©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cleavage at Arginine 145 in Human Blood Coagulation Factor IX Converts the Zymogen into a Factor VIII Binding Enzyme (*)

Peter J. Lenting , Hans ter Maat , Patrick P. F. M. Clijsters , Marie-José S. H. Donath , Jan A. van Mourik , Koen Mertens (§)

From the (1)Department of Blood Coagulation, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, 1066 CX Amsterdam, the Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The transition of the factor IX zymogen into the enzyme factor IXa was investigated. For this purpose, the activation intermediate factors IX and IXa were purified after cleavage of the Arg-Ala and Arg-Val bonds, respectively. These intermediates were compared for a number of functional properties with factor IXa, which is cleaved at both positions. Factor IXa was equal to factor IXa in hydrolyzing the synthetic substrate CHSO-Leu-Gly-Arg-p-nitroanilide (k/K 120 sM) but was less efficient in factor X activation. Factor IX was incapable of generating factor Xa but displayed reactivity toward p-nitrophenol p-guanidinobenzoate and the peptide substrate. The catalytic efficiency, however, was 4-fold lower compared with factor IXa and factor IXa. Factor IX and factor IXa had similar affinity for the inhibitor benzamidine (K 2.5 mM), and amidolytic activity of both species was inhibited by Glu-Gly-Arg-chloromethyl ketone and antithrombin III. Unlike factor IXa, factor IX was unable to form SDS stable complexes with antithrombin III. Moreover, inhibition of factor IXa and factor IX by Glu-Gly-Arg-chloromethyl ketone followed distinct pathways, because factor IX was inhibited in a nonirreversible manner and displayed only minor incorporation of the dansylated inhibitor into its catalytic site. These data demonstrate that the catalytic site of factor IX differs from that of the fully activated factor IXa. Factor IX and its derivatives were also compared with regard to complex assembly with factor VIII in direct binding studies employing the immobilized factor VIII light chain. Factor IX and factor IXa displayed a 30-fold higher affinity for the factor VIII light chain (K 12 nM) than the factor IX zymogen. Factor IXa showed lower affinity (K 50 nM) than factor IX and factor IXa, which may explain the lower efficiency of factor X activation by factor IXa. Collectively, our data indicate that cleavage of the Arg-Val bond develops full amidolytic activity but results in suboptimal binding to the factor VIII light chain. With regard to cleavage of the Arg-Ala bond, we have demonstrated that this results in the transition of the factor IX zymogen into an enzyme that lacks proteolytic activity. Moreover, the same cleavage fully exposes the binding site for the factor VIII light chain, suggesting that cleavage of the Arg-Ala bond serves a previously unrecognized role in the assembly of the factor IX-factor VIII complex.


INTRODUCTION

The blood coagulation pathway comprises a cascade of sequential steps in which proenzymes are converted into active serine proteases (1). The serine protease precursor factor IX (FIX)()circulates in plasma as a single-chain polypeptide (M = 57,000) (2) that comprises a number of discrete domains(3) . At the amino-terminal site of the molecule the so-called ``Gla domain'' is located. This domain contains several glutamic acid residues that have been carboxylated to yield Gla(4) . The presence of Gla residues allows this region to bind metal ions (5) and is essential for surface binding at platelets and endothelial cells(6, 7) . Adjacent to the Gla domain a region is located that shares homology with the epidermal growth factor (EGF) (8). This region consists of two distinct EGF-like domains, which are important for FIX function. The first EGF-like domain contains a single calcium binding site(8) . The second EGF-like domain is connected to the activation peptide, a segment that is liberated during zymogen activation(9) . Finally, the carboxyl-terminal portion comprises the trypsin-like serine protease domain, which contains a single metal ion binding site (10) and the catalytic centre of FIX(11) .

In contrast to most serine proteases, FIX requires two cleavages to yield full enzymatic activity(2, 12, 13, 14, 15) . Dependent on the sequence of cleavage, FIX activation can follow two distinct pathways. Physiological activators first cleave the Arg-Ala bond, resulting in the transient activation intermediate FIX. So far, FIX has been characterized as being enzymatically inactive(16) , which is underscored by the notion that FIX lacks clotting activity(12, 15) . Subsequent cleavage at the amino terminus of the protease domain (i.e. position Arg) then results in the active enzyme FIXa. The nonphysiological activator isolated from Russell's viper venom first cleaves at position Arg(12) . The resulting intermediate FIXa displays proteolytic activity, although its clotting activity is just 20-50% of that of the enzyme FIXa(12, 17) . Subsequent cleavage of the Arg-Ala bond then converts FIXa into the fully active FIXa.

Within the blood coagulation cascade, FIXa activates the zymogen factor X (FX) in a process that requires the presence of phospholipids, calcium ions, and factor VIIIa (FVIIIa)(18, 19) . During FX activation, FIXa is in complex with its protein cofactor FVIIIa(20, 21) . This interaction involves the FVIII light chain, which contains a high affinity binding site for FIXa(22) . Complex formation with FVIIIa results in structural changes within the active site of FIXa(20) . Maximal response requires the presence of the carboxyl-terminal portion of the FVIII heavy chain(20) , indicating that the FVIII heavy chain is involved in complex formation with FIXa as well. Optimal FX activation requires the two-step cleavage of FIX into FIXa, because point mutations at Arg or Arg are both associated with the bleeding disorder hemophilia B(23) . Whereas cleavage of the Arg-Val bond corresponds with the zymogen-activating site that FIX is sharing with many other serine protease precursors(24) , cleavage of the Arg-Ala bond is unique for FIX.

