Long-lasting antithrombotic effects of a single dose of human recombinant, active site-blocked factor VII: insights into possible mechanism(s) of action
2003; Elsevier BV; Volume: 1; Issue: 5 Linguagem: Inglês
10.1046/j.1538-7836.2003.00235.x
ISSN1538-7933
AutoresPlinio Cirillo, Paolo Golino, Maurizio Ragni, Davide D’Andrea, Paolo Calabrò, Nicola Corcione, Francesco Vigorito, Marta Ravera, M Chiariello,
Tópico(s)Hemophilia Treatment and Research
ResumoSummaryTissue factor (TF) is important in initiating intravascular thrombosis. We demonstrated that active-site blocked factor VII (FVIIai) inhibits intravascular thrombosis for at least 6 h following a single injection, despite FVIIai plasma half-life was ∼45 min. The aims of the present study were: (a) to determine the duration of the antithrombotic effects of a single injection of FVIIai; and (b) to assess whether FVIIai prolonged effects can be explained by a slow dissociation rate from TF in the arterial wall. Cyclic flow variations (CFVs), obtained in stenotic rabbit carotid arteries with endothelial injury, were abolished by either FVIIai (100 µg kg−1 min−1 for 10 min) or hirudin (1 mg kg−1). After CFVs were abolished, carotid blood flow velocity was recorded continuously for 24 h. CFVs restored in all hirudin-treated animals after 2.1 ± 0.3 h, while they restored in only four of nine FVIIai-treated rabbits in 10.1 ± 2.2 h. Five animals in this group did not show restoration of CFVs up to 24 h. Immunohistochemistry revealed that FVIIai was still bound to the arterial wall 24 h following its administration, despite at this time FVIIai plasma levels were undetectable. Prothrombin time and partial thromboplastin time did not change significantly. FVIIai exerts potent, long-lasting antithrombotic effects without affecting systemic hemostatic parameters; a possible mechanism is a slow dissociation rate of FVIIai from TF. These proprieties make FVIIai particularly attractive as an antithrombotic intervention. Tissue factor (TF) is important in initiating intravascular thrombosis. We demonstrated that active-site blocked factor VII (FVIIai) inhibits intravascular thrombosis for at least 6 h following a single injection, despite FVIIai plasma half-life was ∼45 min. The aims of the present study were: (a) to determine the duration of the antithrombotic effects of a single injection of FVIIai; and (b) to assess whether FVIIai prolonged effects can be explained by a slow dissociation rate from TF in the arterial wall. Cyclic flow variations (CFVs), obtained in stenotic rabbit carotid arteries with endothelial injury, were abolished by either FVIIai (100 µg kg−1 min−1 for 10 min) or hirudin (1 mg kg−1). After CFVs were abolished, carotid blood flow velocity was recorded continuously for 24 h. CFVs restored in all hirudin-treated animals after 2.1 ± 0.3 h, while they restored in only four of nine FVIIai-treated rabbits in 10.1 ± 2.2 h. Five animals in this group did not show restoration of CFVs up to 24 h. Immunohistochemistry revealed that FVIIai was still bound to the arterial wall 24 h following its administration, despite at this time FVIIai plasma levels were undetectable. Prothrombin time and partial thromboplastin time did not change significantly. FVIIai exerts potent, long-lasting antithrombotic effects without affecting systemic hemostatic parameters; a possible mechanism is a slow dissociation rate of FVIIai from TF. These proprieties make FVIIai particularly attractive as an antithrombotic intervention. Acute thrombosis following ulceration of a coronary atherosclerotic plaque is a major complication of atherosclerosis, which often leads to the abrupt conversion from chronic to acute coronary ischemic syndrome [1Willerson J.T. Golino P. Eidt J. Campbell W.B. Bujia L.M. Specific platelet mediators and unstable coronary artery lesions: experimental evidence and clinical implications.Circulation. 1989; 80: 198-205Crossref PubMed Google Scholar]. One of the key events responsible for intracoronary thrombus formation seems to be the activation of the extrinsic coagulation pathway through exposure of tissue factor (TF) to flowing blood as a consequence of vessel injury [2Furie B. Furie B.C. Molecular and cellular biology of blood coagulation.N Engl J Med. 1992; 326: 800-6Crossref PubMed Google Scholar, 3Rapaport S.I. Rao L.V.M. Initiation and regulation of tissue factor-dependent blood coagulation.Arterioscler Thromb. 1992; 12: 1111-21Crossref PubMed Google Scholar]. TF then binds circulating factor (F)VII and FVIIa, forming an enzymatic complex capable of converting FX and FIX into their active forms, FXa and FIXa, ultimately leading to the generation of thrombin [3Rapaport S.I. Rao L.V.M. Initiation and regulation of tissue factor-dependent blood coagulation.Arterioscler Thromb. 