Inhibition of tissue factor limits the growth of venous thrombus in the rabbit
2003; Elsevier BV; Volume: 1; Issue: 5 Linguagem: Inglês
10.1046/j.1538-7836.2003.00110.x
ISSN1538-7933
AutoresJacques Himber, C. Wohlgensinger, Sébastiên Roux, Lisa A. Damico, John T. Fallon, Daniel Kirchhofer, Yale Nemerson, Markus A. Riederer,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoSummaryAntibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring 125I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation. Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring 125I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation. Tissue factor (TF) is highly concentrated in the vascular wall and is considered the initiator of hemostasis and thrombus formation. TF combines with factor (F)VII/FVIIa thereby forming the catalytic complex that initiates the coagulation cascade [1Nemerson Y. Tissue factor and hemostasis.Blood. 1988; 71: 1-8Crossref PubMed Google Scholar, 2Davie E.W. Fujikawa K. Kisiel W. The coagulation cascade: initiation, maintenance, and regulation.Biochemistry. 1991; 30: 10363-70Crossref PubMed Google Scholar]. TF has been extensively shown to be present in injured arteries [3Jang I.K. Gold H.K. Leinbach R.C. Fallon J.T. Collen D. Wilcox J.N. 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Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques. Effects of tissue factor pathway inhibitor on plaque thrombogenicity under flow conditions.Circulation. 1999; 99: 1780-7Crossref PubMed Google Scholar, 18Corseaux D. Le Tourneau T. Six I. Ezekowitz M.D. McFadden E.P. Meurice T. Asseman P. Bauters C. Jude B. Enhanced monocyte tissue factor response after experimental balloon angioplasty in hypercholesterolemic rabbit: inhibition with dietary l-arginine.Circulation. 1998; 98: 1776-82Crossref PubMed Google Scholar]. Inactivation of the TF/FVIIa complex by TF pathway inhibitor [19Holst J. Lindblad B. Bergqvist D. Nordfang O. Ostergaard P.B. Petersen J.G. Nielsen G. Hedner U. Antithrombotic effect of recombinant truncated tissue factor pathway inhibitor (TFPI1-161) in experimental venous thrombosis − a comparison with low molecular weight heparin.Thromb Haemost. 1994; 71: 214-9PubMed Google Scholar] or active-site blocked FVIIa locally applied [20Holst J. Kristensen A.T. Kristensen H.I. Ezban M. Hedner U. Local application of recombinant active-site inhibited human clotting factor VIIa reduces thrombus weight and improves patency in a rabbit venous thrombosis model.Eur J Vasc Endovasc Surg. 1998; 15: 515-20Abstract Full Text PDF PubMed Scopus (0) Google Scholar] has been shown to reduce thrombus weight and increase patency in a rabbit vein injury model suggesting that TF plays a key role in venous thrombosis. Although TF production by polymorphonuclear (PMN) leukocytes adhering to minimally injured vein wall has been suggested to be a pathogenic mechanism of deep vein thrombosis in man [21Lerner R.G. Goldstein R. Nelson J.C. Production of thromboplastin (tissue factor) and thrombi by polymorphonuclear neutrophilic leukocytes adhering to vein walls.Thromb Res. 1977; 11: 11-22Abstract Full Text PDF PubMed Google Scholar], a role of TF in the growth of a venous thrombus in the absence of a vessel damage has not yet been documented. Therefore, we assessed the ability of AP-1, a monoclonal antirabbit TF antibody [5Pawashe A.B. Golino P. Ambrosio G. Migliaccio F. Ragni M. Pascucci I. Chiariello M. Bach R. Garen A. Konigsberg W.K. Ezekowitz M.D. A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries.Circ Res. 1994; 74: 56-63Crossref PubMed Google Scholar, 8Himber J. Kirchhofer D. Riederer M. Tschopp T.B. Steiner B. Roux S. Dissociation of antithrombotic effect and bleeding time prolongation in rabbits by inhibiting tissue factor function.Thromb Haemost. 