The aim of the present study was to elucidate the role of cleavage of the Arg-Ala bond in the activation of the FIX zymogen. For this purpose FIX was compared with other FIX activation products with regard to a number of parameters that are associated with FIXa enzyme function. These included reactivity toward synthetic and natural substrates and inhibitors and interaction with the cofactor FVIII. This approach allowed us to establish that cleavage at Arg plays a major role in the assembly of the FIX-FVIII complex.


EXPERIMENTAL PROCEDURES

Materials

Protein A-Sepharose CL4B and CNBr-Sepharose CL4B were from Pharmacia LKB Biotechnology AB (Uppsala, Sweden). Microtiter plates (Immulon) were from Dynatech (Plockingen, Germany) unless stated otherwise. Glu-Gly-Arg-chloromethyl ketone (EGR-CK) and dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-CK) were from Calbiochem. CHSO-D-Leu-Gly-Arg-p-nitroanilide (CHSO-LGR-pNA), product name CBS 31.39, was from Diagnostica Stago (Asnières, France). Heparin (grade 1-A) was obtained from Sigma. p-Nitrophenol p-guanidinobenzoate (NPGB) was from BDH Chemicals Ltd. (Poole, United Kingdom).

Antibodies

The anti-FVIII antibodies CLB-CAg 12 and CLB-CAg 69 have been described previously(25, 26) . The murine anti-FIX antibodies CLB-FIX 10 and CLB-FIX 11 were obtained as outlined previously(22) , employing a screening strategy based on binding to immobilized FIX in the presence or absence of calcium ions. Binding of CLB-FIX 10 to FIX was calcium-independent, whereas binding of CLB-FIX 11 was markedly enhanced in the presence of calcium ions. Both antibodies CLB-FIX 10 and CLB-FIX 11 strongly inhibit FIX activity (results not shown). The murine anti-FIX antibody CLB-FIX D4 has been described elsewhere(27) . This antibody is directed against the FIX sequence Asn-Asp, which comprises the Arg-Ala activation site. By virtue of the location of its epitope, antibody CLB-FIX D4 distinguishes between intact FIX and cleaved FIX (i.e. FIX and FIXa)(27) . All FIX antibodies used were from the IgG isotype. Monoclonal antibodies were purified from culture medium employing protein A-Sepharose as recommended by the manufacturer. Polyclonal antibodies against human FIX were obtained as described previously (22). Antibodies were conjugated with horseradish peroxidase as described(28) .

Various Proteins

The human FVIII light chain was purified as described(22) . FX was prepared as described(29) . The factor X-activating protein from Russell's viper venom was purified as described (30) and coupled to CNBr-Sepharose (2 mg/ml) according to the manufacturer's instructions. Purified factor XIa (FXIa) was obtained from Enzyme Research Laboratories. Purified Antithrombin III (ATIII), C-inhibitor, and human serum albumin (HSA) were obtained from the Division of Products of our institute. Bovine serum albumin was from Miles Inc. Purified -antitrypsin and -antiplasmin were gifts from Dr. W. Wuillemin, Department of Autoimmune Diseases of our institute. All proteins used, including FIX and its activation products (see below) were homogeneous as assessed by SDS-polyacrylamide gel electrophoresis (PAGE) (see Figs. 3 and 5).

FIX and FIX Cleavage Products

Human FIX was purified from a concentrate of prothrombin, FIX, and FX (31) obtained from the Division of Products of our institute. NaCl, benzamidine, and sodium citrate (pH 7.4) were added to the concentrate to final concentrations of 0.15 M, 0.01 M, and 0.02 M, respectively, and the mixture was subjected to immunoaffinity chromatography employing the anti-FIX antibody CLB-FIX D4 (5 mg/ml CNBr-Sepharose). After extensive washing with 0.15 M NaCl, 0.01 M benzamidine, 0.02 M sodium citrate (pH 7.4), FIX was eluted in a linear gradient (0-2 M KSCN). FIX-containing fractions were pooled and stored at -20 °C in 0.1 M NaCl, 0.05 M Tris (pH 7.4). The specific activity of the FIX preparations ranged between 300 and 350 units/mg.

FIXa was prepared by incubating purified FIX (4 µM) with human FXIa (0.23 µM) for 2 h at 37 °C in 0.1 M NaCl, 2 mM CaCl, 0.05 M Tris (pH 7.4). After the reaction was terminated by the addition of EDTA (0.01 M final concentration), residual FIX and FIX were removed from the incubation mixture by rechromatography on the CLB-FIX D4 affinity column. In this immunoaffinity step, FIXa and FXIa did not bind to the column, whereas FIX and FIX remained bound (27). Finally, FIXa and FXIa were separated employing anion exchange chromatography as described previously(32) . FIXa was stored at -20 °C in 50% glycerol, 0.1 M NaCl, 0.05 M Tris (pH 7.4). The FIXa preparations were more than 90% active as determined by active site titrations employing NPGB(33) .

