© 1995 American Heart Association, Inc.
Articles |
Effect of Selective Factor Xa Inhibition on Arterial Thrombus Formation Triggered by Tissue Factor/Factor VIIa or Collagen in an Ex Vivo Model of Shear-Dependent Human Thrombogenesis
From Nycomed Pharma AS, Oslo, Norway, and Corvas International (G.P.V.), San Diego, Calif.
Correspondence to Una Ørvim, Nycomed Pharma AS, Gaustadalléen 21, 0371 Oslo, Norway. E-mail una.orvim@nycomed.telemax.no.
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Abstract |
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Abstract Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of factor Xa. TAP has shown good antithrombotic efficacy in experimental animal models of disseminated intravascular coagulation and venous and arterial thrombogenesis. In the present study we evaluated the effect of recombinant TAP (rTAP) on acute thrombus formation in human nonanticoagulated blood triggered either by tissue factor (TF) or by collagen at arterial shear conditions. The main goal was to establish the role of factor Xa in thrombus formation by use of an optimal inhibitory concentration of rTAP. Blood was drawn directly from an antecubital vein by a pump over the respective thrombogenic surfaces, which were positioned in a parallel-plate perfusion chamber. rTAP was mixed homogeneously into the flowing blood by a heparin-coated device positioned proximal to the perfusion chamber. The passage of blood through this device caused minor activation of coagulation but little activation of platelets. Fibrinopeptide A and ß-thromboglobulin levels after 5 minutes of blood perfusion were, on average, 14 ng/mL and 45 IU/mL, respectively. rTAP at a plasma concentration of 0.90 µmol/L completely inhibited TF/factor VIIa–dependent thrombus formation at wall shear rates of 650 and 2600 s-1. These shear conditions are comparable to those in medium-sized arteries and in moderately stenosed small arteries, respectively. In contrast to the TF-coated surface, rTAP was less efficient in reducing collagen-induced thrombus formation. While a significant reduction of 53% was observed at 650 s-1, thrombus formation at 2600 s-1 was not affected by rTAP. Thus, rTAP is an efficient inhibitor of thrombin-driven human thrombus formation on the TF-rich surface but less efficient when thrombus formation is elicited by type III collagen. The lack of antithrombotic effect on collagen type III at 2600 s-1 corroborates earlier findings, showing that collagen-induced thrombus formation in blood from patients with severe factor VIII deficiency is not affected at this blood flow condition and thus is not dependent on the prothrombotic effects of thrombin.
Key Words: arterial thrombogenesis • tissue factor • collagen, factor Xa inhibition • rTAP
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Introduction |
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The biochemical events that compose the blood coagulation cascade play a major role in pathological vascular thrombus formation. Thrombin is the terminal enzyme in this cascade and has been shown to be the primary physiological mediator of fibrin formation and early platelet activation and recruitment to the growing thrombus.1 2 Thrombin also serves to structurally rigidify the growing thrombus through the activation of the transglutaminase factor XIII, which results in fibrin strand cross-linking.3
Based on the importance of thrombin in thrombosis, it is therefore not surprising that many of the new, more selective anticoagulants have been tailored to specifically inhibit the catalytic activity of thrombin4 5 6 or to inhibit the conversion of prothrombin to thrombin by interfering with the catalytic activity of factor Xa in the prothrombinase complex.7 8 9 Both approaches are promising, since each can effectively attenuate the primary thrombogenic source, which is thrombin activity. This has been demonstrated in vivo, where direct inhibitors of thrombin and factor Xa efficiently interrupt experimental thrombogenesis in various animal models.10 11 12 13 14 15 16 17 18 19 20 21 Furthermore, a number of direct and specific thrombin inhibitors are currently being evaluated in clinical trials as acute antithrombotic agents, with encouraging results thus far.22 23
TAP is a 60-amino-acid protein originally isolated from the soft tick Ornithodoros moubata that has been characterized as a potent and selective inhibitor of factor Xa catalytic activity.24 Antithrombotic efficacy greater than that observed with standard heparin has been demonstrated with rTAP14 in a wide variety of experimental preparations, including a primate model of disseminated intravascular coagulation,14 a rabbit model of stasis-induced venous thrombosis,17 canine models of coronary and femoral artery thrombosis,16 21 and a model of occlusive thrombosis in a prosthetic Dacron graft segment positioned in a femoral arteriovenous shunt in baboons.15 18
In the present study, we used an optimal inhibitory concentration of rTAP to establish the role of factor Xa in thrombus formation in nonanticoagulated human blood in an ex vivo model of shear-dependent thrombosis.25 Thrombus formation was elicited by TF or by collagen fibrils at arterial blood flow conditions,26 27 and it was of particular interest to compare the antithrombotic effect of rTAP on these two thrombogenic surfaces under two different arterial shear conditions (650 and 2600 s-1). The role of thrombin generation in collagen-induced thrombus formation at arterial shear conditions has not been characterized, although a previous study indicated that thrombin is partially required only at intermediate arterial shear rate.28
To evaluate the role of factor Xa in arterial thrombus formation, we had to develop a device allowing infusion and homogeneous mixing of rTAP into the flowing blood stream distal to the donor and proximal to the thrombogenic surface. By connecting this system to the parallel-plate perfusion device, we found that inhibition of factor Xa by rTAP virtually abolished thrombus formation on the TF surface. In contrast, thrombus formation was much less affected by rTAP on the collagen surface.