1997; 78: 1142-9Crossref PubMed Scopus (89) Google Scholar, 22Ragni M. Cirillo P. Pascucci I. Scognamiglio A. D'Andrea D. Eramo N. Ezekowitz M.D. Pawashe A.B. Chiariello M. Golino P. Monoclonal antibody against tissue factor shortens tissue plasminogen activator lysis time and prevents reocclusion in a rabbit model of carotid artery thrombosis.Circulation. 1996; 93: 1913-8Crossref PubMed Google Scholar], to prevent thrombus propagation in the rabbit jugular vein and in a silicon vein shunt. In both models, thrombus was initiated by exposure of a collagen-coated thread to flowing blood, thus avoiding proximal vessel damage and direct exposure of TF localized in the vessel wall. Supported by immunostaining of TF we present evidence that functional TF is present in the experimental venous thrombi and that it plays a critical role during thrombus growth. New Zealand rabbits (3–4 kg, BRL, Füllinsdorf, Switzerland) were used for this study. All experiments were performed according to the guidelines of the International Society on Thrombosis and Haemostasis for the use of animals in biomedical research [23Giles A.R. Guidelines for the use of animals in biomedical research.Thromb Haemost. 1987; 58: 1078-84Crossref PubMed Scopus (0) Google Scholar]. Rabbits were anesthetized by intravascular (i.v.) injection into the marginal ear vein of 35 mg kg−1 ketamine-HCl (Ketasol-100, Gräub AG, Bern, Switzerland) and 5 mg kg−1 xylazine (Rompun, Bayer AG, Leverkusen, Germany), the trachea was intubated and the lungs ventilated with room air. Body temperature was thermostatically kept at 38 °C. Then, the right femoral vein was prepared for drug administration and continuous pentobarbital infusion (0.12 mg kg−1 h−1) by the means of a three-way stopcock catheter (TriCath In 18G, Codan, Espergærde, Denmark). The left femoral vein was also dissected and cannulated (TriCath In 18G) for 125I-fibrinogen infusion. A catheter pressure transducer (Millar 3F, Houston, TX) was placed into the left femoral artery for continuous blood pressure and heart rate monitoring (Graphtec Linearcorder VII, model WR 3101, Hugo Sachs, March-Hugstetten, Germany). A cannula (TriCath In 22G) was inserted into the right femoral artery for blood sampling. Standardized surgical procedure was modified from a previously described method [24Agnelli G. Buchanan M.R. Fernandez F. Boneu B. Van Ryn J. Hirsh J. Collen D. A comparison of the thrombolytic and hemorrhagic effects of tissue-type plasminogen activator and streptokinase in rabbits.Circulation. 1985; 72: 178-82Crossref PubMed Google Scholar]. Through a paramedial incision of the neck, both jugular veins were exposed and cleared over a distance of 4 cm each and small side branches were cauterized by bipolar coagulation with an electrosurgical unit (Erbotom ICC50, Erbe, Germany). Two hemostatic clamps temporarily interrupted the blood flow of the right jugular vein. One clamp was placed on the external jugular vein distal to the bifurcation of the internal/external jugular vein. The second clamp was placed on the common jugular vein 2 cm proximal of the bifurcation. Then a cotton thread presoaked in a 1-mg mL−1 fibrillar collagen suspension (Collagen reagent Horm, Nycomed Arzneimittel, München, Germany) was introduced lengthwise into the lumen of the 2 cm segment of the common jugular vein and the internal jugular vein was ligated. The two clamps were then removed to restore blood flow from the external jugular vein through the common jugular vein. In another set of experiments a 3-cm silicon catheter (ID: 1.98 mm OD: 3.175 mm, Medical-Grade Tubing, Dow Corning Corp. Midland, Michigan, USA) already containing an inserted collagen-coated cotton thread was positioned into the common jugular vein, the internal jugular vein was ligated and blood flow from the external jugular vein was restored. An i.v. bolus of 10 µCi 125I-human fibrinogen (specific activity 200 µCi mg−1 fibrinogen, Amersham, Buckinghamshire, UK) was injected in the left femoral vein and maintenance of a constant plasma radioactivity was achieved by an i.v. infusion of 0.8 µCi h−1 as determined in a pilot experiment. As an indicator of thrombus