FIX was prepared by incubating purified FIX (4 µM) with human FXIa (16 nM) in the presence of 6.8 mM MnCl in 0.1 M NaCl, 0.05 M Tris (pH 7.4). After incubation for 2 h at 37 °C, the reaction was terminated by the addition of EDTA and benzamidine (0.01 M final concentrations). Under these conditions, approximately 90% of FIX was converted into FIX as judged by SDS-PAGE. FIX was then separated from FIX and FXIa employing CLB-FIX D4 affinity chromatography. In this step FXIa and possible traces of FIXa passed through the column, while FIX and residual FIX were bound. After extensive washing, FIX and FIX were eluted separately in a linear gradient (0-3 M KSCN)(27) . The FIX-containing fractions were pooled and stored at -20 °C in 0.1 M NaCl, 0.05 M Tris (pH 7.4). The position of cleavage in FIX was assessed by NH-terminal amino acid sequence analysis employing automated equipment (Applied Biosystems, Warrington, UK; Eurosequence, Groningen, the Netherlands). The resulting sequence, Ala-Glu-Thr-Val-Phe, corresponds with the five NH-terminal amino acids of the FIX activation peptide region, demonstrating that indeed cleavage had occurred at Arg-Ala (11). The FIX preparations were more than 95% active as determined by NPGB titration(33) . FIXa was prepared from purified FIX essentially as described (17) but modified in that the purified FX-activating enzyme from Russell's viper venom instead of the crude snake venom was immobilized on CNBr-Sepharose.

Protein Concentrations

Protein was measured by the method of Bradford using HSA as a standard(34) . FIX activity was measured employing a commercially available chromogenic method (Baxter-DADE, Düdingen, Switzerland). Antigen concentrations of FIX or its derivatives were quantified by an immunological assay as described previously (22) but modified in that immunopurified polyclonal anti-FIX antibodies were immobilized (0.2 µg/well) instead of monoclonal antibody CLB-FIX 2. Dose-response curves were transformed by plotting logit absorbance versus log concentration and were linear between 0.07 and 7 nM. Within this range the coefficient of variation was approximately 5%. Antigen values were converted into molar concentrations using the purified FIX derivatives as standards. Molar concentrations were calculated from protein concentrations employing M = 57,000 for FIX, FIX, and FIXa and M = 45,000 for FIXa (2). In experiments in which FIXa or FIX was used as competitor for the binding of FIX to the FVIII light chain, nonbound FIX was assayed by a FIX-specific assay employing the antibody CLB-FIX D4. Samples containing FIX and FIX or FIXa were incubated with the immobilized antibody (0.5 µg/well) in 3 M NaCl, 0.1% (v/v) Tween-20, 1% (w/v) HSA, 0.05 M Tris (pH 7.2). After washing with 0.15 M NaCl, 0.1% (v/v) Tween-20, 0.05 M Tris (pH 7.2), bound FIX was detected employing peroxidase-conjugated polyclonal anti-FIX IgG in the washing buffer. Under these conditions FIX but not FIX or FIXa binds to the immobilized antibody CLB-FIX D4. Dose-response curves were transformed by plotting logit absorbance versus log concentration and were linear between 0.1 and 10 nM.

FX Activation

The ability of FIX or its cleaved derivatives to activate FX was assayed essentially as described previously (35) employing acetylated FX to prevent cleavage of the Arg-Ala bond by the product FXa(16) . FX was acetylated according to Neuenschwander and Jesty(36) . The modified FX zymogen had lost more than 95% of its biological activity, whereas amidolytic activity was fully maintained.

Hydrolysis of CHSO-LGR-pNA

Cleavage of CHSO-LGR-pNA by FIX or its derivatives was assayed in 0.2% (w/v) HSA, 0.1 M NaCl, 0.01 M CaCl, 0.05 M Tris (pH 8.4). Substrate hydrolysis was initiated by the addition of 50 µl of a 2.5 mM solution of CHSO-LGR-pNA to a 50-µl sample in a microtiter plate (Costar, type flat bottom). Initial rates of substrate hydrolysis were measured at 37 °C by monitoring absorbance at 405 nm in time. Kinetic parameters of substrate hydrolysis by FIX cleavage products were determined employing substrate concentrations between 0 and 15 mM at two different enzyme concentrations. Absorbance values were converted into molar concentrations using a molar extinction coefficient of 9.65 10M cm for p-nitroanilide and a pathlength of 0.35 cm for a 100-µl volume. The experimental data were fitted in the Michaelis-Menten equation using EnzFitter software (Elsevier, Amsterdam, the Netherlands) to obtain K and k values.

Protein Binding Assays

The binding of FIX or its cleaved derivatives to the immobilized FVIII light chain and calculation of binding parameters were performed as described(22) .