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Methods |
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Mixing Device
The mixing device consisted of five separate pieces, as shown in Fig 1
. A complex helix made of polypropylene (Extrude,
Kerr) was fitted into a cylinder (Fig 1A) made of polymethyl
methacrylate with a length of 117 mm, OD of 10 mm, and ID of 7 mm. The
dimensions of the helix were 75 mm (length) by 7 mm (ID). To gradually
taper off the diameter of the outlet portal in the long cylinder, a
short cylinder made of polymethyl methacrylate (length, 10 mm; OD, 7
mm; ID, 4 mm) was placed inside the long cylinder distal to the helix.
All materials that came into contact with blood in this disposable
device were coated with covalently bound unfractionated heparin
(Carmeda AB).29 An inlet piece (Fig 1B) made of
polytetrafluoroethylene (Teflon) (Lilaas
Finmekaniske AS) allowed infusion of optional solutions into the
flowing blood. The IDs of the inlet tube and side tube were 1.8 mm. The
inlet tube was coned to 0.8 mm where it entered the mixing device,
while an outlet piece (Fig 1C) of Teflon had an ID of 1.8 mm. The
mixing device was connected to the ex vivo perfusion system proximal to
the parallel-plate perfusion chamber25 by a silicone
elastomer (Silastic) tubing (ID, 2.6 mm; Dow Corning Corp) (Fig 2).
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Mixing Efficiency of the Device
The mixing device was placed vertically to avoid the persistence
of density layers of blood and test agent. The efficiency of mixing was
first determined by a dye (0.2% Ponceau red in 3% trichloroacetic
acid), with maximal absorbance at 518 nm, mixed with water. In another
set of experiments, radiolabeled BSA (125I-BSA; Du
Pont–NEN Products) diluted in saline was mixed with blood
anticoagulated with trisodium citrate. Ponceau red and
125I-BSA were mixed with water or blood, respectively, into
the mixing device by infusion with two separate syringe pumps (Alitea
AB). The mixing agent flow rate and blood/water flow rate were 0.2 and
9.8 mL/min, respectively. A special device that divided the
cross-sectional area of the blood flow into four equal portions
(Fig 1D
) was placed about 140 mm distal to the mixing device. The
fractions were collected, and the absorbance of Ponceau red (DU-70
Spectrophotometer, Beckman Instruments, Inc) or 125I
radioactivity (Cobra II, Auto-Gamma Counting Systems, Packard
Instrument Co) in each fraction was measured and calculated as percent
of total. Homogeneous mixing should give 25% of the
absorbance or 125I activity in each of the four
outlets.
Blood Donors and Blood Sampling
Healthy nonsmoking individuals who denied ingestion of aspirin
or other nonsteroidal anti-inflammatory drugs for at least 10 days
before the experiments gave informed consent to donate blood to
perfusion experiments. Venipuncture of an antecubital vein
was performed with a No. 19 butterfly infusion set (Abbott
Laboratories). The first 4.5 mL of blood was collected for
determination of hematological parameters (Auto Counter AC
920, Swelab Instruments). The successive 50 or 40 mL was used for
perfusion experiments characterizing the mixing device or for thrombus
formation, respectively. Hemoglobin, hematocrit, and platelet count
were within the normal ranges for all volunteers. In the perfusion
experiments with rTAP, the same individuals donated blood for both rTAP
and saline (control) on the same day.