growth, the accumulated radioactivity in the exposed jugular vein or in the silicon shunt was continuously monitored for 4 h. For this purpose, a gamma detector SMM1 (Cd-Te detector for 125I, 30 keV, 15 mm diameter × 18 mm) was positioned 2 cm over the jugular vein and connected to a multichannel analyzer (ND592A) and a computer running under a PCA Ranger v2.01 (Win-95) software (Bächli Instruments, Affoltern, Switzerland). The portion of the jugular vein containing the thrombus was isolated with 3-mm thick lead plates to damp background radioactivity. The radioactive counts were accumulated in 5-min intervals and thrombus growth was quantified for 4 h. The kinetics of the tracer uptake was displayed on the multichannel analyzer and stored on a HP Vectra computer. Monitoring of the left jugular vein that had no manipulation was used to measure background radioactivity. Blood samples were collected before initiation and every hour during the experiment from the femoral artery into 1/10 volume of 108 mmol L−1 sodium citrate. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured by using an ACL™ 300 Coagulation System Analyzer (Instrumentation Laboratory, Milan, Italy). Blood samples were also collected into EDTA solution for measurements of blood cell counts and hematocrit (Cobas Helios VET, F. Hoffmann-La Roche, Switzerland). Plasma radioactivity was measured at hourly intervals to obtain the mean fibrinogen radioactivity in the circulating blood. At the end of the experiment the venous segment or the silastic shunt containing the thrombus was tied off and the remaining thrombus rinsed in phosphate-buffered saline (PBS), blotted, weighed and counted in a gamma-counter (Cobra II Auto-gamma, Packard). Plasma fibrinogen concentration was determined in the ACL™ 300 Coagulation System by using the IL Test™ Fibrinogen-C kit. The ratio of the radioactivity of the thrombus to the circulating fibrinogen radioactivity was used to quantify the total (cold and labeled) fibrinogen accreted into the thrombus. The experiment was initiated by a bolus injection followed by infusion of 125I-human fibrinogen. Increase of tracer uptake within 30 min infusion of more than 30% of background value was indicative of onset of thrombus growth and thus, the counts were set to zero and rabbits received either napsagatran [25Hilpert K. Ackermann J. Banner D.W. Gast A. Gubernator K. Hadvary P. Labler L. Müller K. Schmid G. Tschopp T.B. Van de Waterbeemd H. Design and synthesis of potent and highly selective thrombin inhibitors.J Med Chem. 1994; 37: 3889-901Crossref PubMed Google Scholar] a specific thrombin inhibitor (10 µg kg−1 min−1 in continuous i.v. infusion) or repetitive i.v. bolus of the monoclonal antirabbit TF antibody AP-1 (600 µg kg−1) at hourly intervals. The placebo-treated rabbits (control group) received either saline infused at 1 mL kg−1 or four boli of an irrelevant IgG rabbit mAb. Immunohistochemical detection of rabbit TF within venous thrombi was performed on cryo- and paraffin sections with minor modifications of procedures previously described [5Pawashe A.B. Golino P. Ambrosio G. Migliaccio F. Ragni M. Pascucci I. Chiariello M. Bach R. Garen A. Konigsberg W.K. Ezekowitz M.D. A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries.Circ Res. 1994; 74: 56-63Crossref PubMed Google Scholar, 26Thiruvikraman S.V. Guha A. Roboz J. Taubman M.B. Nemerson Y. Fallon J.T. In situ localization of tissue factor in human atherosclerotic plaques by binding of digoxigenin-labeled factors VIIa and X.Lab Invest. 