© 1995 American Heart Association, Inc.
Articles |
Active Site–Blocked Factors VIIa and IXa Differentially Inhibit Fibrin Formation in a Human Ex Vivo Thrombosis Model
From the Pharma Division, Preclinical Research, F. Hoffmann–La Roche Ltd, Basel, Switzerland.
Correspondence to Daniel Kirchhofer, PhD, Pharma Division, Preclinical Research, F. Hoffmann–La Roche Ltd, Grenzacherstr 124, CH-4002 Basel, Switzerland.
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Abstract |
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Abstract The role of tissue factor/factor VIIa (FVIIa) and factor VIIIa/factor IXa (FVIIIa/FIXa) complexes in thrombus formation was examined in a human ex vivo blood flow system by use of active site–blocked FVIIa (FVIIai) and FIXa (FIXai) as selective inhibitors. Blood was drawn directly from the veins of volunteers into a mixing device where FVIIai and FIXai were mixed with flowing blood. The blood then entered parallel-plate chambers containing coverslips coated with human fibrillar collagen or tissue factor–expressing cell layers of tumor necrosis factor–
–stimulated human endothelial cells, human
smooth muscle cells, and J82 cells. Exposure of stimulated
endothelial cells to blood flowing at a venous shear
rate of 65/s led to fibrin deposition, which was inhibited by infusion
of FVIIai (IC50, 3 nmol/L), as quantified by
microdensitometry of fibrin-stained coverslips. Whereas FIXai (600
nmol/L) was only a weak inhibitor, FVIIai (60 nmol/L)
reduced fibrinopeptide A (FPA) plasma levels from
504±79 to 171±27 ng/mL and concomitantly inhibited platelet
thrombus deposition. Similarly, experiments with smooth muscle
cells and J82 cells showed that FVIIai but not FIXai efficiently
reduced FPA levels. Conversely, with tissue factor–free collagen,
which induces platelet-dependent fibrin formation, infusion of
FIXai but not of FVIIai inhibited fibrin deposition
(IC50, 8 nmol/L) and reduced FPA levels from 55±8
to 9±5 ng/mL. However, FIXai did not affect the number of platelet
thrombi deposited on collagen. The results suggest that fibrin
formation on tissue factor–expressing cellular surfaces is initiated
by tissue factor/FVIIa–dependent direct activation of factor X, while
on the tissue factor–free collagen surface, factor X activation and
subsequent fibrin formation is dependent on the platelet
FVIIIa/FIXa complex.
Key Words: tissue factor • blood platelets • factor VII • factor IX • thrombosis
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Introduction |
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The coagulation system plays an important role in venous as well as arterial thrombosis. The extrinsic pathway of coagulation is initiated by the tissue factor/factor VIIa (FVIIa) complex,1 2 3 which activates factor IX (FIX) to factor IXa (FIXa) and factor X (FX) to factor Xa (FXa) by limited proteolysis. FX can also be converted to FXa by the intrinsic FX activation complex FVIIIa/FIXa, subsequently leading to thrombin generation and fibrin deposition. In different animal thrombosis models, interference with the function of either the tissue factor/FVIIa complex or the FVIIIa/FIXa complex resulted in antithrombotic effects.4 5 6 7 8 9 10 Both enzymatic complexes require cellular surfaces for optimal activity, as shown in studies with isolated cells, such as platelets, monocytes, endothelial cells, and smooth muscle cells.11 12 13 14 15 16 17 18 However, little is known about the role of these different cellular FVIIIa/FIXa and tissue factor/FVIIa assembly sites in thrombus formation under the physiological, more complex conditions of flowing whole blood. In a study in which a perfusion system with human blood was used, Tijburg et al15 demonstrated that fibrin deposition on the extracellular matrix of endothelial cells is mediated by the FVIIIa/FIXa complex. However, this deposition only occurred at a low tissue factor stimulus and was dependent on the presence of platelets, indicating that platelets and the nature of the thrombogenic surface are important determinants for FVIIIa/FIXa activity.
