of both antigen and protein.
Results obtained with DEGR-rFVIIa in several in vitro coagulation assays published by others11,28,44–48 and those presented in the present study demonstrate that DEGR-rFVIIa is a competitive inhibitor of FVII(a) with a very high affinity for TF. It is apparent that DEGR-rFVIIa has similar affinity for TF as it has for FVII and FVIIa, in both the presence and the absence of the natural substrate FX. In contrast, in the binding studies using BIAcore, DEGR-rFVIIa showed a 6-fold to 11-fold higher affinity for TF compared to rFVIIa and FVII. Other groups have reported a similar difference in KD values estimated by functional assays and BIAcore.31 A common denominator for the in vitro assays is that they are very low-shear systems, close to venous shear conditions as used by Kirchhofer et al12 and in part of the present investigation, whereas BIAcore binding studies were performed at high shear (400 s-1). A possible explanation for the discrepancy in KD values estimated by functional assays and plasmon resonance may therefore lie in the different shear rates employed by the different methods. If so, this may imply that less FVIIa is required to initiate extrinsic coagulation at arterial shear than at venous shear.
The present data show that the inhibitory efficacy of DEGR-rFVIIa on the TF/FVIIa catalytic complex and on the resulting thrombus formation is potentiated by increasing shear. The surprisingly high efficacy at the arterial shear condition indicates that such an inhibitor may be of value in acute situations of arterial thrombosis. However, it should be emphasized that a vessel wall lesion possesses other thrombus-promoting components besides TF, such as collagens. Thus, the antithrombotic efficacy of DEGR-rFVIIa remains to be established with thrombus-promoting surfaces consisting of a heterogenous chemical composition.
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Acknowledgments |
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We thank Dr Steinar Bergseth (Nycomed Imaging AS) for valuable help with the BIAcore and Maria J.A.G. Hamers, Anne Engen, and Vigdis Bjerkeli (Nycomed Imaging AS) for excellent technical assistance.
Received February 10, 1997; accepted July 10, 1997.
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References |
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