1996; 75: 451-61PubMed Google Scholar]. Briefly, the whole jugular vein containing the thrombus was tied off and the thrombi were briefly immersed in PBS and then maintained in 15% sucrose/PBS overnight at 4 °C. Cryosections of the thrombus (10 µm) were mounted on polylysine-coated microscope slides and immunohistochemistry was performed by using the monoclonal antirabbit TF antibody (AP-1) as TF marker and the monoclonal antihuman TF antibody HTFI-7B8, which does not stain rabbit TF, as negative control. The cryosections were treated with a blocking solution for endogenous peroxidase activity (Kirkegaard and Perry Laboratories Inc. Gaithersburg, MD, USA). Peroxidase staining was accomplished using HRP mouse 3,3′-diaminobenzidine (DAB) (EnVision™ Kit, Dako Diagnostics AG, Switzerland). Peroxidase was detected using DAB and counterstained with cresyl violet. The thrombi obtained in venous silicon shunts were isolated and fixed in fresh 4% paraformaldehyde for 24 h, then immersed in PBS for 2 days. The cotton thread was left in the thrombus as a position marker. Selected segments were embedded in paraffin and sectioned at 5 µm. For TF staining, the sections were deparaffinized, hydrated and washed in Tris-buffered saline (TBS) for 20 min and blocked with 1% peroxyde in methanol and normal serum. Sections were incubated with 100 nmol L−1 digoxigenin-labeled FVIIa in TBS buffer containing 5 mmol L−1 Ca2+ at 37 °C for 2 h, then washed in TBS and incubated with a 1 : 1000 dilution of sheep Fab antidigoxigenin antibody conjugated with HRP at 37 °C for 1 h, revealed with DAB and counterstained with hematoxylin. In the negative control, digoxigenin-labeled FVIIa was replaced by unlabeled FVIIa and in agreement with previous reports [26Thiruvikraman S.V. Guha A. Roboz J. Taubman M.B. Nemerson Y. Fallon J.T. In situ localization of tissue factor in human atherosclerotic plaques by binding of digoxigenin-labeled factors VIIa and X.Lab Invest. 1996; 75: 451-61PubMed Google Scholar] did not reveal any unspecific binding (data not shown). Thermanox plastic coverslips (Nalge Nunc Intern.) were incubated overnight at 4 C° in a 10-µg/mL fibrillar human collagen type III solution (Sigma), washed with PBS and blocked with 3% bovine serum albumin (BSA) in PBS for 30 min. After one additional wash with PBS, the coverslips were kept in PBS solution. One hour before the collagen-coated coverslips were used for the perfusion experiment, they were washed 3 × 15 min with a 0.9% NaCl solution and 1 × 15 min with a 0.9% NaCl solution containing 0.1% BSA. The collagen-coated coverslips were mounted in a parallel plate perfusion chamber [27Grabowski E.F. Effects of contrast media on endothelial cell monolayers under controlled flow conditions.Am J Cardiol. 1989; 64: 10E-15EAbstract Full Text PDF PubMed Scopus (0) Google Scholar] and the entire system including tubes, mixing devices and parallel plate chambers were filled with PBS containing 0.1% BSA. New Zealand rabbits were anesthetized by an intramuscular injection of xylasin (5 mg kg−1)/ketamine-HCl (35 mg kg−1) and blood drawn from the carotid artery was directly anticoagulated with napsagatran (10 µmol L−1 final concentration). The blood was then drawn through the perfusion system (1 mL min−1) by three individual roller pumps positioned distal to the parallel plate perfusion devices resulting in a wall shear rate of 65 s−1 which corresponds to venous flow conditions. After a 5.5-min perfusion period over the coverslips, the wash solution (PBS) was connected to the perfusion chamber without flow interruption. After three minutes of washing the system was stopped, and the fixative (3% paraformaldehyde in PBS) was perfused at 1 mL min−1 2 min. Then, the coverslips were gently removed from the perfusion chamber and fixation in 3% paraformaldehyde in PBS was continued for 30 min in a six-well plate. The coverslips were then washed three times with PBS and stored at 4 °C. For immunostaining, the coverslips were washed two times with TBS and incubated for 8 min with 40 µL Proteinase K (Dako no. S3004), diluted in 2 mL TBS. After two washes with TBS, the coverslips were blocked with 20% goat serum (Gibco, Invitrogen AG, Basel, Switzerland) in TBS for 20 min. After two washes with TBS, the monoclonal antirabbit TF antibody (AP-1) or the control antibody (mouse IgG1, Dako no. X0931) was added (1 : 20 dilution in TBS). The antibodies were allowed to