RESULTS

Enzymatic Activity of FIX Activation Products

The role of the individual cleavages at Arg and Arg in human FIX was investigated with respect to the development of enzymatic activity. FIX and its cleaved derivatives were compared for their ability to activate FX in the presence of calcium ions, phospholipids, and FVIIIa. As expected, the enzyme FIXa efficiently activated FX under these conditions (Fig. 1A). FXa was also generated by the intermediate FIXa, although at a lower rate than by FIXa. In contrast, both the intermediate FIX and the FIX zymogen were incapable of activating FX. These data demonstrate that cleavage at Arg converts FIX into an active protease but that the additional cleavage at Arg develops full proteolytic activity. To investigate whether limited proteolysis of FIX had a similar effect on amidolytic activity, the reactivity of the zymogen FIX and its cleaved derivatives toward the synthetic substrate CHSO-LGR-pNA was tested. No substrate cleavage occurred in the presence of the FIX zymogen (Fig. 1B). In contrast, all cleaved forms of FIX, including the intermediate FIX, were capable of hydrolyzing this synthetic substrate. The kinetic parameters for the hydrolysis of CHSO-LGR-pNA by FIXa, FIXa, and FIX were determined. As listed in , FIXa and FIXa display similar catalytic efficiency. The catalytic efficiency of FIX, however, appears to be 4-fold lower, which is mainly due to a decreased k. The possibility was considered that the lower catalytic efficiency could be due to FIX being only partially active. However, active site titrations employing the active site titrant NPGB indicated that the extent of the p-nitrophenol burst corresponded to 90-95% of the protein concentrations of both the FIX and FIXa preparations employed (see ``Experimental Procedures''). It was noted that titration of FIXa requires about 4 min to reach completion(33) , whereas the p-nitrophenol burst lasts 7-8 min for FIX (results not shown). This slight difference in reactivity toward NPGB was not further elaborated. Collectively, these results confirm that the zymogen FIX is an inactive species, whereas the enzyme FIXa displays activity toward FX, CHSO-LGR-pNA, and NPGB. With regard to the activation intermediates, FIXa equals the enzyme FIXa in synthetic substrate hydrolysis but is less efficient in FX activation. FIX, however, is extremely inefficient in generating FXa but at the same time hydrolyzes CHSO-LGR-pNA and reacts with the active site titrant NPGB. This demonstrates that cleavage at Arg converts the FIX zymogen into an enzymatic form that lacks proteolytic activity.


Figure 1: Activity of FIX and its cleaved derivatives toward FX and CHSO-LGR-pNA. A, FX activation was assessed by the incubation of various concentrations of FIXa (), FIXa (), FIX (), or FIX () with acetylated FX (0.2 µM), phospholipids (0.1 mM), and FVIIIa (0.4 nM) at 37 °C in 3.0 mM CaCl, 0.1 M NaCl, 0.2 mg/ml bovine serum albumin, 0.05 M Tris (pH 7.4). FXa generation was detected as described (35). B, amidolytic activity was assayed by the addition of CHSO-LGR-pNA (1.25 mM final concentration) to solutions containing various concentrations of FIXa (), FIXa (), FIX (), or FIX () in 0.2% (w/v) HSA, 0.1 M NaCl, 0.01 M CaCl, 0.05 M Tris (pH 8.4). Hydrolysis of CHSO-LGR-pNA was monitored continuously at 405 nm as described under ``Experimental Procedures.'' The data shown represent the mean values ± S.D. of three to five experiments. pNA, p-nitroanilide.



Interaction of FIX and FIXa with Serine Protease Inhibitors

Although the concentration of active sites was in good agreement with the protein concentrations of purified FIXa and FIX, these experiments do not fully exclude the possibility that traces of other serine proteases could contribute to the observed CHSO-LGR-pNA hydrolysis by FIX or FIXa. Therefore, the effect of a number of serine protease inhibitors was tested. Amidolytic activity of FIX or FIXa appeared to be unaffected by the presence of serine protease inhibitors including hirudin, soybean trypsin inhibitor, -antitrypsin, -antiplasmin, and C-inhibitor (data not shown). This demonstrates that the amidolytic activity is not likely to be associated with the presence of a variety of potential contaminants. In contrast, CHSO-LGR-pNA hydrolysis was effectively inhibited in the presence of the monoclonal anti-FIX antibody CLB-FIX 10 (see Fig. 5). Control experiments demonstrated that this antibody does not inhibit CHSO-LGR-pNA hydrolysis by thrombin, FXa, or FXIa. This strongly suggests that the observed CHSO-LGR-pNA hydrolysis originates from the enzyme FIXa or the activation intermediate FIX.


Figure 5: Effect of monoclonal anti-FIX antibodies on FIX amidolytic activity and FVIII light chain binding. Amidolytic activity (closed symbols) by FIX (300 nM) was determined as described under ``Experimental Procedures'' in the presence of various concentrations of antibody CLB-FIX 10 () or CLB-FIX 11 (). Binding of EGR-CK-inactivated FIX (40 nM) to the immobilized FVIII light chain (open symbols) was investigated in the presence of varying concentrations of antibody CLB-FIX 10 () or CLB-FIX 11 () as described (22). Plotted is the percentage of residual activity or binding versus the molar excess of antibody over the FIX concentration. An inhibition constant of 0.7 ± 0.3 nM was calculated for antibody CLB-FIX 11 by analyzing the data for the FIX-FVIII light chain interaction employing a model of competitive inhibition (22). Inset, FIX was reduced and subjected to 12.5% (w/v) SDS-PAGE. Total protein was visualized by silver staining (lane 1). Protein blots were incubated with antibody CLB-FIX 10 (lane 2), antibody CLB-FIX 11 (lane 3), or the FVIII light chain (lane 4). The bound antibodies were detected employing peroxidase-labeled goat anti-mouse antibodies, and the bound FVIII light chain was detected employing peroxidase-labeled anti-FVIII antibody CLB-CAg 69.