To gain further insight into the roles of tissue factor/FVIIa and
FVIIIa/FIXa complexes in thrombus formation mediated by the
procoagulant surfaces of intact cells, we used a recently described
human ex vivo blood flow system.19 This experimental
system allowed us to neutralize the enzymatic activity of tissue
factor/FVIIa and FVIIIa/FIXa complexes in native (nonanticoagulated)
human blood and to examine the effects on thrombus formation. Human
vascular cells, such as tumor necrosis factor (TNF)––stimulated
endothelial cells and smooth muscle cells, were used as
tissue factor–expressing cellular surfaces, as was the carcinoma cell
line J82. Human fibrillar collagen served as a tissue factor–free
thrombogenic surface. To selectively neutralize the function of tissue
factor/FVIIa or FVIIIa/FIXa complexes, we infused the prototype
inhibitors active site–blocked FVIIa (FVIIai) or FIXa
(FIXai). The inhibitors were prepared by covalently binding
D-Phe-L-Phe-Arg-chloromethylketone
to the active site of FVIIa or
dansyl-Glu-Gly-Arg-chloromethylketone to the active
site of FIXa, thus rendering FVIIa and FIXa enzymatically inactive.
By use of these tools, it could be demonstrated that under venous blood flow conditions FVIIai and FIXai strongly differed in their ability to inhibit fibrin formation mediated by different thrombogenic surfaces. The findings suggest a role of the FVIIIa/FIXa complex primarily in platelet-mediated coagulation; this complex seems less important when coagulation is initiated by the tissue factor/FVIIa complex on the surface of tissue factor–expressing cells.
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Methods |
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Cell Cultures
Smooth muscle cells isolated from a human aorta by enzymatic digestion were obtained from Dr Jürgen Fingerle (F. Hoffmann–La Roche). The cells were cultured in Dulbecco's modified Eagle's/F-12 (DMEM/F-12) medium (1:4) containing 20% (vol/vol) fetal bovine serum (GIBCO), penicillin, streptomycin, glutamine, and a cocktail of essential amino acids (GIBCO). For the perfusion experiments the smooth muscle cells were used between passages 5 and 8 with split ratios of 1:2.
Endothelial cells were isolated from umbilical veins as described by Jaffe et al20 by use of a solution of 0.1% collagenase (CLS; Worthington Biochemical Corp) in medium M199 (Sigma Chemical Co). The endothelial cells were grown in medium M199 supplemented with 10% (vol/vol) newborn calf serum (GIBCO), 10% (vol/vol) fetal calf serum (GIBCO), penicillin, streptomycin, glutamine (GIBCO), 50 µg/mL endothelial cell growth factor (Collaborative Research), and 100 µg/mL heparin (Sigma Chemical Co). Immunofluorescence staining showed that confluent cultures expressed the endothelial cell marker von Willebrand factor (polyclonal antiserum directed against FVIII-related antigen; Dakopatts). Endothelial cells were used for perfusion experiments between passages 2 and 5.
The human bladder carcinoma cell line J82 (ATCC HTB1) was from the American Type Culture Collection. The cells were cultured in DMEM supplemented with 10% (vol/vol) fetal bovine serum (GIBCO), penicillin, streptomycin, glutamine, and a cocktail of essential amino acids (GIBCO).
Antibodies and Proteins
Monoclonal antifibrin antibody was from American
Diagnostica. The antibody used for immunogold-silver
staining of platelets was the monoclonal antibody pl-62 directed
against the platelet-specific glycoprotein GPIIb/IIIa
(obtained from Dr B. Steiner, F. Hoffmann–La Roche). The antibody
pl-62 is complex specific and recognizes GPIIb/IIIa on resting as well
as on activated platelets.21 22
Bovine FIXai was obtained from Dr David Stern (Columbia University). For some experiments human FIXai was used instead of bovine FIXai. Human FIXai was prepared by incubating purified human FIXa (Enzyme Research Inc) at a concentration of 15 µmol/L in Tris-buffered saline (TBS; 50 mmol/L Tris buffer, pH 7.5, containing 100 mmol/L NaCl) with a threefold molar excess of dansyl-Glu-Gly-Arg-chloromethylketone (Calbiochem-Behring) for 7 hours at room temperature and then for 17 hours at 4°C. FIXai was separated from excess dansyl-Glu-Gly-Arg-chloromethylketone by extensive dialysis against TBS at 4°C. The remaining enzymatic activity of human FIXai was measured in an activated partial thromboplastin time clotting test and found to be less than 0.05% of noninhibited FIXa.