Activation of Coagulation and Platelets and Heparin Release by
the Mixing Device
Heparin-coated and noncoated mixing devices were compared to
determine the effects on coagulation and platelet activation.
Plasma levels of FPA, ß-TG, and heparin were measured proximal and
distal to the mixing device. After venipuncture of an
antecubital vein, 0.9 mL of blood was collected into three Eppendorf
tubes, each prefilled with 0.1 mL of appropriate anticoagulant
mixtures, for plasma measurement of FPA, ß-TG, or heparin (all from
Diagnostica Stago). The butterfly infusion set was
subsequently connected to the mixing device prefilled with saline
(37°C), and the blood was drawn directly from the antecubital vein
through the device by an occlusive roller pump (model M312, Gilson).
The pump was placed distal to the mixing device, and the blood flow
rate was maintained at 10 mL/min. At 3 and 4.5 minutes of perfusion
time, blood samples (0.9 mL) were collected immediately distal to the
mixing device into a syringe prefilled with 0.1 mL of the anticoagulant
mixture of the FPA kit (Diagnostica Stago). The blood
sampling was performed in a rubber-coated area of the tubing to
prevent leakage after puncture of the Silastic tubing.30
After 5 minutes of perfusion, the roller pump was switched off, and two
0.9-mL blood samples for determination of ß-TG and heparin were
immediately collected into syringes prefilled with 0.1 mL of the ß-TG
and heparin anticoagulants (Diatube H, Diagnostica
Stago).
Preparation of rTAP
rTAP was prepared from fed-batch fermentation media after
expression in the methylotrophic yeast Pichia pastoris as
described.31 The purified inhibitor was
determined to be >98% pure by a number of analytical criteria,
including reverse-phase high-performance liquid
chromatography, electrospray mass spectrum
analysis, quantitative amino acid analysis, and
amino-terminal sequence analysis. The specific content of
the lyophilized trifluoroacetic acid salt was determined by
quantitative amino acid analysis. The inhibition of human
factor Xa amidolytic activity by rTAP was determined and yielded a
Ki of 0.187 nmol/L.
All experiments were done from a single lot of rTAP diluted in sterile saline. For perfusion experiments, rTAP was diluted to a concentration of 26 µmol/L (0.21 mg/mL).
Coagulation Assay With rTAP
The anticoagulant activity of rTAP was assessed by a
one-stage clotting assay using a Thrombotrack coagulometer
(Thrombotrack 4, Nycomed Pharma). Pooled citrated human plasma (100
µL) was preincubated at 37°C for 3 minutes. Different
concentrations of rTAP (0 to 33 µmol/L; final concentrations, 0 to
1065 nmol/L) were added to the plasma after 2.5 minutes. Thromborel
(Thromborel S; Behringwerke AS), which consists of a lyophilized TF
preparation from human placenta, was diluted in 0.9% saline, and 100
µL of this dilution (37°C) was added to the plasma-rTAP
mixture. Subsequently, 100 µL of prewarmed (37°C) 25 mmol/L
CaCl2 was added, and the clotting time was automatically
measured by the coagulometer. Detailed biochemical characterization of
this Thromborel preparation was reported previously.26
Preparation of TF Surface
One vial of Thromborel was dispersed in 2 mL 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 
17 hours in 2 mL
of the Thromborel dilution. The coated coverslips were rinsed six times
with sterile PBS and stored in PBS for a maximum of 7 hours before use
in perfusion experiments.26 Thrombus formation triggered
by this surface is dependent on TF, since inclusion of a monoclonal
antibody that is directed against human TF and blocks complexing of TF
and factor VIIa interrupts the thrombotic response
completely.26
Preparation of Collagen Surface
Type III collagen was purified from a pepsin digest of human
placenta by selective salt precipitation.32 Purification,
fibril formation, and coating of coverslips were performed as
previously reported.27 The collagen-coated coverslips
were kept at 21°C for 16 hours before use in perfusion
experiments. This surface does not activate
coagulation.27
Ex Vivo Perfusions With rTAP, Fixation, and Embedding
Ex vivo perfusions33 were performed at 37°C. The
mixing device was placed vertically between the donor and a
parallel-plate perfusion chamber (Fig 2). 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).34 Human nonanticoagulated blood was drawn
directly from an antecubital vein and mixed with rTAP or saline
(control) in the heparin-coated mixing device as described above.