1992; 12: 1111-21Crossref PubMed Google Scholar]. Several experimental and clinical studies indicate that TF is involved in the pathophysiology of intravascular thrombus formation following arterial injury [4Pawashe 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, 5Wilcox J.N. Smith K.M. Schwartz S. Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque.Proc Natl Acad Sci USA. 1989; 86: 2839-43Crossref PubMed Google Scholar, 6Annex B.H. Denning S.M. Channon K.M. Sketch M.H. Stack R.S. Morrisey J.H. Peters K.G. Differential expression of tissue factor protein in directional atherectomy specimens from patients with stable and unstable coronary syndromes.Circulation. 1995; 91: 619-22Crossref PubMed Google Scholar, 7Ragni M. Cirillo P. Pascucci I. Scognamiglio A. D'Andrea D. Eramo N. Ezekowitz M.D. Pawashe A.B. Chiariello M. Golino P. A 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: 1913Crossref PubMed Google Scholar]. Recently, development of human recombinant, active site-blocked FVII (FVIIai) has made it possible to further characterize the role of inhibiting TF-procoagulant activity as an antithrombotic intervention. We have recently shown that FVIIai administration resulted in potent antithrombotic effects in a rabbit model of recurrent thrombus formation, without any significant change in hemostatic parameters, such as prothrombin time (PT) and partial thromboplastin time (PTTs) [8Golino P. Ragni M. Cirillo P. D'Andrea D. Scognamiglio A. Ravera A. Buono C. Ezban M. Corcione N. Vigorito F. Condorelli M. Chiariello M. Antithrombotic effects of recombinant human, active site-blocked Factor VIIa in a rabbit model of recurrent arterial thrombosis.Circ Res. 1998; 82: 39-46Crossref PubMed Google Scholar]. In that study, we observed that the antithrombotic effects of a single intravenous injection of FVIIai lasted for at least 6 h, despite a plasma half-life of about 45 min [8Golino P. Ragni M. Cirillo P. D'Andrea D. Scognamiglio A. Ravera A. Buono C. Ezban M. Corcione N. Vigorito F. Condorelli M. Chiariello M. Antithrombotic effects of recombinant human, active site-blocked Factor VIIa in a rabbit model of recurrent arterial thrombosis.Circ Res. 1998; 82: 39-46Crossref PubMed Google Scholar]. Thus, the major aims of the present study were: (a) to determine the duration of the antithrombotic effects of a single dose of FVIIai in a conscious rabbit model of carotid artery thrombosis. This was achieved by extending the experimental period to 24 h; and (b) to clarify the possible mechanism(s) responsible for these prolonged antithrombotic effects. In particular, we hypothesized that the prolonged antithrombotic effects of FVIIai might be explained, at least in part, by a slow dissociation rate of this compound from its ligand, TF, in the arterial wall. Thus, immunohistochemistry studies were performed on carotid sections to assess persistence of FVIIai in the damaged vessels 24 h following its administration. Cyclic flow variations (CFVs), the expression of recurrent intravascular thrombus formation, were induced in rabbit carotid arteries as previously described [9Golino P. Ambrosio G. Pascucci I. Ragni M. Russolillo E. Chiariello M. Experimental carotid stenosis and endothelial injury in the rabbit: an in vivo model to study intravascular platelet aggregation.Thromb Haemost. 1992; 67: 302-5Crossref PubMed Scopus (41) Google Scholar, 10Golino P. Cappelli-Bigazzi M. Ambrosio G. Ragni M. Russolillo E. Condorelli M. Chiariello M. Endothelium-derived relaxing factor modulates platelet aggregation in an in vivo model of recurrent platelet activation.Circ Res. 1992; 71: 1447-56Crossref PubMed Google Scholar]. To permit a longer experimental period following instrumentation and drug administration, animals were allowed to recover from the surgical procedure while continuously recording carotid blood flow velocity through a computerized system (see below). This technique allowed us to extend the experimental period to 24 h and to precisely establish the time of spontaneous restoration of CFVs after they were abolished by drug treatment. Two groups of animals were included in the study: a first group received FVIIai as previously described [8Golino P. Ragni M. Cirillo P. D'Andrea D. Scognamiglio A. Ravera A. Buono C. Ezban M. Corcione N. Vigorito F. Condorelli M. Chiariello M. Antithrombotic effects of recombinant human, active site-blocked Factor VIIa in a rabbit model of recurrent arterial thrombosis.Circ Res. 1998; 82: 39-46Crossref PubMed Google Scholar] and a second group was treated with hirudin. The purpose of including the latter group was to determine whether another antithrombotic compound with a similar half-life of FVIIai would result in a prolonged antithrombotic effect. At the end of the experiment, carotid sections were immunohistochemically stained for FVIIai. The hypothesis to be tested was that the prolonged antithrombotic effects of FVIIai might be explained, at least in part, by a slow dissociation rate of FVIIai from its ligand, TF, in the damaged arterial wall. FVIIa and FVIIai were obtained from Novo Nordisk A/S (Gentofte, Denmark). FVIIai was produced by blocking the active site of recombinant human FVIIa, obtained as previously described [11Thim L. Bioern S. Christiensen M. Nicolaisen E.M. Lund T. Pedersen A.H. Hedner U. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells.Biochemistry. 1988; 27: 7785-93Crossref PubMed Google Scholar] by the addition of twofold molar excess of Phe-Phe-Arg chloromethyl ketone (FFRcmk) [12Sorensen B.B. Persson E. Freskgard P.O. Kjalke M. Ezban M. Williams T. Rao L.V.M. Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor.J Biol Chem. 1997; 272: 11863-8Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar]. The residual FVIIa activity in the FVIIai solution was < 0.1% when measured in a FVIIa-specific amidolytic assay [12Sorensen B.B. Persson E. Freskgard P.O. Kjalke M. Ezban M. Williams T. Rao L.V.M. Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor.J Biol Chem. 1997; 272: 11863-8Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar]. The present study was performed in a chronic rabbit model of recurrent carotid artery thrombosis which represents a modification of the acute model previously described by our group [9Golino P. Ambrosio G. Pascucci I. Ragni M. Russolillo E. Chiariello M. Experimental carotid stenosis and endothelial injury in the rabbit: an in vivo model to study intravascular platelet aggregation.Thromb Haemost. 1992; 67: 302-5Crossref PubMed Scopus (41) Google Scholar, 10Golino P. Cappelli-Bigazzi M. Ambrosio G. Ragni M. Russolillo E. Condorelli M. Chiariello M. Endothelium-derived relaxing factor modulates platelet aggregation in an in vivo model of recurrent platelet activation.Circ Res. 1992; 71: 1447-56Crossref PubMed Google Scholar]. Briefly, New Zealand White rabbits of either sex (2.7–3.2 kg body weight) were anesthetized with a mixture of ketamine (35 mg kg−1) and xylazine (5 mg kg−1) administered intramuscularly. Anesthesia was maintained during the course of the experiment by an intravenous infusion of ketamine sufficient to abolish the corneal reflex. Under sterile conditions, a median incision of the neck was made and either the left or right common carotid artery was exposed and carefully isolated from the surrounding tissue. A polyethylene catheter was inserted into a jugular vein for drug administration. A segment of the exposed vessel was injured by gently squeezing the artery between a pair of rubber-covered forceps. An external plastic constrictor was placed around the damaged site to produce an arterial stenosis of about 75% of the luminal diameter. Carotid blood flow velocity was measured continuously by a Doppler flow probe positioned proximal to the constrictor. A small polyethylene catheter (25 G outer diameter) was placed into the carotid artery for the local infusion of FVIIai. After instrumentation, the animals developed a typical pattern of carotid blood flow, called CFVs and characterized by gradual decreases of flow to almost zero values followed by spontaneous or induced (by gently shaking constrictor) restorations of flow. Previous studies have shown that CFVs are due to recurrent episodes of thrombus formation followed by its dislodgment [11Thim L. Bioern S. Christiensen M. Nicolaisen E.M. Lund T. Pedersen A.H. Hedner U. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells.Biochemistry. 1988; 27: 7785-93Crossref PubMed Google Scholar, 12Sorensen B.B. Persson E. Freskgard P.O. Kjalke M. Ezban M. Williams T. Rao L.V.M. Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor.J Biol Chem. 1997; 272: 11863-8Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 13Folts J.D. Crowel Jr, E.B. Rowe C.G. Platelet aggregation in partially obstructed vessels and its elimination by aspirin.Circulation. 1976; 54: 365-70Crossref PubMed Google Scholar, 14Golino P. Ragni M. Cirillo P. Pascucci I. Ezekowitz M.D. Pawashe A. Scognamiglio A. Pace L. Guarino A. Chiariello M. Aurintricarboxylic acid reduces platelet deposition in stenosed and endothelially injured rabbit carotid arteries more effectively than other antiplatelet interventions.Thromb Haemost. 