Recombinant FVIIa was from Novo Nordisk A/S and was inactivated by use of D-Phe-L-Phe-Arg-chloromethylketone (Calbiochem-Behring) as described by Waxman et al.23 FVIIa (final concentration, 20 µmol/L) was incubated with D-Phe-L-Phe-Arg-chloromethylketone (final concentration, 40 µmol/L) for 2 hours on ice and then dialyzed extensively against TBS containing 5 mmol/L CaCl2 at 4°C. The remaining procoagulant activity of FVIIai was assessed in a prothrombin time assay with FVII-depleted human plasma (Behringwerke AG) and relipidated recombinant tissue factor (obtained from Dr Yale Nemerson, Mount Sinai School of Medicine) used as a clotting initiator. The residual procoagulant activity of FVIIai varied slightly between different batches but was always less than 0.01% of the activity of untreated FVIIa. Both FIXai and FVIIai at 1 µmol/L did not inhibit the amidolytic activity of FXa, trypsin, and thrombin, indicating that no residual reactive chloromethylketone was present in the FIXai and FVIIai preparations.
Human collagen type III was purified by salt precipitation as described previously.24 Fibril formation was induced by dialysis of a solution of 1 mg/mL collagen type III in 0.1 mol/L acetic acid against 20 mmol/L Na2HPO4, pH 7.5, at 4°C for 24 hours. The activities of the preparations were tested in the aggregometer with human plasma, and they were stored at 4°C until being used in the perfusion experiments.
Native Blood Perfusion Experiments With Different
Thrombogenic Surfaces
Human collagen type III was sprayed in fibrillar form onto
Thermanox plastic coverslips (Miles Lab) at a density of 20
µg/cm2. The collagen-coated coverslips were dried for
several hours at room temperature, washed with 0.9% NaCl solution over
a period of 1 hour, and kept in 0.9% (wt/vol) NaCl–0.1% (wt/vol) BSA
until they were used for the perfusion experiments. Human smooth muscle
cells were grown in six-well culture plates (Costar) containing
sterilized Thermanox plastic coverslips. The cells were washed with
DMEM/F-12 medium containing 0.1% (wt/vol) BSA 2 to 3 days after
reaching confluence and were used for the perfusion experiments.
Endothelial cells were grown on gelatin-coated
Thermanox coverslips. Tissue factor expression was induced 1 to 3 days
after the endothelial cells reached confluence by
stimulation of the endothelial cells with 2 nmol/L
TNF- (Genzyme) for 4 hours. J82 cells were grown on Thermanox
coverslips and used 1 to 3 days after reaching confluence.
The coated coverslips were then positioned in the three parallel-plate perfusion chambers and the entire system, including tubings, mixing devices, and parallel-plate chambers, was filled with PBS 0.1% (wt/vol) BSA (collagen), DMEM/F12 1% (wt/vol) BSA (smooth muscle cells), M199–1% (wt/vol) BSA (endothelial cells), or DMEM–1% (wt/vol) BSA (J82 cells). The details of the experimental system were described recently.19 Blood was then drawn from the antecubital vein of a healthy donor directly into a Plexiglas distribution block, where the blood was separated into four tubings. One tubing was connected to the accessory pump, serving as a safety measure in case of blood backflow. In the remaining three tubings the blood flowed in parallel at a rate of 1 mL/min into a mixing device consisting of three individual mixing chambers. The blood flow was controlled by three individual roller pumps positioned at the distal end of the parallel-plate perfusion devices. Immediately before entering the mixing chambers the flowing blood was supplemented with inhibitor solution at a rate of 50 µL/min, resulting in a final concentration of 5% (vol/vol) in the blood. In standard experiments human FVIIai and bovine FIXai were used. For some experiments with endothelial cells we also tested human FIXai. The inhibitors were diluted in 0.9% (wt/vol) NaCl–0.1% (wt/vol) BSA and were infused by three 1-mL Hamilton glass syringes (Hamilton Bonaduz AG) by means of an infusion pump (Infu 362, Datex AG). The inhibitors were mixed with the blood in the three mixing chambers, each containing a rotating magnetic stir bar. The homogenous blood-inhibitor mixture then entered three parallel-plate perfusion devices containing the coated coverslips. The blood flow of 1 mL/min resulted in a shear rate of 65/s on the coverslips, which corresponded to venous blood flow conditions. After a 3.5-minute perfusion period for smooth muscle cells, endothelial cells, and J82 cells and a 5.5-minute perfusion period for collagen, the wash solution (PBS for collagen, DMEM/F-12 for smooth muscle cells, M199 for endothelial cells, and DMEM for J82 cells) was connected to the distribution block without interruption of flow. During the 3-minute washing period the inhibitor infusion of 50 µL/min was maintained. The mixing device was then disconnected from the parallel-plate devices, which were subsequently perfused at 1 mL/min for 2 minutes with 3% (wt/vol) paraformaldehyde in PBS (for immunochemical staining) after a brief interruption of flow of approximately 5 seconds. For morphometric examinations the coverslips were perfused at 1 mL/min for 2 minutes with 2.5% (vol/vol) glutaraldehyde in 0.1% (wt/vol) cacodylate buffer, pH 7.4, containing 2.5 mmol/L CaCl2 and 0.9 mmol/L MgCl2. The coverslips were then removed from the chambers, incubated in fresh fixative for an additional 30 minutes, and stored in PBS–0.03% azide and cacodylate buffer containing 7% (wt/vol) sucrose for immunochemical staining and morphometric analysis, respectively.