The blood was subsequently exposed to either a TF- or
collagen-coated coverslip positioned in the perfusion
chamber.25 27 The blood flow rate was maintained at 10
mL/min for 4 minutes by an occlusive roller pump placed distal to the
perfusion chamber. Perfusion chambers with different geometrical
dimensions of the blood flow channel were used, such that a flow rate
of 10 mL/min produced shear rates of 650 and 2600
s-1 at the thrombogenic surface. These
wall shear rates are encountered in medium-sized arteries and
moderately stenosed small arteries,35 respectively. The
reactive surface was exposed to blood for 3.5 minutes because of the
0.5-minute residence time of blood in the mixing device. Either rTAP
dissolved in saline or saline (control) was infused with a syringe pump
at a flow rate of 0.2 mL/min. The plasma levels of rTAP were calculated
from the hematocrit values, and the mean final plasma concentration of
rTAP was 0.90 µmol/L (range, 0.83 to 1.09 µmol/L).
The total volume in the tubing from the mixing device to the donor was 0.8 mL, which restricted the possibility of backflow of the injected compound into the antecubital vein of the blood donor during the 4 minutes of perfusion time.
After 4 minutes of blood perfusion, the mixing device and the blood donor were disconnected from the parallel-plate perfusion chamber device, which then immediately was perfused with Buffer C (37°C) for 20 seconds 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 was then stored in 0.1 mol/L cacodylate/7% sucrose at 4°C until being embedded in epoxy resin.36
FPA Plasma Levels During Blood Perfusions
Plasma FPA levels were measured proximal to the mixing device
before perfusion and distal to the perfusion chamber after 3 minutes of
perfusion. Blood samples for quantification of plasma FPA levels were
processed further according to the manufacturer of the assay kit
(Diagnostica Stago).
Thrombus Morphometry
Microscopic evaluations of thrombotic deposits were performed on
epoxy resin–embedded sections 1 µm thick prepared perpendicular
to the direction of the blood flow 1 mm downstream from the upstream
edge of the coverslip.37 The sections were stained with
toluidine blue and basic fuchsin.38
Percent surface coverage with fibrin (% fibrin deposition), percent platelet-fibrin adhesion (TF surface), and percent platelet-collagen adhesion (collagen surface) were assessed by standard morphometry.38 The evaluations were carried out at x1000 magnification with 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.37 The evaluations were performed by a Kontron Vidas image analyzing unit (Zeiss) at magnifications of x500 or x2000, depending on the size of the thrombi.
Statistical Analysis
Results are expressed as mean±SEM. Significance for paired data
was calculated with a two-tailed Student's t test.
Values of P<.05 were considered significant.
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Results |
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Mixing Device
Mixing Efficiency
The efficiency of mixing was studied with two different sets of experiments, in which either a dye (0.2% Ponceau red in 3% trichloroacetic acid) or 125I-BSA was used as a mixing agent. The device was found to efficiently mix the dye with water (data not shown) and 125I-BSA with citrated blood. Percentages of 125I radioactivity from the four test outlets were 24.1±0.9%, 23.5±1.1%, 26.9± 4.5%, and 25.5±2.5% (mean±SEM, n=6). The theoretical percentage of mixed label was 25% of the signal in each of the four outlets.
The minimal length of the helix required for homogeneous mixing was 75 mm.
Activation of Coagulation (FPA) and Platelets
(ß-TG)
The mixing device caused some activation of platelets and
coagulation after 5 minutes of perfusion (10 mL/min) of
nonanticoagulated blood. The plasma levels of FPA increased, on
average, from 2.8 ng/mL at the flow inlet at time 0 to 14 ng/mL at the
outlet of the heparin-coated device after 5 minutes of perfusion
(P<.05) (Table 1). The increase in FPA was
higher in the noncoated device (from 2.8 to 24 ng/mL), although the
plasma levels measured at 5 minutes in coated and noncoated devices
were not statistically different. The corresponding ß-TG values
increased, on average, from 25 to 45 IU/mL (P<.05).