1995; 74: 974-80Crossref PubMed Scopus (22) Google Scholar]. Once CFVs were obtained, they were observed for 30 min. Animals were then randomly assigned to receive an intracarotid infusion of FVIIai at a dose of 100 µg kg−1 min−1 for 10 min, as previously described [8Golino P. Ragni M. Cirillo P. D'Andrea D. Scognamiglio A. Ravera A. Buono C. Ezban M. Corcione N. Vigorito F. Condorelli M. Chiariello M. Antithrombotic effects of recombinant human, active site-blocked Factor VIIa in a rabbit model of recurrent arterial thrombosis.Circ Res. 1998; 82: 39-46Crossref PubMed Google Scholar] (group I, n= 11), or an intravenous bolus of hirudin at the dose of 1 mg kg−1 (group II, n= 11). If CFVs were inhibited at the end of drug infusion, the animals were followed for additional 60 min Then, with the arterial constrictor left in place, the surgical incisions were sutured, an antibiotic was administered (cefuroxim, 15 mg kg−1 intramuscularly), and the animals were allowed to recover. The Doppler flow wires were externalized from the posterior base of the neck and connected via a swivel to a computerized system (Mac Laboratory). The swivel allowed the animals to freely move in the cage while the Doppler signal was continuously recorded. When CFVs were inhibited, animals in each group were monitored for 24 h. To determine the effect of FVIIai and of hirudin administration on PT and PTT, blood was obtained at the end of the CFV period (baseline) and after administration of FVIIai or hirudin. Blood was collected in sodium citrate (3.8%) and centrifuged at 2000 × g for 10 min at 4 °C to separate the plasma. PTs and PTTs were measured in duplicate within 2 h from blood collection. To evaluate the pharmacokinetics of FVIIai and correlate its plasma levels with the antithrombotic effects, blood samples were obtained at baseline, at the end of FVIIai infusion, and 1, 2, 3 and 24 h after administration of FVIIai. The concentration of FVIIai in rabbit plasma was determined in triplicate by an ELISA method using the FVII EIA kit from Dako Corp according to the manufacturer's instructions. To evaluate whether the prolonged inhibition of CVFs was associated with a persistence of FVIIai in the damaged arterial wall, immunohistochemistry for FVIIai was performed on carotid arteries harvested from animals in both groups. At the end of the experiment (i.e. 24 h after CFVs were inhibited), the injured carotid arteries, as well as the normal, uninjured, contralateral arteries were harvested, cleaned from the surrounding tissue, and briefly washed in phosphate-buffered saline (PBS). Arterial segments were then fixed in 4% buffered neutral formalin and embedded in paraffin. Serial sections (5 µm) were mounted on coated slides and, once deparaffinized, were immunostained using indirect immunoperoxidase method as follows. Sections were blocked with 10% rabbit serum for 30 min at 37 °C and then incubated for 1 h at 37 °C with a 1 : 1000 dilution of a goat polyclonal antibody directed against human FVIIa (see below). Sections were then washed three times with PBS and afterwards incubated with 1 : 200 dilution of a peroxidase-conjugated donkey antigoat IgG (Sigma Chemicals, St. Louis, MO, USA) at room temperature for 30 min. Endogenous peroxidase activity was inhibited with methyl alcohol, while antigoat IgG peroxidase activity was developed using 3-aminoethylcarbazole (AEC) in PBS containing 0.3% H2O2 for 10 min at room temperature. Section were counterstained with hematoxylin, dehydrated in ethanol, displaced in xylene, and mounted with Permount™ (Fisher Scientific, Glostrop, Denmark). Internal control immunohistochemistry of carotid vessels was performed as described above, except that the first incubation with the primary antibody directed against human FVIIa, was omitted. Antihuman FVIIa polyclonal antibody was raised in goats. The antigen used for immunization was recombinant human FVIIa. The immunization protocol has been described elsewhere [15Harlow E. Lane D. Antibodies: a Laboratory Manual. Cold Spring Harbor Laboratory, 1988Google Scholar]. At the end of the immunization period, the antiserum was collected and specific goat antihuman FVIIa IgG were purified by immunoaffinity chromatography using a commercial purification kit, following the manufacturer instructions (QuickPure affinity column, Sterogene Bioseparations, Inc., Arcadia, CA, USA). Purified antibody was dialyzed against PBS, filter sterilized and stored in aliquots at −80 °C. The purified antibody recognized a single band of about 50 kDa in human plasma, but not in rabbit plasma, in Western blot experiments (data not shown). All values are expressed as mean ± SEM. The rate of inhibition of CFVs by FVIIai was evaluated by Fisher's exact test. One- and two-way anova with a design for repeated measurements were used to compare hemodynamic