Determination of Fibrinopeptide A
Levels
Fibrinopeptide A (FPA) levels were measured in
the blood leaving the perfusion device (ie, postchamber blood). For
that purpose a second mixing device was positioned at the distal end of
the parallel-plate perfusion chambers. This second device was used to
mix the blood with an anticoagulant cocktail (32 mg/mL trisodium
citrate, 1000 IU/mL heparin, 1 TIU/mL aprotinin) to prevent further FPA
generation. The anticoagulant cocktail was supplied by an additional
roller pump at a flow rate of 0.1 mL/min, resulting in a mixing ratio
of 1:10 (anticoagulant cocktail:blood). The blood flow rate at the
distal end of the mixing device was 1.1 mL/min, which resulted in a
blood flow rate of 1 mL/min (shear rate 65/s) over the cover slip,
consistent with the perfusion experiments described above.
Three different conditions were tested: blood perfusion with mixing
device only, blood perfusion with the entire system and use of
noncoated coverslips in the parallel-plate devices, and blood perfusion
with the entire system and use of collagen-coated or cell-coated
coverslips in the parallel-plate devices. The anticoagulated blood was
collected from the roller pumps into polypropylene tubes over a period
of 2.5 minutes (for smooth muscle cells, endothelial
cells, and J82 cells) or 4.5 minutes (for collagen). FPA concentrations
were also measured from blood that was not in contact with any part of
the perfusion system. For that purpose blood was drawn from the veins
of volunteers directly into a syringe by means of a butterfly device
(Butterfly-19; Abbott Ireland Ltd). After
centrifugation the platelet-poor plasma was stored
at -20°C until the FPA concentrations were determined according to
the manufacturer's instructions (ELISA FPA; Boehringer
Mannheim GmbH).
Determination of ß-Thromboglobulin
The plasma levels of ß-thromboglobulin were
determined as markers of platelet activation. To avoid platelet
activation due to the passage of the blood through the roller pumps,
blood was collected into silicone elastomer tubing (Dow Corning Corp)
proximal to the roller pumps. For measurement of platelet
activation in the mixing device, the tubing was positioned between the
mixing device and the roller pumps. For measurement of platelet
activation in the entire system (including the mixing device), the
tubing was positioned between the roller pumps and the parallel-plate
perfusion chamber containing a noncoated or collagen-coated plastic
coverslip. The total perfusion period was 5.5 minutes, and the blood
was collected during the 2.5 minutes from 2.5 minutes to 5.0 minutes
after the start of perfusion. The 2.5-mL blood content of the tubing
(840 mm in length and 2 mm in inner diameter) was immediately poured
into a tube containing cooled inhibitor cocktail (Kodak
Clinical Diagnostics, Amersham) and put on ice.
ß-Thromboglobulin concentrations were also measured
from blood that was not flowing through any part of the perfusion
system. In this case blood was drawn from the vein directly into a
syringe by means of a butterfly device. Plasma was prepared and stored
at -20°C. The concentration of ß-thromboglobulin
was determined in a radioimmunoassay as described by the manufacturer
(Kodak Clinical Diagnostics).
Quantification of Platelets and Fibrin on Semithin
Sections
After the perfusion experiments, the coverslips were embedded in
Epon 812 (Fluka Chemie) and semithin sections perpendicular to the
blood flow direction were prepared as described
previously.25 The semithin sections were stained with
0.01% (wt/vol) toluidine blue and 0.01% (wt/vol) fuchsin, and the
deposition of platelets and fibrin along the 8-mm length of the
semithin section was determined morphometrically by use of a Zeiss
Standard microscope.25 Fibrin coverage was quantified by
determination of the presence or absence of fibrin at 10-µm intervals
along the cross section. Platelet thrombi were defined as three or
more cohesive platelets that underwent shape change and were in
contact with collagen or fibrin (experiments with collagen-coated
coverslips) and with the cell layer or fibrin (experiments with
endothelial cell–coated coverslips). We quantified the
platelet thrombi by counting their number along the cross section.