Additional experiments were performed to determine the affinity of FIX and FIXa for the inhibitor benzamidine. This reversible inhibitor is known to inhibit the various coagulation enzymes with K values ranging from 0.04 to 11 mM(37, 38, 39) and as such could contribute to the identification of the active species in FIX. Under the same experimental conditions as in Fig. 1B, FIX inhibition proved to be similar to that of FIXa, with Kvalues of 2.9 ± 0.3 and 2.0 ± 0.2 mM, respectively. These data demonstrate that FIX and FIXa share similar inhibition characteristics by benzamidine.

The enzymatic properties of FIXa and FIX were examined in more detail using the synthetic serine protease inhibitor EGR-CK. Binding of EGR-CK to FIX or FIXa was determined by continuously monitoring CHSO-LGR-pNA hydrolysis by FIX or FIXa in the presence of various concentrations of EGR-CK. As expected, substrate hydrolysis by FIXa displayed progress curves that are typical for irreversible enzyme inhibition; in the steady state situation, all FIXa had been inactivated by EGR-CK as p-nitroanilide formation was completely inhibited (Fig. 2A)(40, 41) . In contrast, progress curves of CHSO-LGR-pNA hydrolysis by FIX displayed residual substrate hydrolysis in the steady state (Fig. 2B), suggesting that FIX inhibition, unlike that of FIXa, is not irreversible. Apparently, FIX and FIXa are both capable of binding EGR-CK but are dissimilar with respect to the mechanism of inhibition. This notion was further explored by incubating FIX and FIXa with the dansyl-labeled derivative DEGR-CK. Irreversible binding of DEGR-CK to FIXa was clearly visualized by incorporation of the DEGR-moiety into the FIXa heavy chain, whereas only a faint band was observed for FIX (Fig. 2, inset). This is compatible with the view that FIX and EGR-CK associate in a nonirreversible manner.


Figure 2: Effect of EGR-CK on amidolytic activity of FIX and FIXa. 25 µl of FIXa (A) or FIX (B) was added to a mixture containing CHSO-LGR-pNA and EGR-CK in 0.2% (w/v) HSA, 0.1 M NaCl, 0.01 M CaCl, 0.05 M Tris (pH 8.4) to a final volume of 100 µl. Final concentrations were 250 nM FIXa, 500 nM FIX, 4 mM CHSO-LGR-pNA and 0 (), 25 µM (), 50 µM (▾), or 100 µM () EGR-CK. After the addition of FIXa or FIX, p-nitroanilide formation was monitored as described under ``Experimental Procedures.'' Data represent the mean of three independent experiments. Inset, the interaction between EGR-CK and FIX or FIXa was examined by incubating FIX or FIXa (both 1 µM) with dansyl-modified EGR-CK (50 µM) in 0.1 M NaCl, 0.01 M CaCl, 0.05 M Tris (pH 8.4) for 16 h at room temperature. Subsequently, 1.5 µg of FIX and 1.5 µg of FIXa were reduced and subjected to 12.5% (w/v) SDS-PAGE. Incorporation of the dansyl-L-glutamyl-L-glycyl-L-arginine moiety in the respective active sites was detected by UV light illumination. FIXa and FIX treated with DEGR-CK are presented in lanes 1 and 3, respectively. FIX treated with the unmodified EGR-CK is presented in lane 2. pNA, p-nitroanilide.



Finally, the interaction between FIX or FIXa and the macromolecular inhibitor ATIII was addressed. In preliminary experiments it was observed that binding was most efficient in the presence of heparin, resulting in complete inhibition within 2 min. In the ATIII inhibition experiments, 20-min incubation periods were maintained to ensure that residual activities represent true end points. As shown in Fig. 3, ATIII readily inhibited cleavage of CHSO-LGR-pNA by both FIX and FIXa. FIXa and FIX were similar in their interaction with ATIII in that the stoichiometry was approximately 1:1 in both cases (Fig. 3). The same reaction mixtures were analyzed by SDS-PAGE. ATIII formed a SDS-resistant complex with FIXa, whereas no complex could be visualized for FIX or the FIX zymogen (Fig. 3, inset). Apparently, the association of the activation intermediate FIX with ATIII is different from that of mature serine proteases such as FIXa.