bind for 75 min at room temperature. Then, the coverslips were washed twice with TBS and incubated for 30 min with goat antimouse IgG (Dako no. Z0420) diluted 1 : 25 in TBS. After two washes, the coverslips were incubated for 30 min with APAAP-mouse monoclonal antibody (Dako no. D0651), diluted 1 : 50 in TBS. The color development was performed for 20 min using Fast Red TR/Naphthol AS-MX (Sigma Fast, Sigma F-4523). The coverslips were then washed twice in PBS and cell nuclei were stained for 4 min in hematoxylin. After a 10-min wash step in tap water and a short wash step in distilled water, the coverslips were embedded. AP-1, the monoclonal antirabbit TF antibody was purchased from Dr M.D. Ezekowitz (Yale University, New Haven, CT, USA). Napsagatran [N-(N4-{[(S)-1-amidino-3-piperidinyl]methyl}-N2-(2-naphthalene-sulfonyl)-l-asparaginyl)-N-cyclopropylglycine was from F. Hoffmann-La Roche, Basel, Switzerland. Results are expressed as mean ± SEM. Statistical comparisons were carried out by Student's t-tests and a P-value <0.05 was considered as significant. All rabbits treated either with AP-1, napsagatran or placebo did not differ in their heart rate, blood pressure, plasma fibrinogen concentrations, hematocrit and blood cell counts over time (data not shown). The baseline PT and APTT were 10 ± 0.2 s and 15 ± 1.8 s, respectively. AP-1 treatment did not affect the PT and APTT levels while a 1.3-fold prolongation of the PT and a 1.7-fold prolongation of the APTT were observed in the napsagatran-treated animals. A collagen-coated cotton thread was inserted into the jugular vein of anesthetized rabbit, blood flow was restored and incorporation of 125I-fibrinogen into the thrombus was monitored. After this procedure the animal was allowed to stabilize for 30 min before administration of placebo, function-blocking antirabbit TF antibody AP-1 or the specific thrombin inhibitor napsagatran was started. The radioactivity already incorporated into the thrombus after 30 min confirmed that the system is properly established and was set as the baseline signal at start of the experiment. After the first hour of drug administration (from 0 to 60 min), the incorporated radioactivity into thrombi increased from baseline by 73 ± 13, 67 ± 22 and 32 ± 10% in placebo, AP-1 and napsagatran-treated rabbits, respectively (Fig. 1a). Statistical analysis confirmed that thrombus growth in the placebo and AP-1 treated rabbits was not different (Student's t-test, not significant). In contrast, reduction of 125I-fibrinogen incorporation by napsagatran was statistical different from the placebo group (P < 0.01). During the second hour (60 min to 120 min) thrombus growth in the placebo group continued to increase by 68% up to a total of 141 ± 16% whereas the presence of AP-1 only allowed a small increase of 17% up to 84 ± 2%. In the napsagatran-treated group incorporated radioactivity only increased by 11% up to 43% ± 0.7%. The inhibitory effect of AP-1 and napsagatran relative to the placebo were both statistical significant (P < 0.01). During the last 2 h of the experiment (from 120 min to 240 min), the incorporation of radioactivity increased in the placebo group by 248% up to a total of 389 ± 24%. In contrast, in the AP-1 treated animals radiolabel incorporation only increased by 12% up to 96 ± 1%, and in the napsagatran-treated animals only increased by 36% up to 79 ± 3%. The observed difference between placebo and AP-1 or napsagatran treated rabbits were highly significant (Student's t-test, P < 0.001). The contralateral jugular vein showed no changes of radioactivity over the entire duration of the experiment and thus confirmed that the measured incorporation of radioactivity in the vein with the inserted thread is specific for the growing thrombus (data not shown). At the end of the experiment thrombi were removed from the jugular vein and total fibrin in the thrombi was quantified. Thrombus associated fibrin was 4200 ± 900 µg, 140 ± 60 µg and 90 ± 50 µg in