Because the length of cross sections varied slightly between the
specimens, the numbers were normalized to a standard length of 10
mm.
Immunogold-Silver Staining of Fibrin and Platelets and
Microdensitometry
After the perfusion experiments, the coverslips were incubated
with either 2.5 µg/mL monoclonal antifibrin antibody (for fibrin
staining) or 10 µg/mL monoclonal anti-GPIIb/IIIa antibody pl-62 (for
platelet staining) in PBS. After being washed with PBS the
coverslips were incubated at room temperature for 30 minutes with
gold-labeled antimouse antibody (Auro Probe LM, 5-nm gold particle
diameter; Amersham) diluted 1:50 in PBS for fibrin staining and 1:10
for platelet staining. The coverslips were then washed with PBS,
treated for 10 minutes with 2% (vol/vol)
glutaraldehyde in PBS, and washed with PBS and
distilled water. After incubation with silver enhancer for 10 to 15
minutes (IntenSE M; Amersham) the coverslips were fixed with Rapidfix
(Kodak) and thoroughly washed in distilled water. After air drying, the
coverslips were embedded in Merckoglass (Merck) and examined under the
microscope (Zeiss Axiophot).
To quantify the relative amounts of fibrin deposited on the coverslips, we determined the relative optical densities of the immunogold-silver stained fibrin with a computerized image analysis system (MCID; Imaging Research Inc) by use of a Zeiss Axiophot microscope as described recently.26 The values were obtained from three measurements of areas of 1 mm2 in the center of the coverslip. The fibrin staining on all coverslips of a perfusion experiment (nine to 12 coverslips per experiment) was carried out simultaneously, and for each experiment the value of each coverslip was expressed as percent of the average value of the control coverslips.
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Activation of Platelets and the Coagulation System in the Perfusion System
To examine whether the perfusion system itself induced platelet activation, we measured ß-thromboglobulin levels in blood collected for 2.5 minutes in experiments with the standard blood flow rate of 1 mL/min. The ß-thromboglobulin level determined in blood that was not exposed to any part of the perfusion system was 34.5±4.6 ng/mL (mean±SEM). The mixing device alone increased ß-thromboglobulin levels to 64.4±8.8 ng/mL. The entire system, consisting of a mixing device and a parallel-plate device containing an uncoated coverslip, further elevated ß-thromboglobulin levels to 151.9±51.1 ng/mL (Table 1
), which was about 10% of the ß-thromboglobulin
levels that were generated when collagen-coated coverslips were used
(1677.9±331.3) (Table 1). FPA level, indicative of
activation of the coagulation system, was determined in blood collected
for 4.5 minutes. Table 1 shows that there was minimal FPA generation
during the passage of the blood through the mixing device (6.0±0.7
ng/mL) and the entire perfusion system with uncoated coverslips
(8.2±1.6 ng/mL). However, this increase in FPA levels did not result
in any measurable platelet and fibrin deposition on uncoated
coverslips. In comparison, the FPA level in blood that was drawn from
the vein directly into a syringe by means of a butterfly device was
3.9±0.3 ng/mL (Table 1).
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Effects of FVIIai and FIXai on Fibrin Deposition on Stimulated
Endothelial Cells and Collagen
Exposure of TNF-–stimulated endothelial cells
for 3.5 minutes to native human blood flowing at a venous shear rate of
65/s resulted in the deposition of fibrin on the
endothelial cell surface. Infusion of FVIIai inhibited
fibrin deposition in a concentration-dependent manner with an
IC50 of 3 nmol/L (blood concentration), as quantified by
optical density measurements of immunogold-silver stained fibrin (Fig 1a). Complete inhibition was achieved at a blood
concentration of 60 nmol/L. In contrast, FIXai at the 10-fold higher
concentration of 600 nmol/L inhibited fibrin deposition less than 50%
(Fig 1a). In agreement, morphometric quantification of fibrin
deposition on semithin sections perpendicular to the blood flow
direction showed that 60 nmol/L FVIIai reduced the coverage of the
endothelial surface with fibrin from 69±5%
(mean±SEM) to 14±11%, whereas 600 nmol/L FIXai reduced it to
29±8%.