variables and PTs and PTTs. When applicable, differences between groups were tested by Student's t-test for paired or unpaired samples with the Bonferroni's correction. A P-value less than 0.05 defined significant differences between populations. After arterial injury and placement of the constrictor, CFVs developed in all rabbits in each group. The intracarotid infusion of FVIIai resulted in a complete inhibition of CFVs in nine of the initial 11 animals; similarly, hirudin administration resulted in inhibition of CVFs in nine of 11 animals. Thus, the acute antithrombotic effects of FVIIai and hirudin were identical (Fig. 1). In contrast, a striking difference between the two groups of animals was observed regarding the duration of the antithrombotic effects: in the hirudin treated group, CFVs recurred spontaneously in all animals within 2.1 ± 0.3 h, while in FVIIai-treated rabbits, CFVs spontaneously restored in only four of nine animals, as at the end of the study period, i.e. 24 h, five rabbits were still inhibited. In the remaining four rabbits, the time of spontaneous restoration of CFVs averaged 10.1 ± 2.2 h (Fig. 2).Figure 2Time of spontaneous restoration of CFVs in FVIIai- and hirudin-treated animals after administration of the two compounds. Although FVIIai and hirudin treatment produced similar acute antithrombotic effects, CFVs spontaneously returned in all hirudin-treated animals in which CFVs were initially inhibited (n = 9) in 2.1 ± 0.3 h, while they restored in only four of nine rabbits treated with FVIIai in 10.1 ± 2.2 h. In the remaining five rabbits, CFVs were still inhibited at 24 h.View Large Image Figure ViewerDownload Hi-res image Download (PPT) FVIIai plasma levels were measured at baseline, at the end of FVIIai infusion and 1, 2, 3, and 24 h after FVIIai administration. Plasma FVIIai levels increased from undetectable levels before starting the infusion to 29.2 ± 3.8 µg mL−1 immediately after stopping its infusion (Fig. 3). Plasma FVIIai levels progressively decreased to undetectable levels 24 h after its administration (Fig. 3). These data strongly suggest that the prolonged antithrombotic effects of FVIIai cannot be explained by its pharmacokinetic properties. To study the possible systemic effects of FVIIai which may predispose to an increased risk of bleeding, and to compare these effects with the systemic effects of hirudin, PT and aPTT were measured in blood samples collected at 30 min of CFVs and after FVIIai or hirudin administration. In FVIIai-treated group, at the end of the 30-min CFV period, PTs and PTTs averaged 8.2 ± 0.6 and 25 ± 3 s, respectively. FVIIai administration did not affect PTTs, while a slight increase in PTs to 10.1 ± 0.7 s was observed. This increase, however, did not reach statistical significance (P = 0.07, Fig. 4). On the contrary, in group II rabbits, hirudin administration caused a significant increase in PTs to 13 ± 0.1 s and a striking prolongation of PTTs to greater than 360 s (Fig. 4). To evaluate whether the prolonged inhibition of CFVs was associated with a persistence of FVIIai in the damaged arterial wall, carotid vessels were subjected to immunohistochemical staining for FVIIai to assess its presence in the vessel wall. In carotid vessels obtained from rabbits in which CFVs were still inhibited after 24 h, FVIIai could be detected predominantly in the media and in the adventitia. In contrast, FVIIai could not be detected in the carotid arteries obtained from rabbits treated with FVIIai, but in which CFVs restored before the end of the experiment, nor in histological sections obtained from rabbits treated with hirudin (Fig. 5). The effectiveness of FVIIai as an antithrombotic intervention in different experimental model of arterial thrombosis has been previously reported [8Golino P. Ragni M. Cirillo P. D'Andrea D. Scognamiglio A. Ravera A. Buono C. Ezban M. Corcione N. Vigorito F. Condorelli M. Chiariello M. Antithrombotic effects of recombinant human, active site-blocked Factor VIIa in a rabbit model of recurrent arterial thrombosis.Circ Res. 1998; 82: 39-46Crossref PubMed Google Scholar, 16Harker L.A. Hanson S.R. Wilcox J.N. Kelly A.B. Antithrombotic and antilesion benefits without hemorragic risks by inhibiting tissue factor pathway.Haemostasis. 