placebo, AP-1- and napsagatran-treated animals, respectively. The difference in fibrin content between AP-1 or napsagatran treatment relative to placebo was significant (P < 0.01 relative to control). Furthermore, the total amount of incorporated fibrin correlated with the amount of incorporated radiolabeled fibrinogen and thus confirms the inhibitory effect of AP-1 and napsagatran on venous thrombus growth in vivo. In order to understand the contribution of the vessel wall to thrombus formation, incorporation of 125I-fibrinogen into a thrombus on a collagen-coated cotton thread placed in a silicon venous shunt was measured (Fig. 1b). After the first hour of drug administration (from 0 to 60 min), radioactivity incorporation in thrombi increased from baseline by 76 ± 10, 56 ± 9 and 30 ± 9% in rabbits receiving placebo, AP-1 and napsagatran, respectively. The difference in thrombus growth between placebo and AP-1 was not statistical significant. In contrast, the observed reduction of thrombus growth due to administration of napsagatran was already different from placebo (P < 0.01). In the second hour of the experiment (from 60 min to 120 min), thrombus growth in the placebo-treated group increased by 84% up to 160 ± 9%. In contrast, in the AP-1 treated group thrombi increased only by 33% up to 89 ± 9% and in the napsagatran-treated group increase was only 3% up to 33 ± 1%, The last 2 h of the drug administration (from 120 to 240 min), the radioactivity incorporation in the thrombus increased by 188% up to 346 ± 38% in the placebo group. For the AP-1-treated animals increase was only by 52% up to 141 ± 2%. Infusion of napsagatran only allowed an increase of 16% up to a total of 49 ± 5%. For AP-1 and napsagatran, the inhibitory effect relative to placebo was highly significant (P < 0.01). At the end of the 240 min-experiment the total fibrin measured from the post mortem thrombi removed from the silicon shunt was 1800 ± 500, 280 ± 130 and 130 ± 90 µg for placebo, AP-1 and napsagatran-treated animals, respectively. Therefore, this data confirms that quantification of radiolabeled fibrinogen parallels total fibrin incorporation and thus thrombus mass. In conclusion, these data demonstrate that thrombus growth in a silicon shunt can be inhibited by a function blocking anti-TF antibody and by a specific inhibitor of thrombin activity. Thrombi generated under blood flow conditions by exposure of a collagen-coated cotton thread in the rabbit jugular vein were stained using the TF-specific antibody AP-1. Figure 2(a) shows numerous TF-positive leukocytes. Because cryosections do not preserve the integrity of the cellular structure we were unable to identify clearly the type of TF-bearing leukocytes. To obtain more information about the involved TF-positive cell types another detection method was applied. Paraffin sections of thrombi generated in the venous silicon shunt after 4 h of blood flow were incubated with digoxigenin-labeled FVIIa. The TF-specific signal revealed the presence of TF associated with PMN leukocytes mainly colocalized with erythrocytes in the collagen coated thread area (Fig. 2c). To establish that TF observed in thrombi could arise from blood, studies were performed using a system that contains no exogenous TF, namely collagen-coated coverslips placed in a parallel plate flow chamber [27Grabowski E.F. Effects of contrast media on endothelial cell monolayers under controlled flow conditions.Am J Cardiol. 1989; 64: 10E-15EAbstract Full Text PDF PubMed Scopus (0) Google Scholar] and perfused with rabbit blood for 5.5 min. Blood was anticoagulated with napsagatran to prevent massive fibrin formation and thus interference with the staining procedure. Figure 2(d) shows deposition of TF-positive PMN leukocytes onto the platelet-collagen surface. Because this experiment was performed for only 5 min, significant de novo synthesis of TF is not possible suggesting that the TF
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