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) and FIXai (
) were
infused into the flowing blood by use of a mixing device. After
washing, fixation, and immunogold-silver staining, the deposited fibrin
was quantified by optical density measurements. Fibrin deposition on
TNF-
The initiation of fibrin deposition on collagen, compared with
that on stimulated endothelial cells, has a longer lag
phase. Therefore, the perfusion period of experiments with human
fibrillar collagen as a thrombogenic surface was extended to 5.5
minutes. Infusion of 100 nmol/L FVIIai had no effect on fibrin
deposition on the collagen surface, as determined by microdensitometry
(Fig 1b
). In contrast, infusion of FIXai very efficiently inhibited
fibrin deposition, with an IC50 value of 8 nmol/L and full
inhibition at 50 nmol/L (Fig 1b). Similarly, fibrin coverage determined
on semithin sections was reduced from 92±8% to 0% by 50 nmol/L
FIXai, whereas 100 nmol/L FVIIai was not inhibitory
(100±0%).
Effects of FVIIai and FIXai on FPA Levels
As another measurement of fibrin formation, we determined the
plasma concentration of FPA, which is generated by the
thrombin-dependent proteolytic conversion of fibrinogen to fibrin. For
FPA measurements the perfusion experiments were carried out in a manner
identical to that for fibrin quantification. FPA levels in postchamber
blood were determined for human smooth muscle cells and the human
bladder carcinoma cell line J82 in addition to collagen and stimulated
endothelial cells. The results are summarized in Table 2. They show that infusion of FVIIai efficiently
inhibited FPA generation by all the tested tissue factor–expressing
cell surfaces. With J82 cells, FVIIai at a concentration of 60 nmol/L
inhibited the generation of FPA only weakly (744±103 ng/mL; six
donors); therefore, a higher concentration was used, which resulted in
a stronger inhibition (Table 2). Infusion of 600 nmol/L FIXai only
moderately reduced FPA levels for endothelial cells,
smooth muscle cells, or J82 cells. Conversely, when collagen was used
as a thrombogenic surface, FIXai almost completely inhibited FPA
formation at the low concentration of 50 nmol/L, whereas FVIIai at 100
nmol/L was not inhibitory and even increased FPA levels
(Table 2).
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To establish that the inability of the bovine FIXai to effectively
inhibit fibrin formation was not due to its lack of cross-reactivity
with the human cells, an experiment with active site–blocked human
FIXa (human FIXai) was performed. This experiment with
TNF-–stimulated human endothelial cells showed that
human FIXai (400 nmol/L), like bovine FIXai, reduced FPA levels only
weakly from 701±91 to 465±70 ng/mL (mean±SEM; four donors).
Morphometric Determination of Platelet Deposition on Stimulated
Endothelial Cells and Collagen After Infusion of FVIIai
and FIXai
To examine whether FVIIai- and FIXai-dependent inhibition of
fibrin deposition also affected platelet deposition, we examined
semithin sections of the coverslips perpendicular to the flow
direction. We found that neither FIXai (50 nmol/L) nor FVIIai (100
nmol/L) reduced the number of deposited platelet thrombi on
collagen (Table 3). In contrast, FVIIai at a
concentration of 60 nmol/L reduced the number of thrombi deposited on
the surface of stimulated endothelial cells by 81%
compared with controls (buffer infusion), a result similar to the
observed inhibition of fibrin deposition. FIXai showed only moderate
inhibition at a 10-fold higher concentration of 600 nmol/L (Table 3).
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Fig 2 shows the corresponding semithin sections and
further illustrates that in perfusions with stimulated
endothelial cells, most platelet thrombi were
associated with fibrin and were not in direct contact with the cell
surface (Fig 2a and 2e). Accordingly, reduction of fibrin deposition by
infusion of FVIIai (Table 3) also caused a reduction of platelet
thrombus deposition (Fig 2c). On collagen, the platelet thrombi
were mainly found in direct contact with the collagen matrix with or
without infusion of inhibitors (Fig 2b, 2d, and 2f).
Although platelet thrombus deposition on collagen was not reduced
by infusion of FIXai, the thrombi appeared morphologically different in
that the platelets were loosely packed (Fig 2f), unlike the dense
platelet thrombi found in controls (Fig 2b).