1996; 26: 76-82PubMed Google Scholar, 17Harker L.A. Mann K.G. Thrombosis and fibrinolysis.in: Fuster V Verstraete M Thrombosis in Cardiovascular Disorders. W.B. Saunders Co., 1992: 1-16Google Scholar]. In particular, we have previously shown that a single injection of FVIIai inhibited CFVs in an anesthetized rabbit model of carotid artery thrombosis similar to the conscious model used in the present study for at least 6 h following its administration, despite very low FVIIai plasma levels at this point [8Golino P. Ragni M. Cirillo P. D'Andrea D. Scognamiglio A. Ravera A. Buono C. Ezban M. Corcione N. Vigorito F. Condorelli M. Chiariello M. Antithrombotic effects of recombinant human, active site-blocked Factor VIIa in a rabbit model of recurrent arterial thrombosis.Circ Res. 1998; 82: 39-46Crossref PubMed Google Scholar]. This observation clearly indicated that a discrepancy exists between FVIIai plasma levels and persistence of its antithrombotic effects, suggesting possible different mechanisms of action for this compound, and prompted us to design and perform the present study. Indeed, based on our previous observations, the aim of the present study was to extend the experimental period to 24 h after inhibition of CFVs in order to determine the duration of the antithrombotic effects of FVIIai. In addition, we also wanted to evaluate the possible mechanisms involved in the prolonged effects of FVIIai, ultimately leading to significant local antithrombotic effects, even in the absence of measurable levels of FVIIai in the plasma. The novel findings of the present study are: (1) the antithrombotic effects of a single injection of FVIIai persisted in five of nine treated animals for at least 24 h, while in the remaining four rabbits CFVs spontaneously restored after 10.1 ± 2.2 h. Measurement of FVIIai plasma levels at the end of the experiment showed that at this time point FVIIai in the plasma was unmeasurable. This was in striking contrast to the duration of the antithrombotic effects of hirudin: in this group of rabbits CFVs restored spontaneously in all animals in 2.1 ± 0.3 h, indicating that the prolonged effects were specific for FVIIai. (2) Immunohistochemical staining specific for FVIIai of carotid sections obtained at the end of the 24-h period, when plasma levels of FVIIa were undetectable, showed that FVIIai was still bound to the vascular wall despite the plasma levels were undetectable, suggesting that persistence of the compound in the vessel wall might explain, at least in part, its prolonged biological effects. Recent studies have suggested that interfering with TF procoagulant activity represents an attractive target for antithrombotic therapy. For example, administration of a monoclonal antibody against rabbit TF resulted in inhibition of CFVs in an acute experimental model similar to the chronic one used in the present study [4Pawashe 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]. More recently, the availability of recombinant human, active site-blocked FVIIa (FVIIai) has permitted further characterization of the TF-FVII axis in the pathophysiology of intravascular thrombus formation. Studies from our laboratory, as well as from other groups, have demonstrated that FVIIai may exert potent antithrombotic effects in different experimental models with a relative lack of systemic effects [8Golino P. Ragni M. Cirillo P. D'Andrea D. Scognamiglio A. Ravera A. Buono C. Ezban M. Corcione N. Vigorito F. Condorelli M. Chiariello M. Antithrombotic effects of recombinant human, active site-blocked Factor VIIa in a rabbit model of recurrent arterial thrombosis.Circ Res. 1998; 82: 39-46Crossref PubMed Google Scholar, 16Harker L.A. Hanson S.R. Wilcox J.N. Kelly A.B. Antithrombotic and antilesion benefits without hemorragic risks by inhibiting tissue factor pathway.Haemostasis. 1996; 26: 76-82PubMed Google Scholar]. The possible mechanisms responsible for the strikingly prolonged antithrombotic effects of FVIIai require further discussion. These prolonged effects are specific for FVIIai, as hirudin-treated animals showed spontaneous restoration of CFVs in a much shorter time with respect to FVIIai-treated rabbits. In addition, immunohistochemistry data showed persistence of FVIIai in the arterial wall in animals with CFVs still inhibited at 24 h, despite unmeasurable FVIIai plasma levels. The data of the present study do not allow us to draw a firm conclusion about this finding. However, possible explanations for the prolonged FVIIai antithrombotic effects are discussed below. Previous studies have shown that that inactivation of FVIIa active site with FFR cmk (the same method used to obtain the FVIIai preparation used in the present study) resulted in a 5-fold higher affinity for TF and a 2-fold slower dissociation rate from TF [12Sorensen B.B. Persson E. Freskgard P.O. Kjalke M. Ezban M. Williams T. Rao L.V.M. Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor.J Biol Chem. 1997; 272: 11863-8Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar]. Considering this observation, it can be speculated that FVIIai, once bound to TF, dissociates very slowly from it, such that significant antithrombotic effects can be still achieved even when FVIIai is almost completely cleared from the circulation. A second hypothesis is that human FVIIai possesses a much slower dissociation rate from rabbit TF, as compared with human TF. Unfortunately, at present there are no data available on this issue and this hypothesis, although possible, remains entirely speculative. It can be speculated that differences between native FVIIa- and FVIIai-dependent intracellular TF metabolism might play a determinant role in mediating the prolonged FVIIai antithrombotic effects observed in the present study. It is known, in fact, that TF procoagulant activity is down-regulated by translocation of the complex TF/FVIIa into non-coated plasmalemma vesicles, but that this phenomenon is dependent upon formation of the quaternary complex TF/FVIIa/Xa/TFPI [18Sevinsky J.R. Rao L.V.M. Rul W. Ligand protease receptor translocation into caveolae mechanism for regulating cell surface proteolysis of the tissue factor-dependent coagulation pathway.J Cell Biol. 1996; 133: 293-304Crossref PubMed Scopus (0) Google Scholar]. Considering the strict requirement of TFPI for TF internalization, this mechanism does not seem to play an important role under the experimental conditions of the present study, as FVIIai is not able to bind TFPI. However, in a more recent paper, Iakhiaev et al. have demonstrated that FVIIai, after binding TF, can be internalized and degraded with kinetics similar to native FVIIa in a TFPI-independent manner [19Iakhiaev A. Pendurthi U.R. Voigt J. Ezban M. Rao L.V.M. Catabolism of FVIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor.J Biol Chem. 1999; 274: 36995-7003Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Most importantly, this group of investigators has also showed that a significant fraction of internalized FVIIai recycles back to the cell surface, where it may become available for binding new TF [20Iakhiaev A. Pendurthi U.R. Rao L.V.M. Active site blockade of Factor VIIa alters its intracellular distribution.J Biol Chem. 2001; 276: 45895-901Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. In this regard, immunohistochemistry data obtained in the present study, i.e. persistence of FVIIai immunohistochemical staining at 24 h, might be in line with the data of Iakhiaev et al. [20Iakhiaev A. Pendurthi U.R. Rao L.V.M. Active site blockade of Factor VIIa alters its intracellular distribution.J Biol Chem. 2001; 276: 45895-901Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. On the other hand, the finding that, in the animals in which CFVs returned before 24 h, FVIIai was not detectable by immunohistochemistry could be explained by a faster recycling of FVIIai, which in turn might determine a faster metabolism of the compound. On these grounds, it can be speculated that the prolonged antithrombotic effects of FVIIai observed in the present study might be due to a three-step mechanism in which, first, FVIIai binds TF and inhibits its procoagulant activity; second, FVIIai promotes TF translocation from the cell surface to the cytoplasm; and, third, FVIIai recycles back to cell surface where it may be available for binding other TF molecules. Another possible explanation for the prolonged antithrombotic effects of FVIIai is that this compound, interfering with the coagulation pathway at an early step, inhibits thrombin generation, possibly resulting in a reduction of thrombin-related TF up-regulation in the cells of the arterial wall [21Bartha K. Brisson C. Archipoff G. De La Salle C. Lanza F. Cazenave J.P. Beretz A. Thrombin regulates tissue factor and thrombomodulin mRNA levels and activities in human saphenous vein endothelial cells by distinct mechanisms.J Biol Chem. 1993; 268: 421-9Abstract Full Text PDF PubMed Google Scholar]. In contrast, hirudin, interfering only with thrombin activity rather than its formation, might not be able to fully inhibit this phenomenon. Thus, thrombin-mediated induction of TF might be partially responsible for the early return of CFVs in hirudin-treated animals. It should be emphasized, however, that the data of our study do not allow us to support this hypothesis, which remains entirely speculative. Taken together, the findings of the present study indicate that FVIIai might be particularly attractive for clinical use. Studies about the pathophysiological mechanisms involved in the formation of intravascular thrombi have shown that thrombin is a key enzyme of the coagulation cascade involved in this phenomenon. For this reason, in addition to heparin, new direct thrombin inhibitors have been recently identified, including hirudin and hirulog [22Lefkovits J. Topol E.J. Direct thrombin inhibitors in cardiovascular medicine.Circulation. 