Ixolaris: a Factor Xa heparin-binding exosite inhibitor

Proteins and chemicals

Recombinant Ixolaris was produced in High Five cells (Invitrogen, San Diego, CA, U.S.A.), purified and quantified as described previously [24]. Recombinant GDFXa (Gla-domainless FXa, i.e. FXa from which N-terminal residues 1–45 have been removed) forms (wild type and mutants R93A, K96A, R125A, R165A, K169A, K236A and R240A) were expressed and purified as described elsewhere [14]. AT was a gift from Dr Paulo A. Mourão (Federal University of Rio de Janeiro General Hospital-HUCFF/UFRJ). Human prothrombin and thrombin were purified from frozen human plasma following previously described procedures [25,26]. Protein concentration was estimated using the molar absorption coefficients (A^1%_1 cm,280) of human prothrombin (1.47) and human thrombin (1.83). S-2238 (H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline dihydrochloride) and S-2765 (N-α-benzyloxycarbonyl-D-Arg-Gly-Arg-p-nitroanilide) were from Chromogenix (Mölndal, Sweden). Human FVa and human FXa were purchased from Haematologic Technologies (Essex Junction, VT, U.S.A.). Pentasaccharide (Fondaparinux sodium) was obtained from Organon Sanofi-Synthelabo LLC (West Orange, NJ, U.S.A.).

PC (L-α-phosphatidylcholine) and PS (L-α-phosphatidylserine) were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Phospholipid vesicles composed of 75% (w/w) PC and 25% (w/w) PS were prepared by sonication. Briefly, phospholipids in chloroform were dried with a stream of N₂, resuspended in 50 mM Tris/HCl, 150 mM NaCl, pH 7.5, and sonicated for 2 min. The solution was adjusted further to 500 μM (final concentration).

Gel filtration

Gel filtration chromatography was performed using a Superdex G-75 HR column (Pharmacia, Uppsala, Sweden) attached to a Shimadzu HPLC system. The column was equilibrated with 50 mM Tris/HCl, 150 mM NaCl, pH 7.5, at a flow rate of 0.5 ml/min. [F]-Ixolaris (Ixolaris labelled at the N-terminus by fluorescein) was prepared by incubating the protein with fluorescein succinimidyl ester (PanVera, Madison, WI, U.S.A.), according to the manufacturer's instructions. Samples containing [F]-Ixolaris or [F]-Ixolaris–FXa were preincubated at room temperature for 20 min prior to 20 μl injections. Elution was followed using two detectors: one for absorbance at 280 nm and another for fluorescein fluorescence (excitation at 490 nm, emission at 510 nm).

Amidolytic activity of FXa

Amidolytic activity was measured by hydrolysis of the synthetic substrate S-2765 in 50 mM Tris/HCl, 150 mM NaCl, 10 mM CaCl₂ and 0.1% PEG [poly(ethylene glycol)] 6000, pH 7.5. FXa (1 nM final concentration) or GDFXa forms (0.3 nM final concentration) were incubated with various Ixolaris concentrations for 15 min at 37 °C, and the reaction was initiated by the addition of S-2765 (200 μM final concentration). Substrate hydrolysis was detected using a Thermomax Microplate Reader (Molecular Devices, Menlo Park, CA, U.S.A.) equipped with a microplate mixer and heating system as described [27]. Reactions were recorded continuously at 405 nm for 10 min at 37 °C. The total volume of the reactions was 100 μl. The apparent dissociation constants (K_d) were determined from the saturable Ixolaris-dependent increase in the amidolytic activities of FXa derivatives using the quadratic eqn (1) as described [28]:

(1)

where K_d is the apparent dissociation constant for the enzyme–ligand (Ixolaris) complex, V_s is the steady-state velocity in the presence of Ixolaris, V_o is the control velocity, L is the total Ixolaris concentration, and E is the total FXa concentration.

Binding to heparin–Sepharose

FXa (7.5 μg) was incubated for 2 min in the absence or presence of Ixolaris (3 μg) in 20 mM Tris/HCl (pH 7.5) buffer containing 5 mM CaCl₂ and 0.1% PEG 6000, and then applied on a 1 ml HiTrap heparin–Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ, U.S.A.) FPLC column pre-equilibrated with the same buffer. The column was washed with 5 ml of this buffer followed by elution with a 30 ml gradient of 0–1.0 M NaCl. Fractions (1 ml) were collected and their activity towards S-2765 was determined.