Figure 3: Interaction of ATIII with FIX and FIXa. FIX () and FIXa (), both 600 nM, were incubated with various concentrations of ATIII for 30 min at 37 °C in the presence of heparin (2 mg/ml). Residual amidolytic activity was then measured by the addition of 50 µl of CHSO-LGR-pNA to 50-µl samples as described under ``Experimental Procedures.'' Data represent mean values ± S.D. of three experiments. Final concentrations (mean ± S.D.) of FIXa and FIX were 320 ± 14 nM and 303 ± 12 nM, respectively, as determined employing an immunological assay, and final concentrations of ATIII are those indicated. Inset, complex formation between ATIII and FIX, FIX, or FIXa was examined by the incubation of FIX, FIX, or FIXa (all 1 µM) with ATIII (1.8 µM) in the presence of heparin (2 mg/ml). Subsequently, 2 µg of unreduced FIX, FIX, or FIXa was subjected to 7.5% (w/v) SDS-PAGE. Protein was visualized by Coomassie staining. The lanes represent the following proteins. Lane 1, ATIII; lane 2, FIX; lane 3, ATIII + FIX (no complex); lane 4, FIXa; lane 5, ATIII + FIXa (complex); lane 6, FIX; lane 7, ATIII + FIX (no complex).



Effect of FIX Cleavage on Binding to the FVIII light chain

Optimal FX activation by FIXa requires complex formation with the protein cofactor FVIIIa. One of the sites that might be exposed during FIX activation is the binding site for the FVIII light chain. Therefore, the contribution of the individual cleavages at Arg and Arg to the affinity for the FVIII light chain was tested employing a previously established method(22) . As shown in Fig. 4, binding of FIXa to the FVIII light chain was less effective than observed for FIXa. In contrast, the interaction of FIX with the FVIII light chain was similar to that of FIXa. The interaction between the FVIII light chain and FIX displayed a K of 11.9 ± 1.0 nM and a stoichiometry of 0.8 ± 0.2 mol/mol of FVIII light chain. These parameters are similar to those of FIXa (Fig. 4)(22) . For FIXa the K and stoichiometry were 49.9 ± 7.4 nM and 0.9 ± 0.1 mol of FIXa/mol of FVIII light chain, respectively. This indicates that cleavage at Arg alone is insufficient for full exposure of the FVIII light chain binding site.


Figure 4: Interaction of FVIII light chain with FIX and its cleaved derivatives. Various amounts of FIXa (), FIXa (), or FIX () were inactivated with EGR-CK and subsequently incubated with the immobilized FVIII light chain (1.2 pmol/well) in 1% (w/v) HSA, 0.1% (v/v) Tween-20, 0.1 M NaCl, 5 mM CaCl, 25 mM histidine (pH 6.2). Binding was quantified as described elsewhere (22). Binding parameters for the interaction with the FVIII light chain were derived employing a model describing the interaction with one single class of binding sites (22). The calculated dissociation constants were 14.8 ± 3.2, 11.9 ± 1.0, and 49.9 ± 7.4 nM for FIXa, FIX, and FIXa, respectively. Inset, EGR-CK-inactivated FIX (40 nM) was incubated with the immobilized FVIII light chain (1.2 pmol/well) in the presence of various concentrations of EGR-CK-inactivated FIXa () or FIX (). FIX binding was measured as described under ``Experimental Procedures'' and expressed as , which represents the ratio of FIX bound in the presence and the absence of competitor. The drawn lines were obtained from a model describing competitive inhibition (22). The calculated inhibition constants for FIXa and FIX were 10.1 ± 3.2 and 282 ± 19 nM, respectively. Data represent mean values ± S.D. of three experiments.



The interaction of FIX with the FVIII light chain was examined more closely in competition experiments. As shown in the inset of Fig. 4, FIXa inhibited binding of FIX to the FVIII light chain in a dose-dependent manner. By analyzing these experimental data in a model of competitive inhibition(22) , the K was calculated to be 10.1 ± 3.2 nM (mean ± S.D.), which is similar to the K for the interaction of the FVIII light chain with FIXa obtained in direct binding studies (Fig. 4)(22) . The same experimental approach was used to determine the Kfor the interaction of the FVIII light chain with the zymogen FIX. As presented in the inset of Fig. 4, only minor inhibition became apparent at the higher concentrations of FIX tested, which was calculated to reflect a K of 282 ± 19 nM. This demonstrates that the affinity of FIX for the FVIII light chain is considerably lower compared with that of FIX and FIXa. In conclusion, these findings imply that full exposure of the FVIII binding site requires cleavage of the Arg-Ala bond.

Monoclonal Antibodies Distinguish between Amidolytic Activity and FVIII Light Chain Binding