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Identification of the Sites of FIXa-Mediated Fibrin
Formation
To determine whether adherent platelets were the main sites of
FIXai inhibitory activity in the collagen perfusion system,
we used a platelet- and fibrin-specific
immunostaining on en face preparations. For these
studies, coverslips were derived from perfusion experiments with
infusion of 12.5 nmol/L FIXai. This concentration of FIXai reduced the
fibrin density on the coverslip, making it more suitable for a detailed
analysis. Platelets were selectively stained by use of the
monoclonal anti-GPIIb/IIIa antibody pl-62.21 22 Both
stained platelets and nonstained fibrin could be visualized
simultaneously by phase-contrast microscopy (Fig 3a). Bright-field microscopy of the same field showed
only material reactive with the anti-GPIIb/IIIa antibody, such as
platelets (Fig 3b). Using this technique, we found that fibrin
fibers originated from single platelets or small platelet
aggregates and extended along the blood flow direction (Fig 3a and 3b).
In Fig 3c and 3d, stained fibrin on coverslips from a different
experiment is shown, further illustrating that adherent platelets
were the nuclei of fibrin fiber generation and that they were often
encapsulated by a distinct fibrin layer (Fig 3c and 3d, arrow).
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Time Course of Platelet and Fibrin Deposition on
Collagen
To further establish that fibrin deposition on collagen is
dependent on the preceding platelet deposition, the perfusion
period was shortened. After a perfusion period of 2.5 minutes,
significant numbers of platelets and platelet thrombi were
deposited on collagen, but no fibrin was detectable by
immunostaining (Fig 4a and 4b). In
agreement, the determined FPA concentration in postchamber blood
(4.8±0.8 ng/mL; three donors) was very low. After 5.5 minutes of
perfusion, fibrin was deposited (Fig 4d) and the FPA concentration was
concomitantly elevated 11-fold (Table 2).
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Discussion |
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For this study we used a human ex vivo flow system that allowed us to measure the effects of selective inhibition of tissue factor/FVIIa and FVIIIa/FIXa complexes on thrombus formation in flowing nonanticoagulated human blood. Thrombus formation was initiated by cellular and noncellular constituents of the vessel wall. Smooth muscle cells and TNF-
–stimulated endothelial cells express
tissue factor,16 17 27 28 which might play a role in
thrombotic events after angioplasty29 and in disseminated
intravascular coagulation,30 respectively. To additionally
examine fibrin formation under conditions of a strong tissue factor
stimulus, we used the human carcinoma cell line J82, which is known to
express tissue factor at high density.31 32 Fibrillar
collagen, the other surface used, represents a major
thrombogenic component of the vessel wall33 known to
induce platelet activation and thrombus
formation.33 34 Exposure of these surfaces to native human
blood under venous blood flow conditions (shear rate of 65/s) resulted
in the deposition of fibrillar fibrin and led to an elevation in FPA
levels, the degree of which was dependent on the type of surface (Table 2). The observed fibrin depositions were specific, because we
demonstrated that the passage of blood through the perfusion system
without a thrombogenic surface led only to a minor activation of the
coagulation system and platelets. This was probably due to blood
activation in the mixing device and the low flow rate (1 mL/min), which
led to a prolonged interaction of platelets with the Plexiglas
surface that resulted in platelet activation. Fibrin formation on fibrillar collagen was strongly inhibited by infusion of FIXai, which, however, did not affect the deposition of platelet thrombi. Compared with controls, the platelet thrombi formed in the presence of FIXai appeared loosely packed, which might have been caused by the FIXai-mediated suppression of thrombin generation leading to inhibited platelet activation. Similarly, Roald et al35 demonstrated that inhibition of platelet activation resulted in less densely packed thrombi under flow conditions. As expected, FVIIai interfered with neither fibrin nor platelet thrombus formation on collagen fibrils, which have been shown to be devoid of tissue factor.24
Because collagen is a strong platelet-activating surface, we assumed that fibrin formation under our experimental conditions was mediated by adherent platelets and therefore represented the cellular sites of FIXai inhibitory activity. Two sets of experiments were carried out to verify our assumption. First, consistent with a platelet-mediated fibrin formation, we showed that platelet deposition preceded the formation and deposition of fibrin, confirming observations made by Sakariassen et al36 and Diquélou et al37 with similar perfusion systems. Second, using an immunogold-silver staining specific for platelets and fibrin, we demonstrated that collagen-adherent platelets accumulated fibrin on their surfaces and formed the nuclei for the deposition of fibrin fibers on the collagen surface. Because infusion of FIXai inhibited fibrin deposition on both collagen and the platelet surface, these results suggest that platelets were the main promoters of fibrin formation, although adherent leukocytes24 might also