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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Thromb Haemost. Author manuscript; available in PMC 2010 July 9.
Published in final edited form as:
PMCID: PMC2900789



Ixolaris is a two-Kunitz tick salivary gland protein identified in Ixodes scapularis that presents extensive sequence homology to TFPI. It binds to FXa or FX as scaffolds and inhibits extrinsic Xnase. Differently from TFPI, however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite, which may also results in decreased FXa activity into the prothrombinase complex. In this report, we show that recombinant 125I-Ixolaris interacts with rat and human FX in plasma and prolongs the prothrombin time (PT) and activated partial thromboplastin time (aPTT) in vitro. We have also investigated the effects of Ixolaris in vivo, using a venous thrombosis model. Subcutaneous (s.c.) or intravenous (i.v.) administration of Ixolaris in rats causes a dose-dependent reduction in thrombus formation, with complete inhibition attained at 20 μg/kg and 10 μg/kg, respectively. Remarkably, antithrombotic effects were observed 3 h after s.c. administration of Ixolaris and lasted for 24 h thereafter. Ex vivo experiments also showed that Ixolaris (up to 100 μg/kg) did not affect the aPTT, while the PT was increased by ~0.4-fold at the highest Ixolaris concentration. Remarkably, effective antithrombotic doses of Ixolaris (20 μg/Kg) was not associated with bleeding which was significant only at higher doses of the anticoagulant (40 μg/Kg). Our experiments demonstrate that Ixolaris is an effective and possibly safe antithrombotic agent in vivo.

Keywords: Anticoagulant, Ixolaris, venous thrombosis, factor Xa, tissue factor, tick saliva, Ixodes scapularis


Following tissue injury, exposition of membrane-bound tissue factor (TF) is a crucial step in the initiation of blood coagulation. TF binds to blood coagulation factor VIIa (FVIIa), and the binary FVIIa/TF complex then generates FIXa and FXa (13). Generation of FIXa by the FVIIa/TF complex results in formation of the Xnase complex following binding to the nonenzymatic co-factor, activated factor VIII (FVIIIa). The Xnase complex, along with FVIIa/TF, converts FX to activated factor X (FXa), which assembles with activated factor V (FVa) into the prothrombinase complex that is directly responsible for the formation of thrombin (13).

Once generated, and depending on the particular vascular bed and rheologic environment, thrombin will convert fibrinogen to fibrin monomers, activate platelets, and eventually lead to thrombus development. Under pathologic conditions, clot formation will present clinically as deep vein thrombosis, pulmonary embolism, acute coronary syndrome, cerebrovascular events, peripheral arterial thrombosis, and even disseminated intravascular coagulation. Therefore, efforts to develop potent and specific antithrombotic agents are still a priority in cardiovascular medicine (4, 5).

In addition to physiologic blood coagulation inhibitors tissue factor pathway inhibitor (TFPI), antithrombin, and heparin cofactor II (6, 7), a number of specific inhibitors from exogenous sources have been identified from the salivary gland of blood-sucking arthropods (8, 9). We have recently characterized Ixolaris, a two Kunitz-like TFPI from the tick Ixodes scapularis (10). Ixolaris binds to FXa or FX as scaffolds and inhibits extrinsic Xnase. Differently from TFPI, however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite (11).

In the present study, we evaluated the in vivo effects of Ixolaris on thrombus formation using a thrombosis model in rats. After intravenous (i.v.) or subcutaneous (s.c.) administration, Ixolaris showed effective and long-lasting antithrombotic activity that was not associated with hemorrhage or bleeding. Therefore, Ixolaris may be used as a prototype to develop new anticoagulants targeting the extrinsic pathway.

Material and methods


Human FX was purchased from Hæmatologic Technologies (Essex Junction, VT). Activated partial thromboplastin time (aPTT) (cephalin plus kaolin) and prothrombin time (PT) (thromboplastin with calcium) reagents were from BioMériaux (Rio de Janeiro, Brazil). Anasedan (Xylazin) and Dopalen (Ketamin) were from Agribrands (Rio de Janeiro, Brazil). NU-PAGE, MES buffer, and molecular weight markers were obtained from Invitrogen (Carlsbad, CA).


Adult Wistar rats (both sexes) weighing 200–250 g were housed under controlled conditions of temperature (24 ± 1°C) and light (12 h light starting at 07:00 h), and all experiments were conducted in accordance with standards of animal care defined by the Institutional Committee (Institute of Medical Biochemistry, Federal University of Rio de Janeiro).