1994; 90: 1522-36Crossref PubMed Google Scholar, 23Topol E.J. Bonan R. Jewitt D. Sigwart U. Kakkar V.V. Rothman M. De Bono D. Ferguson J. Willerson J.T. Strony J. Ganz P. Cohen M.D. Raymond R. Fox I. Maraganore J. Adelman B. Use of a direct antithrombin, hirulog, in place of heparin during coronary angioplasty.Circulation. 1993; 87: 1622-9Crossref PubMed Google Scholar]. These new thrombin inhibitors have the advantage over heparin in that they are antithrombin III-independent, inactivate clot-bound thrombin, and prevent thrombin-induced platelet aggregation [22Lefkovits J. Topol E.J. Direct thrombin inhibitors in cardiovascular medicine.Circulation. 1994; 90: 1522-36Crossref PubMed Google Scholar]. However, a potential limitation of these agents is that new formation of thrombin is not affected. This may cause persistence of thrombin activity despite the presence of an inhibitor [22Lefkovits J. Topol E.J. Direct thrombin inhibitors in cardiovascular medicine.Circulation. 1994; 90: 1522-36Crossref PubMed Google Scholar, 23Topol E.J. Bonan R. Jewitt D. Sigwart U. Kakkar V.V. Rothman M. De Bono D. Ferguson J. Willerson J.T. Strony J. Ganz P. Cohen M.D. Raymond R. Fox I. Maraganore J. Adelman B. Use of a direct antithrombin, hirulog, in place of heparin during coronary angioplasty.Circulation. 1993; 87: 1622-9Crossref PubMed Google Scholar]. In addition, these compounds may carry an increased risk of bleeding when administered in conjunction with thrombolytic therapy, which represents the most concerning adverse effect [23Topol E.J. Bonan R. Jewitt D. Sigwart U. Kakkar V.V. Rothman M. De Bono D. Ferguson J. Willerson J.T. Strony J. Ganz P. Cohen M.D. Raymond R. Fox I. Maraganore J. Adelman B. Use of a direct antithrombin, hirulog, in place of heparin during coronary angioplasty.Circulation. 1993; 87: 1622-9Crossref PubMed Google Scholar]. Inhibition of the extrinsic coagulation pathway at an early step by FVIIai offers two advantages as compared to compounds that block later steps in the coagulation pathway, such as hirudin. First, FVIIai interrupts thrombus formation and prevents the activation of the extrinsic coagulation cascade, by inhibiting thrombin formation. Second, this substance binds to TF only where arterial damage is present. As a consequence, FVIIai does not exert significant effects on blood coagulation and platelet aggregation. This should ultimately translate into a lower risk of bleeding compared with other antithrombotic interventions. Moreover, the observation that the antithrombotic effects of FVIIai can be promptly reversed by administration of recombinant human FVIIa [8Golino P. Ragni M. Cirillo P. D'Andrea D. Scognamiglio A. Ravera A. Buono C. Ezban M. Corcione N. Vigorito F. Condorelli M. Chiariello M. Antithrombotic effects of recombinant human, active site-blocked Factor VIIa in a rabbit model of recurrent arterial thrombosis.Circ Res. 1998; 82: 39-46Crossref PubMed Google Scholar], underline that this drug could have a safe profile for clinical use, as its effects could be rapidly reverted in case of bleeding complications. Finally, data of the present work show that another potential advantage of FVIIai is represented by its persistence in the damaged vascular wall, ultimately resulting in long-lasting antithrombotic effects, such that significant antithrombotic activity can be achieved even when the compound is completely cleared from the circulation. In conclusion, the administration of a single dose of human recombinant FVIIai completely inhibited thrombus formation in this rabbit model of recurrent carotid artery thrombosis without affecting coagulation parameters. These antithrombotic effects were prolonged for 24 h in the majority of the treated animals, and persisted in spite of FVIIai plasma levels that were almost undetectable. Thus, the administration of FVIIai seems to be an effective antithrombotic strategy in vivo with fewer side effects (i.e. risk of bleeding) as compared with other antithrombotic interventions, such as hirudin. Additional studies are needed to clarify the mechanism(s) responsible for the prolonged antithrombotic effects of FVIIai and its potential usefulness in human disease. Drs Plinio Cirillo, Paolo Golino and Massimo Ragni contributed to the conception, design and interpretation of data, and drafting of the manuscript; Drs D'Andrea, Calabrò, Corcione, Vigorito and Ravera, contributed to the interpretation of data and drafting of the manuscript; Dr Chiariello contributed to the drafting of the manuscript and gave final approval of the manuscript.
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