have contributed. The role of activated platelets in mediating coagulation is in accordance with their ability to support the assembly of the FVIIIa/FIXa complex.18 38 39 Ahmad et al38 40 showed that in the presence of FVIIIa and FX, both FIXa and active site–blocked FIXa bound to activated platelets with a significantly higher affinity than the zymogen FIX. Assuming that in our experimental system only a small fraction of the zymogen FIX in plasma was converted to FIXa, these data imply that subzymogen concentrations of FIXai should suffice to inhibit FIXa activity. In support of this view, we found that FIXai inhibited fibrin deposition on collagen by 50% at a blood concentration of 8 nmol/L, corresponding to a plasma concentration of about 14 nmol/L. These results are also in accord with the findings from a canine thrombosis model that active site–blocked FIXa inhibited thrombus formation at subzymogen concentrations.4
At present it remains unknown what mechanism initially generated FIXa in our experimental system. Although fibrillar collagen seems incapable of triggering coagulation by activating FXII to FXIIa,24 41 42 it might be that the deposited platelets themselves initiated coagulation.41 43 Alternatively, tissue factor exposed at the site of venipuncture could have generated FIXa, as suggested by Sakariassen et al.24
Unlike the results of the experiments with collagen as a thrombogenic
surface, fibrin formation initiated by the tissue factor–expressing
cellular surfaces studied was effectively prevented by infusion of
FVIIai but not of FIXai. It is interesting to note that FVIIai
inhibitory activity was strongest on smooth muscle cells,
which generated the lowest FPA levels, and it was weakest on J82 cells,
which produced the highest FPA levels (Table 2), indicating that FVIIai
inhibitory efficacy may be related to the density of
cell-expressed tissue factor. The role of endothelial
and smooth muscle cell tissue factor in fibrin formation under blood
flow conditions was also demonstrated by Zwaginga et al44
with the extracellular matrix of these cells, which is a condition
somewhat different from that in our experimental system with intact
cell layers. Moreover, we found that on TNF-–stimulated
endothelial cells, fibrin deposition was also reduced
by infusion of FVIIai, as was platelet thrombus deposition. This
suggested that deposited fibrin, rather than the
endothelial cells themselves, served as an adhesive
surface for anchoring the platelet thrombi, which contrasted with
the fibrin-independent thrombus deposition on collagen.
The FIXa independence of fibrin formation by cellular surfaces was
somewhat surprising because two of these surfaces, those of
endothelial cells and smooth muscle cells, have been
shown to express FIX/FIXa binding sites13 45 46 47 48 and
mediate FX activation by the FVIIIa/FIXa complex under nonflow
conditions.13 14 15 47 It is possible that these sites become
important under conditions that are different from those in our
experiments, such as different shear stress or very low tissue factor
density on the cell surfaces, as suggested by others.15 In
a similar flow system Tijburg et al15 showed that FIXai
inhibited fibrin formation on the exposed extracellular matrix of
endothelial cells. Inhibition was only observed when
the TNF- stimulus and, consequently, tissue factor expression were
low. Our results, however, show that even when tissue factor activity
was low, as on smooth muscle cells (Table 2), FIXai lacked efficient
inhibitory activity. This apparent difference might be due
to the different roles of platelets in these systems, in that they
are important in mediating fibrin formation on the collagen-containing
extracellular matrix of endothelial
cells15 but are not important for the monolayers of intact
cells in our study.
In conclusion, the present study demonstrates that fibrin formation by tissue factor–expressing intact cell layers is largely independent of FIXa activity and is mediated by direct activation of FX by the tissue factor/FVIIa complex. The results suggest further that, in vivo, interference with FIXa and FVIIa enzymatic activity might inhibit thrombus formation under different conditions. Inhibition of FVIIIa/FIXa function could be antithrombotic when platelet procoagulant activity is important for thrombus formation, such as after exposure of subendothelial collagen to flowing blood. The subendothelium also contains tissue factor,49 which makes our experimental conditions with isolated, tissue factor–free collagen somewhat different from the in vivo situation. Nevertheless, platelet procoagulant activity also plays an important role in thrombus formation when the entire subendothelium is used as a thrombogenic surface, as shown in experiments with platelets from a patient with Scott syndrome.50 On the other hand, when thrombus formation involves extrinsic, direct activation of FX, as might be the case for venous thrombosis, interference with the tissue factor/FVIIa complex may be useful.
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Acknowledgments |
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We express our thanks to Isabelle Guillaumat, Hildegund Mösch, and Olivier Kuster for their excellent technical help.
Received May 25, 1994; accepted April 10, 1995.
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References |
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