Ixolaris expression and purification

Recombinant Ixolaris was expressed in High Five cells, using Baculovirus expression system (Invitrogen, San Diego, CA). Ixolaris was purified through affinity chromatography on a FX-Sepharose column (Pharmacia, Uppsala, Sweden) followed by a Macrosphere octadecylsilica column (Altech, Deerfield, IL). One single band was observed in 4% to 12% NU-PAGE gel with MES buffer.

Iodination of Ixolaris

125I-labeled Ixolaris (125I-Ixolaris) was prepared by iodination with [125I] sodium iodide (CNEN, São Paulo, Brazil) in 200 μCi/mg of protein, using Iodogen (Sigma Chemical, St. Louis, MO) (100 μg/mg of protein) following the manufacturer’s instructions. Free iodide was removed by extensive dialysis against Tris-buffered saline, pH 7.4.

In vitro effect of Ixolaris on aPTT and PT

The in vitro effect of Ixolaris on coagulation tests aPTT and PT was evaluated on an Amelung KC4A coagulometer (Labcon, Heppenheim, Germany). Human blood samples were collected from healthy donors in 3.8% trisodium citrate (9:1, v/v), and platelet-poor plasma were obtained by further centrifugation at 2000×g for 10 min. Rat plasma samples were obtained following the same procedure. Plasma (50 μl) was incubated with Ixolaris (10 μl) for 2 min at 37°C, followed by addition of aPTT reagent (50 μl, 1 min) and then 25 mM CaCl2 (100 μl) or PT reagent (100 μl). Time for clot formation was then recorded.

Polyacrylamide gel electrophoresis

Electrophoresis was performed in polyacrylamide gels (PAGE) under non-denaturing conditions. 125I-Ixolaris (4.0 × 104 cpm in 320 ng) and purified FX (4.5 μg) or human or rodent plasma were incubated at room temperature for 15 min and loaded onto a PAGE (10% acrylamide). Gels were further exposed to phosphorimaging (Storm, Amersham Biosciences, Uppsala, Sweden).

Stasis-induced thrombosis after injection of tissue thromboplastin

Thrombus formation by a combination of stasis and hypercoagulability was induced as described by Vogel et al. (12) with slight modifications (13). Wistar rats were anesthetized with xylazin (16 mg/kg, intramuscularly) followed by ketamin (100 mg/kg, intramuscularly). The abdomen was surgically opened, and after careful dissection the vena cava was exposed and dissected free from surrounding tissues. Two loose ligatures were prepared 1 cm apart on the inferior vena cava just below the left renal vein. Ixolaris at the indicated doses was administered i.v. (below the distal loose suture) or s.c. at different times before thrombosis induction. Tissue thromboplastin (3 mg/kg body weight) was injected into the vena cava, and stasis was immediately established by tightening the proximal suture. Tightening of the distal suture was performed 20 min after administration of thromboplastin, and the ligated segment was removed. The formed thrombus was removed from the segment, rinsed, blotted on filter paper, dried for 1 h at 60°C, and weighed. The protocol received official approval with regard to the care and use of laboratory animals.

Determination of radioactivity of 125I-Ixolaris in rat blood

The in vivo distribution of 125I-Ixolaris in rat blood was evaluated after s.c. administration. Samples containing ~5.5 × 106 cpm (4 μg Ixolaris) were resuspended in 200 μl of phosphate-buffered saline (PBS) and administered by s.c. route. After 3, 20, 40, or 60 h, blood was collected by cardiac puncture in EDTA (5 mM, final concentration). Platelet-poor plasma was obtained by centrifugation, and aliquots of 500 μl were placed in glass test tubes. Radioactivity was determined in a gamma counter (LKB, Wallac, Finland).

Ex vivo effect of Ixolaris on aPTT and PT

The ex vivo effect of Ixolaris on aPTT and PT coagulation tests was evaluated on an Amelung KC4A coagulometer (Labcon). Ixolaris was administered s.c. to rats and after 24-h blood was collected by cardiac puncture in 3.8% trisodium citrate in (9:1, v/v). Platelet-poor plasma was obtained by centrifugation at 2000×g for 10 min. Plasma (50 μl) was incubated for 1 min at 37°C followed by addition of aPTT reagent (50 μl, 1 min) and then 25 μM CaCl2 (100 μl) or PT reagent (100 μl). Time for clot formation was then recorded.