However, the 5-minute ß-TG sample was significantly increased by the
noncoated system (85 versus 45 IU/mL; P<.05) (Table 1).
Apparently, thrombus formation on collagen at 2600
s-1 was not affected by the slight
activation of coagulation and platelets by the mixing device, since
the platelet-collagen adhesion of 43.1±3.7% and the
platelet thrombus volume of 7.0±2.9
µm3/µm2 in the absence of the mixing device
were not significantly different from thrombus formation in the
presence of the mixing device (Fig 3A and 3B).
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Heparin release from the heparin-coated device into the perfused blood stream was not detected after 5 minutes of perfusion with nonanticoagulated blood.
Inhibitory Efficacy of rTAP in Plasma
The anticoagulant activity of rTAP was measured in a one-stage
clotting assay. rTAP had a dose-dependent inhibitory
effect (data not shown), and at 1.07 µmol/L rTAP (final
concentration), the clotting time increased from 32 to >590
seconds.
rTAP Perfusion Experiments
Thromborel (TF/phospholipids) or collagen fibrils were exposed to
nonanticoagulated human blood mixed with rTAP or saline (control) for
3.5 minutes. The mean final plasma concentration of rTAP was 0.90
µmol/L, and the shear rates at the thrombogenic surfaces were 650 or
2600 s-1.
Effect of rTAP on TF/Factor VIIa–Induced Thrombus
Formation
Infusion of rTAP reduced fibrin deposition by 99% at both wall
shear rates (650 and 2600 s-1,
P<.01) (Fig 4A). Platelets adhered
exclusively to the fibrin mesh. rTAP reduced the
platelet-fibrin adhesion by 94% at 650
s-1 (P<.05) and by 98% at
2600 s-1 (P<.01) (Fig 4B).
Thrombus volume was reduced by 99% at both wall shear rates
(P<.001) (Fig 4C).
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Effect of rTAP on Collagen-Induced Thrombus Formation
rTAP had no effect on platelet adhesion (Fig 3A) at either 650
or 2600 s-1. However, rTAP reduced the
average thrombus volume by 53% at 650
s-1 (P=.03) but had no effect
on thrombus volume at 2600 s-1. There was
no detectable fibrin deposition on the collagen surface after 3.5
minutes of perfusion at these blood flow conditions.
Effect of rTAP on FPA Plasma Levels
The TF surface resulted in substantial FPA generation. rTAP
reduced the FPA levels by 92% at 650 s-1
(P<.05) and by 93% at 2600
s-1 (Table 2). However,
the latter reduction was not significant because of the large variation
in the control group. In contrast, the collagen surface resulted in
little FPA generation, which was not affected by rTAP (Table 2).
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Discussion |
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Among the various strategies taken in the development of novel antithrombotic therapies, the direct inhibition of factor Xa activity has been shown to be an effective approach. Inhibition of factor Xa has gained particular attention, since it can terminate the amplified burst of thrombin generation that plays an important role in arterial thrombus development. rTAP is a potent and selective inhibitor of factor Xa that has been shown to be superior to unfractionated heparin in animal models of in vivo thrombogenesis15 and shows a better efficacy than hirudin in dogs at doses that may limit the risk of concomitant hemorrhage often encountered in antithrombotic therapy.16 However, the antithrombotic effect of TAP in humans has not been studied either in TF/factor VIIa– or collagen-induced arterial thrombogenesis. The latter issues are of particular importance, since both TF and collagens are exposed to the bloodstream at sites of vascular lesions. Therefore, it was relevant to investigate and compare the antithrombotic effects of rTAP on thrombogenic surfaces consisting of either TF or collagen in nonanticoagulated human blood flowing at different arterial shear conditions. The main goal was to establish the role of factor Xa in thrombus formation. Therefore, a plasma concentration of rTAP known to block thrombus formation in baboons15 was chosen.
rTAP at a plasma concentration of 0.90 µmol/L efficiently inhibited TF/factor VIIa–dependent platelet-rich thrombus formation at shear conditions of both normal (650 s-1) and atherosclerotic (2600 s-1) coronary arteries. However, the collagen-induced thrombus formation was less affected by rTAP, since significant reduction of thrombus formation was observed only at shear rates corresponding to healthy coronary flow but not at a shear rate found at atherosclerotic lesions.