Inactivation by AT

The ability of Ixolaris to alter the inhibitory effect of the AT–heparin complex on FXa or α-thrombin, as well as that of the AT–pentasaccharide complex on FXa, was evaluated as follows. Various amounts of unfractionated heparin or pentasaccharide in 50 mM Tris/HCl, 150 mM NaCl, 10 mM CaCl₂ and 0.1% PEG 6000, pH 7.5, were incubated for 10 min at 37 °C with AT (20 nM) plus FXa (0.5 nM) or α-thrombin (0.5 nM) in the presence or absence of Ixolaris. Residual FXa or thrombin activities were determined by the addition of S-2765 or S-2238 (200 μM) respectively, and substrate hydrolysis was detected using a Thermomax Microplate Reader. Reactions were recorded continuously at 405 nm for 10 min at 37 °C. The total volume of the reactions was 100 μl.

Prothrombin activation by FXa

Activation of prothrombin to thrombin by FXa was performed in 50 mM Tris/HCl, 150 mM NaCl, 10 mM CaCl₂ and 0.1% PEG 6000, pH 7.5, using a discontinuous assay as described [29]. FXa (10 nM final concentration) was incubated with different Ixolaris concentrations for 15 min at 37 °C. The reaction was started by the addition of human prothrombin (1 μM final concentration), and aliquots of 25 μl were removed every 10 min into microplate wells containing 25 μl of 50 mM Tris/HCl, 150 mM NaCl, 50 mM EDTA and 0.1% PEG 6000, pH 7.5. After addition of 50 μl of 200 μM S-2238, absorbance at 405 nm was recorded at 37 °C for 10 min at 6 s intervals using a Thermomax Microplate Reader (Molecular Devices). Velocities (milli-absorbance units/min) obtained in the first few minutes of reaction were used to calculate the amount of thrombin formed using a standard curve.

Experiments in the presence of FVa were performed as follows. FXa (1 nM final concentration) was incubated in the presence of FVa (50 nM final concentration) and different Ixolaris concentrations for 15 min at 37 °C. The reaction was started by addition of human prothrombin (1 μM final concentration), and aliquots of 10 μl were removed every 1 min into microplate wells containing 40 μl of 50 mM Tris/HCl, 150 mM NaCl, 50 mM EDTA and 0.1% PEG 6000, pH 7.5. The amount of thrombin formed was determined as described above.

Experiments in the presence of phospholipids were conducted as follows. FXa (10 pM final concentration) was incubated with different Ixolaris concentrations in the presence of FVa (1 nM final concentration) and PC/PS vesicles (10 μM) for 15 min at 37 °C. The reaction was started by the addition of human prothrombin (1 μM final concentration). Then aliquots of 5 μl were removed every 20 s into microplate wells containing 45 μl of 50 mM Tris/HCl, 150 mM NaCl, 50 mM EDTA and 0.1% PEG 6000, pH 7.5. The amount of thrombin formed was determined as described above.

Prothrombin activation as monitored by SDS/PAGE

Activation of purified human prothrombin by the prothrombinase complex was monitored by SDS/PAGE as follows. Assay medium (50 mM Tris/HCl, 100 mM NaCl, 10 mM CaCl₂, 0.1% PEG 6000, pH 7.5) containing FXa (1 nM final concentration), FVa (3 nM final concentration) and 30 μM PC/PS vesicles was incubated in the absence (control) or presence of Ixolaris (40 nM final concentration) for 15 min at room temperature. The reaction was started by the addition of prothrombin (1.4 μM final concentration), giving a total volume of 500 μl. To reduce the formation of cleavage products by thrombin, reactions were carried out in the presence of 6 μM DAPA [dansylarginine N-(3-ethyl-1,5-pentanediyl) amide]. Samples (50 μl) were taken at various times, the reaction was stopped with 15 mM EDTA and 5 μM D-Phe-Pro-Arg-chloromethyl ketone (20 μl), and the samples were subjected to SDS/PAGE (12% acrylamide) under non-reducing conditions and gels were stained with Coomassie Blue.

Complex formation between Ixolaris and FXa

Binding of Ixolaris to human FXa was examined first by Superdex G-75 gel filtration, using [F]-Ixolaris to monitor elution. In a typical experiment, the retention time of [F]-Ixolaris was ∼24.2 min (15.5 kDa) (Figure 1A, solid line), and that of FXa was ∼21.7 min (46 kDa) (results not shown). When Ixolaris and FXa were incubated in stoichiometric proportions and loaded into the column, a peak at ∼19.4 min was obtained (Figure 1A, broken line). These results were consistent with formation of a 1:1 complex of ∼60 kDa between [F]-Ixolaris (15.5 kDa) and FXa (46 kDa). Ixolaris behaves as a tight-and-fast-binding ligand of FXa without inhibiting the proteinase catalytic site [24]. On the other hand, Figure 1(B) shows that Ixolaris bound FXa tightly with an apparent K_d of 520±18 pM that led to an increase in chromogenic substrate hydrolysis.