Bleeding effect

A rat tail-transection model was used to evaluate the effect of Ixolaris on bleeding time. Ixolaris at appropriate doses was administered s.c. and animals were anesthetized after 3 or 24 h, as described above. The rat tail was cut 3 mm from the tip and carefully immersed in 40 ml of distilled water at room temperature. The hemoglobin content in water solution (absorbance at 540 nm) was used as an estimated of blood loss (14). Appropriate controls (s.c injection of PBS) were run in parallel.


All data presented represent mean ± SD. Differences in mean values were analyzed using Student’s t-test. P-values <0.05 were considered to be statistically significant.


The in vitro anticoagulant effect of Ixolaris on human plasma is shown in Figure 1A. Ixolaris caused a dose-dependent increase in both PT and aPTT; however, Ixolaris showed a more pronounced effect toward the extrinsic pathway. This was not surprising since previous observations employing purified proteins showed that Ixolaris is potent inhibitor of the extrinsic Xnase complex (10). We further evaluated the anticoagulant properties of Ixolaris on rat plasma. Figure 1B shows a similar pattern of inhibition towards PT and aPTT. However, Ixolaris concentrations above 0.75 μg/ml seems to produce a more pronounced effect in both clotting tests with a more remarkable increase in the PT.

Figure 1
In vitro anticoagulant activity of Ixolaris

Ixolaris forms a complex with FX, as demonstrated by non-denaturing PAGE (10). In an attempt to detect Ixolaris binding to FX in plasma, we used 125I-Ixolaris followed by identification of complex formation using non-denaturing PAGE. Figure 2, lane 1, shows the migration patterns obtained for 125I-Ixolaris; and lane 2 shows 125I-Ixolaris bound to purified FX, which displays a slower migration pattern. Figure 2 also shows that incubation of 125I-Ixolaris with human (lane 3) or rat (lane 4) plasma produces a band with a similar migration pattern observed for 125I-Ixolaris and purified FX. We have also tried to identify the Ixolaris-FX complex in plasma using monoclonal antibodies against the zymogen. This was not possible due to the highly heterogenous migration pattern exhibited by FX, under non-denaturing conditions, either in the absence or in the presence of Ixolaris.

Figure 2
Detection of FX-125I-Ixolaris complexes by PAGE

To determine whether Ixolaris exerts antithrombotic action in vivo, we used a thrombosis model in rats that combines stasis and hypercoagulability (see Materials and methods). The control group that received tissue thromboplastin (3 mg/kg) showed 100% of thrombus formation, with a mean thrombus weight of 5.1 ± 0.26 mg (n = 4). In contrast, i.v. administration of Ixolaris produced a progressive decrease on thrombus formation, with a maximum effect observed at 10 μg/kg (Figure 3).

Figure 3
Effect of Ixolaris on stasis-induced venous thrombosis in rats

Next, the antithrombotic effect of Ixolaris was tested after s.c. administration. Figure 4A shows that Ixolaris was highly effective in preventing venous thrombosis by the s.c route, with complete inhibition attained with 20 μg/kg Ixolaris. We also evaluated the time dependence of antithrombotic activity of Ixolaris. Figure 4B shows that Ixolaris (20 μg/kg) completely abolished thrombus formation after 3 h of s.c. administration. The effect persisted for 24 h and was progressively reversed thereafter. To confirm the prolonged half-life of Ixolaris in vivo, a semiquantitative estimate of Ixolaris pharmacokinetics was obtained using 125I-Ixolaris. Figure 5 shows that 125I-Ixolaris concentration in plasma reached a peak after 3 h of s.c. administration and was 40% of the maximum value 20 h after administration of the anticoagulant.

Figure 4
Effect of Ixolaris on venous thrombosis in rats after s.c. administration
Figure 5
Determination of blood distribution of 125I-Ixolaris after s.c. administration

The effects of Ixolaris on ex vivo clotting assays were then tested. Figures 6A shows that aPTT was not affected by 40 or 100 μg/kg Ixolaris. In contrast, PT values were statistically higher in relation to control for Ixolaris doses higher than 20 μg/kg, showing a ~0.4-fold increase at 100 μg/kg.

Figure 6
Ex vivo anticoagulant activity of Ixolaris

Finally, the bleeding effect of Ixolaris was evaluated using a tail-transection model (Fig. 7); no significant bleeding was observed 3 or 24 h after Ixolaris administration (20 μg/kg). On the other hand, a statistically significant blood loss was observed 24 h after administration of 40 μg/kg Ixolaris (see Discussion).