Previous experiments with collagen-induced thrombus formation in blood from patients with severe factor VIII deficiency28 have indicated that the thrombotic response at a shear rate of 650 s-1 is partly thrombin dependent, whereas this is not the case at 2600 s-1. The findings with rTAP are consistent with these results, and it is interesting to note that rTAP reduced the thrombus volume by 53% at 650 s-1, whereas the reduction of thrombus volume in blood from severe hemophiliacs was 69%. In contrast, neither rTAP nor deficiency of factor VIII affected the collagen-induced thrombus formation at 2600 s-1. Thus, thrombus formation triggered by a surface of type III collagen is apparently driven by a platelet-dependent mechanism that does not involve thrombin.
The striking inhibition of TF/factor VIIa–dependent thrombus formation by rTAP is also consistent with previous findings in which preincubation of the same TF-rich surface with a monoclonal antibody directed against human TF abolished thrombus formation as well.26 Therefore, thrombus formation on a TF-coated surface appears to be completely driven by thrombin and as such is dependent on the activity of factor Xa in the prothrombinase complex. These findings demonstrate for the first time that potent and specific factor Xa inhibition is an extremely efficient means for interrupting human, platelet-dependent thrombus formation on a procoagulant surface consisting of TF/phospholipids but is less so on a nonprocoagulant surface consisting of pure type III collagen fibrils.
The TF-dependent thrombus formation was blocked at an average plasma concentration of 0.90 µmol/L rTAP, thus at approximately a sevenfold higher concentration than the factor X concentration present in normal plasma (130 to 140 nmol/L). The antithrombotic efficiency of rTAP at this concentration was simultaneously reflected by the reduction of the plasma levels of FPA sampled downstream of the site of thrombus formation. However, the reduction in thrombus formation on the collagen surface at 650 s-1 was not paralleled by a reduction in FPA. Apparently this was due to the "background noise" triggered by the perfusion chamber device, which yields 15 to 20 ng/mL FPA without the mixing device.39 40 It should also be mentioned that the efficacy of rTAP in this human system remains to be established, since no dose response of the inhibitor was investigated in the present study. This is of interest, because rTAP plasma concentrations ranging from 0.5 to 1.0 µmol/L were previously reported to protect against thrombus formation in baboons.15
Development and characterization of a device that facilitated homogeneous mixing of an antithrombotic test agent into flowing native blood were a prerequisite for the successful completion of this study. Another type of mixing device other than the one we used was recently described,41 but no data on homogeneous mixing were reported. The present device, which makes use of a complex helix with covalently bound unfractionated heparin, mixes the test agent homogeneously in flowing native blood. However, this is at the expense of moderate activation of platelets and coagulation. Fortunately, heparin coating of the surfaces substantially reduces the level of activation. Although the ß-TG plasma levels were lowered close to the upper limit of the normal range, the FPA plasma levels still remained significantly higher than the normal range. Nevertheless, these levels are within the range of those frequently encountered in patients suffering from thromboembolic disorders,42 and there was no evidence that this activation affected thrombus formation in the perfusion chamber, at least not on collagen. It is apparent that investigation of experimental antithrombotic agents in native blood in this novel human model of thrombogenesis offers great advantages at the expense, however, of modest upstream activation of the coagulation cascade.
The antithrombotic effect of rTAP on TF/factor VIIa–dependent arterial thrombus formation in humans is striking. The strategy of inhibiting the formation of thrombin and thus thrombus formation at the level of factor Xa seems to be a most efficient approach. However, as demonstrated by Fressinaud et al28 and the present investigation, collagen-induced thrombus formation at high arterial shear conditions (2600 s-1) appears to be thrombin-independent. Since both TF and a variety of collagen types are exposed to the blood flow at vascular lesions, inclusion of a selective inhibitor of platelet adhesion or aggregation in addition to a selective factor Xa inhibitor may be necessary.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank Maria J.A.G. Hamers (Nycomed Pharma AS) for excellent technical assistance. Professor Helge Stormorken and Dr Steinar Bergseth (Nycomed Pharma AS) are acknowledged for stimulating discussions.
Received February 28, 1995; accepted August 23, 1995.
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