Ixolaris interacts with the HBE of FXa

Kinetic data strongly suggest that full-length heparin may effectively support the interaction between FXa and AT through a template mechanism [30]. This phenomenon occurs at physiological levels of calcium and is modulated by specific residues that form the FXa HBE [14]. Therefore we tested the ability of Ixolaris to interfere with inactivation of FXa by the AT–heparin complex. Figure 2(A) shows that increasing the concentration of heparin enhanced the inhibitory effect of AT on FXa, with complete inhibition observed above 1 μg/ml heparin; however, in the presence of Ixolaris, inhibition of FXa by the AT–heparin complex was only 20% at 1 μg/ml and 40% at 100 μg/ml heparin. Accordingly, Ixolaris decreased the inactivation of FXa by the AT–heparin complex (1 μg/ml) in a dose-dependent fashion, suggesting that Ixolaris and heparin compete for the same binding site on FXa (Figure 2B). Figure 2(C) shows that the effect of the inhibitor is specific for FXa, as thrombin inhibition by AT–heparin occurred to the same extent regardless of the presence of Ixolaris. Unlike AT–heparin, the mechanism of inhibition of FXa by the AT–pentasaccharide complex is independent of FXa HBE [31]. Thus, as expected, the presence of Ixolaris had only a minor effect on the pentasaccharide-accelerated reaction of AT with FXa (Figure 2D).

Finally, the ability of Ixolaris to compete with heparin for binding to FXa was documented further by affinity chromatography using a heparin–Sepharose column. FXa showed a high affinity for the resin and could be recovered by a linear gradient of NaCl, with a major peak eluted at ∼0.4 M NaCl (Figure 3, solid line). In contrast, FXa incubated with a 1.2-fold molar excess of Ixolaris was mostly eluted before the NaCl gradient started, indicating that interaction of the enzyme with heparin was markedly impaired (Figure 3, broken line).

Ixolaris interacts with basic residues that comprise the FXa HBE

Studies using site-directed mutagenesis showed that seven basic residues in the heavy chain of FXa constitute the HBE of the enzyme, known to bind to heparin [14,30]. We used GDFXa mutants and chromogenic substrate hydrolysis to estimate the relative importance of the residues of the HBE for the Ixolaris–FXa interaction. Analysis of these results indicated a similar, although apparently tighter, dissociation constant for wild-type GDFXa (K_d 78±15 pM) compared with native FXa (K_d 520±18 pM) (Figure 1B), suggesting that the Gla domain may somehow modulate the Ixolaris–FXa interaction. Table 1 shows the relative affinities obtained for the interaction between the various GDFXa mutants and Ixolaris, as compared with wild-type GDFXa. All mutations interfered with complex formation, with relative affinities ranging from 0.07 to 0.21. Most strikingly, however, was the observation that mutation of residue Arg-93 abolished the interaction of GDFXa with Ixolaris (Table 1).

Effect of Ixolaris on prothrombin activation

Mutagenesis experiments have identified several basic residues in the FXa HBE that are critical for substrate recognition by the prothrombinase complex [14]. Therefore we initially attempted to determine whether Ixolaris affects the ability of FXa to convert prothrombin into thrombin in the absence of phospholipids and cofactors [32]. Figure 4(A) shows that premixing of Ixolaris with FXa (10 nM) produced a concentration-dependent inhibition of prothrombin activation by the enzyme. The inhibitory effect of Ixolaris was then tested and confirmed when zymogen activation was promoted by FXa (1 nM) in the presence of FVa (50 nM) (Figure 4B). Figure 4(C) shows that Ixolaris was also effective in preventing prothrombin activation by fully assembled prothrombinase complex, i.e. FXa (10 pM) in the presence of FVa (1 nM), phospholipids (30 μM) and Ca²⁺ (see Discussion).

The effects of Ixolaris were documented further by monitoring prothrombin activation using SDS/PAGE, in the presence of PC/PS vesicles, cofactor and Ca²⁺. As shown in Figure 4(D) (upper panel), conversion of prothrombin into thrombin was almost complete after 20 min of zymogen incubation with the prothrombinase complex. On the other hand, incubation of FXa with Ixolaris prior to the addition of prothrombin significantly delayed the activation process, as shown by weaker bands corresponding to thrombin (Figure 4D, lower panel) as compared with the control (upper panel).

Figure 5 shows a working hypothesis for the mechanism of inhibition of the prothrombinase complex by Ixolaris (See Discussion and Figure legends).