Figure 7
Determination of the bleeding effect of Ixolaris


Ixolaris is a two-Kunitz salivary gland protein that has been previously characterized as a potent inhibitor of the extrinsic Xnase (FVIIa-TF) complex (10). However, although Ixolaris presents extensive primary sequence homology with TFPI, it does not bind to the FXa catalytic site. On the contrary, complex formation is mediated by a number of surface-charged residues that constitute the FXa heparin-binding exosite (11). Another striking difference between Ixolaris and TFPI is the ability of the former to form a tight complex with zymogen FX. In fact, interaction of Ixolaris with FXa or FX is a prerequisite for inhibition of the extrinsic Xnase complex (10).

In the present study, we demonstrated that Ixolaris displays potent anticoagulant and antithrombotic activities. In vitro assays performed with human plasma demonstrated a robust prolongation of PT and, to a lesser extent, of aPTT. The prominent effect toward the extrinsic pathway (PT) clearly indicates the potent and preferential inhibitory activity toward the FVIIa-TF complex. The finding that Ixolaris at higher concentrations also affects the intrinsic pathway (aPTT) suggests that the inhibitor interferes with protrombin conversion by FXa. In fact, we have recently demonstrated that Ixolaris is a specific ligand of the FXa heparin-binding exosite and impairs the productive assembly of the prothrombinase complex (11). These findings are corroborated by site-directed mutagenesis studies demonstrating that the FXa heparin-binding exosite contains a number of specific residues implicated in zymogen recognition (15, 16).

As observed with purified FX, Ixolaris forms a stable, noncovalent complex with zymogen either in human or rat plasma. These observations lead us to test the inhibitor using an in vivo model of venous thrombosis. Ixolaris showed a potent antithrombotic activity following i.v. or s.c. administration in rats. Remarkably, Ixolaris displays very high potency (IC50 = 7.5–15 μg/kg) compared with that of other antithrombotic molecules tested in this same animal model. A number of sulfated polysaccharides including unfractionated heparin that potentialize either antithrombin or heparin cofactor II present antithrombotic activity in the range of 250–3000 μg/kg (17, 18). In addition, a number of direct thrombin inhibitors, including argatroban and hirudin variants, inhibit venous thrombosis with IC50 ranging from 170 to 250 μg/kg and with half-lives below 60 min (19). It

Analysis of the time-dependence pattern of the antithrombotic action of Ixolaris (Figure 4) as well as the disappearance of 125I-Ixolaris radioactivity in rat plasma (Figure 5) indicates a prolonged half-life. This is not surprising since Ixolaris is a tight ligand of FX which plasma half life is about 34–40 hours (20). Notably, no significant bleeding was observed 3h or 24 h after Ixolaris administration at concentrations that effectively blocked thrombus formation (20 μg/kg). In addition, blood loss was not observed 3 h after administration of Ixolaris at 40 μg/kg but it was significant at 24 h. The slower distribution of Ixolaris after S.C. administration at higher doses as compared to the distribution observed with 4 μg/kg 125I-Ixolaris (Fig. 5) appears to accounts for the bleeding effects detected at later time points (Fig. 7).

This tight binding of Ixolaris to FX may represent an advantage when compared with other small molecular weight anticoagulants that display a shorter half life (5). A similar pharmacokinetic property has been observed in humans after administration of NAPc2, a hookworm anticoagulant that targets the extrinsic pathway and that has recently undergone clinical trials (21). It is important to recognize that administration of anticoagulants targeting FVIIa/TF such as Ixolaris, NAPc2, and TFPI appears to be effective in vivo and, notably, is most often not associated with major manifestations of bleeding (5, 6, 21, 22).

In conclusion, Ixolaris displays potent antithrombotic effect in vivo, with prolonged half life and minor significant bleeding. Therefore, Ixolaris appears to be a promising anticoagulant for the prevention and treatment of venous or arterial thrombosis and/or in pathologic conditions with abnormal expression of TF (2325).


We thank NIAID intramural editor Brenda Rae Marshall for assistance. This research was supported in part by a grant from “Programa Interinstitucional de Ensino, Pesquisa e Extensao em Biologia do Cancer” by Fundacao Ary Frauzino para Pesquisa e Controle do Câncer (FAF) and Fundação Educacional Charles Darwin (FECD). Additional support was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro Carlos Chagas Filho (FAPERJ), Fundação Universitária José Bonifácio and from the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA.


activated partial thromboplastin time
factor X
factor Xa
factor VIIa
Electrophoresis was performed in polyacrylamide gel
phosphate-buffered saline
prothrombin time
tissue factor
tissue factor pathway inhibitor


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