Cleavage of the Arg-Ala bond develops both amidolytic activity (Fig. 1B) and exposure of the binding site for the FVIII light chain (Fig. 4). To identify regions involved in these processes, the monoclonal antibodies CLB-FIX 10 and CLB-FIX 11, which strongly inhibit FIX activity (see ``Experimental Procedures''), were investigated for their effect on the FVIII light chain binding and amidolytic activity. With regard to amidolytic activity, it appeared that CHSO-LGR-pNA hydrolysis by FIX was unaffected by the presence of antibody CLB-FIX 11 but strongly inhibited by antibody CLB-FIX 10 (Fig. 5). With respect to FVIII light chain binding, the opposite was observed; CLB-FIX 11 strongly interfered in the interaction of FIX with the FVIII light chain, whereas CLB-FIX 10 did not (Fig. 5). FIXa was identical to FIX in that its amidolytic activity and FVIII light chain binding also were inhibited by the same antibodies (not shown). In conclusion, our data demonstrate that FVIII light chain binding and amidolytic activity are inhibited by two different monoclonal antibodies, indicating that distinct regions contribute to these processes. In immunoblotting experiments (Fig. 5, inset), both antibody CLB-FIX 10 and CLB-FIX 11 were found to be directed against the light chain of FIX. The notion that the latter antibody also interferes with binding of FIX to the FVIII light chain suggests that the light chain of FIX is involved in the interaction with the light chain of factor VIII. This possibility was confirmed in ligand blotting experiments employing purified FVIII light chain. By this method, the FVIII light chain was observed to bind to the light chain but not the heavy chain of FIX (Fig. 5, inset, lane 4). Collectively, these findings suggest that distinct FIX light chain regions are involved in amidolytic activity and binding to the FVIII light chain.


DISCUSSION

In the proteolytic activation of human FIX, cleavage of the Arg-Ala bond is the first of two consecutive cleavages catalyzed by the physiological activators FXIa or factor VIIa(2, 12, 13, 14, 15) . Cleavage at Arg alone gives rise to the activation intermediate FIX(12, 15) . This intermediate is known to lack clotting activity(15) , which is in agreement with our observation that FIX is incapable of activating FX (Fig. 1A). It was surprising, however, that FIX did display enzymatic activity toward the active site titrant NPGB and the synthetic substrate CHSO-LGR-pNA (Fig. 1B and ). Although we considered the possibility that traces of contaminating serine proteases contributed to the observed amidolytic activity, several lines of evidence led us to conclude that substrate hydrolysis indeed originates from FIX. First, a series of serine protease inhibitors did not affect FIX or FIXa amidolytic activity, although these inhibitors should interfere with the activity of a variety of potential contaminants. Second, benzamidine inhibits FIX and FIXa activity with a similar K of 2-3 mM, whereas the K values for other enzymes are significantly lower (thrombin, FXa, FXIa, factor XIIa, kallikrein, trypsin, plasmin) or higher (factor VIIa)(37, 38, 39) . Third, a 1:1 stoichiometry was observed in active site titrations of the intermediate FIX employing the active site titrant NPGB or the serine protease inhibitor ATIII (Fig. 3). Finally, a monoclonal anti-FIX antibody specifically inhibited amidolytic activity of FIX and FIXa (Fig. 5). Together with our finding that FIX differed from FIXa by 4 orders of magnitude in activating FX (Fig. 1A), our results provide strong evidence that the observed amidolytic activity is an intrinsic property of the intermediate FIX.

Because conversion of FIX into FIX promotes development of amidolytic activity, cleavage of the Arg-Ala bond is apparently associated with changes within the FIX protease domain. In this respect, FIX seems similar to the homologous intermediate prethrombin-2, which exposes binding sites for the inhibitors dansylarginine-N-(3-ethyl-1, 5-pentanediyl)-amide and hirugen, whereas these sites are not exposed in the prothrombin zymogen (42, 43). With regard to the interaction of FIX with the inhibitors ATIII and EGR-CK, however, we obtained some intriguing data. In contrast to FIXa, FIX displayed minor incorporation of the dansyl-labeled EGR-CK into its active site (Fig. 2, inset). In concordance with this observation, FIX and FIXa were also found to be dissimilar in EGR-CK inhibition studies. FIXa was inhibited in the expected irreversible manner, whereas FIX showed nonirreversible inhibition (Fig. 2). Although the latter seems unusual, this type of inhibition is compatible with the general mechanism by which chloromethyl ketone inhibitors inactivate serine proteases(44, 45) . Inhibition is initiated by the formation of a noncovalent complex, after which the methyl carbon of the inactivator is attached to the active site serine hydroxyl group to form a hemiketal intermediate. After subsequent conversion of this intermediate into an epoxyether intermediate, two distinct pathways have been distinguished. First, the serine-bound epoxyether intermediate may alkylate the active site histidine residue. This is the generally known pathway that results in the irreversible, covalent complex as observed for FIXa. Alternatively, the epoxyether intermediate may be hydrolyzed by the water solvent, leading to the formation of a free hydroxymethyl ketone and to regeneration of the active enzyme(44, 45) . Generally, serine protease inhibition by chloromethyl ketones follows both pathways simultaneously(44) . Our finding that FIX displays residual activity in the steady state indicates that for FIX the alternative pathway is predominant over the irreversible pathway. As spatial alignment of the active site histidine and serine residues is critical for irreversible inhibition(45) , we speculate that the intermediate FIX lacks the optimal alignment within its catalytic site.

With respect to the interaction with the serine protease inhibitor (``serpin'') ATIII, FIX was strikingly different from FIXa, because no stable complexes were formed in the presence of SDS (Fig. 3, inset). Usually, serpin-serine protease complexes are driven into a stable complex upon incubation with denaturing agents such as SDS(46, 47) . However, FIX is not the sole exception to this general mechanism in lacking such complex formation. Anomalous behavior has been found to be associated with serpin variants with mutations in the reactive site loop(48, 49, 50) , or with modifications in the active site of the target protease(51, 52) . The observation that FIX and ATIII are unable to form a stable complex in the presence of SDS thus indicates that the active site of FIX differs from that of FIXa. In this respect, the data for ATIII and EGR-CK are fully compatible, because they both suggest that the FIX active site is not fully developed. This is in support of the notion that FIX is an intermediate in the transition of the FIX zymogen into the mature enzyme FIXa.

Proteolytic activation of the FIX zymogen is associated with the exposure of the cofactor binding site, because the activation intermediates FIX and FIXa both display increased affinity for the FVIII light chain compared with uncleaved FIX (Fig. 4). The FIX zymogen thus is similar to the serine protease precursors FX and prothrombin in that proteolytic activation is associated with the exposure of the respective cofactor binding sites of these zymogens as well(53, 54) . Whereas FX and prothrombin require cleavage at the protease domain amino terminus for full exposure of the cofactor binding site(53, 54) , this seems to be untrue for FIX. Cleavage of the Arg-Val bond alone results in a 5-fold lower affinity for the FVIII light chain compared with the enzyme FIXa (Fig. 4). This may explain the observation that FIXa is less efficient than FIXa in generating FXa in the presence of FVIIIa (Fig. 1A)(12, 17) , whereas FIXa and FIXa are equally potent in generating FXa in the absence of FVIIIa(17) . Cleavage of the Arg-Ala bond alone is sufficient for full exposure of the binding site for the FVIII light chain, because FIX and FIXa were similar in binding the FVIII light chain (Fig. 4). This indicates that cleavage at Arg plays a major role in the formation of the FIX-FVIII complex. With respect to the FIX zymogen it is important to note that its plasma concentration of 35-70 nM(3) is well below the K of approximately 300 nM for the interaction with the FVIII light chain and thereby not in favor of complex assembly. This limitation is overcome after cleavage of FIX at Arg, which decreases the K to approximately 12 nM. Because cleavage of the Arg-Ala bond may be catalyzed by FXa (16), this may provide a previously unrecognized mechanism that promotes assembly of the FIX-FVIII complex.

In an attempt to identify the region involved in binding the FVIII light chain, we employed monoclonal antibodies that inhibit FIX activity. One antibody, designated CLB-FIX 11, was observed to interfere with the FIX-FVIII light chain interaction (Fig. 5). It is of interest to note that Bajaj and co-workers (55) have also described an anti-FIX monoclonal antibody that interferes with FVIII-dependent FX activation by FIXa and probably also with binding of FIXa to FVIIIa. However, that antibody is directed against the FIX heavy chain(10) , whereas our antibody is directed against the FIX light chain (Fig. 5, inset). The presence of an independent binding site on the FIX light chain would be in agreement with our observation that the FVIII light chain interacts with the FIX light chain when tested by ligand blotting (Fig. 5, inset). However, we cannot exclude the possibility that the FVIII light chain binds also to a FIX heavy chain site that is not resistant to the immunoblotting technique. Alternatively, it seems conceivable that binding of the FVIII light chain to the FIX heavy chain is dependent on the conformation of the FIX light chain. In this respect it should be noted that the antibody CLB-FIX 10 is directed against the FIX light chain but inhibits amidolytic activity (Fig. 5), which obviously is associated with the FIX protease domain. Further studies will be needed to resolve the mutual interrelation between the various FIX domains with regard to the interaction with the complete FVIII heterodimer.

  
Table: Kinetic parameters for the hydrolysis of CHSO-LGR-pNA by FIX, FIXa, or FIXa

Hydrolysis of CHSO-LGR-pNA was monitored in the presence of varying concentrations of CHSO-LGR-pNA (0-15 mM) as described under ``Experimental Procedures.'' Kinetic parameters were determined at enzyme concentrations of 200 and 300 nM (FIXa and FIXa) or at 300 and 400 nM (FIX). Mean values ± S.D. of 3-4 experiments are presented. Catalytic efficiency k/K is calculated from K and k.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Blood Coagulation, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, the Netherlands. Tel.: 31-20-5123120; Fax: 31-20-5123332.

The abbreviations used are: FIX, factor IX; ATIII, antithrombin III; DEGR-CK, dansyl-L-glutamyl-L-glycyl-L-arginine chloromethyl ketone; EGR-CK, L-glutamyl-L-glycyl-L-arginine chloromethyl ketone; HSA, human serum albumin; FVIII, factor VIII; FVIIIa, factor VIIIa; FIX, factor IX; FIXa, factor IXa; FIXa, factor IXa; FX, factor X; FXa, factor Xa; FXIa, factor XIa; CHSO-LGR-pNA, CHSO-D-leucyl-L-glycyl-L-arginine-p-nitroanilide; NPGB, p-nitrophenol p-guanidinobenzoate; PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor.


ACKNOWLEDGEMENTS

We thank F. Meijer-Huizinga, J. Rentenaar, and A. Blok for preparing and characterizing the anti-FIX antibodies and Dr. J. Voorberg for critically reading the manuscript.


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