©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Sequence GluLys of Human Blood Coagulation Factor VIII Comprises a Binding Site for Activated Factor IX (*)

(Received for publication, July 18, 1995; and in revised form, November 10, 1995)

Peter J. Lenting Jan-Willem H. P. van de Loo Marie-José S. H. Donath Jan A. van Mourik Koen Mertens (§)

From the Department of Blood Coagulation, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In previous studies we have shown that the interaction between factors IXa and VIII involves the light chain of factor VIII and that this interaction is inhibited by the monoclonal antibody CLB-CAg A against the factor VIII region Gln-Asp (Lenting, P. J., Donath, M. J. S. H., van Mourik, J. A., and Mertens, K.(1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu and Met. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the factor VIII region Tyr-Ala. Factor IXa binding was found to be particularly efficient to peptides corresponding to the factor VIII sequences Lys-Lys and Glu-Gln. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys-Lys and Glu-Gln interfere with binding of factor IXa to immobilized factor VIII light chain (K approx 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit factor X activation by factor IXa in the presence of factor VIIIa (K approx 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the factor IX zymogen and its activated form, factor IXa, with the same affinity (apparent K approx 0.2 mM), whereas the complete factor VIII light chain displays preferential binding to factor IXa. In conclusion, our results demonstrate that peptides consisting of the factor VIII light chain residues Lys-Lys and Glu-Gln share a factor IXa binding site that is essential for the assembly of the factor X-activating factor IXa-factor VIIIa complex. We propose that the overlapping sequence Glu-Lys comprises the minimal requirements for binding to activated factor IX.


INTRODUCTION

Human blood coagulation factor VIII (FVIII) (^1)is an essential protein of the hemostatic system, which is evident from the severe bleeding disorder hemophilia A that is associated with FVIII deficiency or dysfunction (1) . FVIII is synthesized as a single chain polypeptide containing a number of discrete domains arranged in the sequence A1-A2-B-A3-C1-C2 (2, 3) . Examination of its primary structure reveals that FVIII shares considerable homology with the plasma proteins factor V (FV) and ceruloplasmin(4, 5, 6) . Whereas ceruloplasmin comprises a triple A domain structure (A1-A2-A3), FV displays the same domain structure(7, 8) . In contrast to FV, FVIII predominantly circulates as a heterodimeric protein, consisting of a Me-linked light and heavy chain (9, 10, 11) . The heavy chain contains the A1-A2-B domains and is heterogeneous (M(r) 90,000-200,000) due to limited proteolysis at a number of positions within the B domain. The light chain of FVIII (M(r) 80,000) comprises the domains A3-C1-C2(10, 12) .

In the intrinsic pathway of blood coagulation, FVIII functions as a nonenzymatic cofactor in the factor X (FX)-activating complex(13) . Within this complex, the serine protease factor IXabeta (FIXa) activates FX in the presence of calcium ions, phospholipids, and activated FVIII. In order to play its role in the generation of FXa, FVIII has to be activated(14, 15) . Activation is achieved by limited proteolysis in both the FVIII heavy and light chain by FXa or thrombin(12) , which results in the formation of a heterotrimeric product, FVIIIa(16, 17) . The relatively labile FVIIIa heterotrimer is known to be stabilized by the enzyme FIXa in the presence of phospholipids(18) . In addition, it has been reported that the phospholipid-FIXa complex enhances the reassociation of isolated FVIIIa subunits into the FVIIIa heterotrimer (19) , indicating that FVIIIa is capable of directly interacting with FIXa.

Several studies have been performed in order to characterize the assembly of the FIXbulletFVIII complex in more detail(19, 20, 21, 22) . The FVIII heavy chain regions Ser-Gln and Arg-Ser have been recognized to represent FIXa interactive sites(22, 23) . Previously, we have shown that FVIII light chain comprises an exosite that binds FIXa with high affinity(21) . In the same study, we found that the FIXa-FVIII light chain interaction was inhibited by the anti-FVIII antibody CLB-CAg A, which is known to bind to the FVIII A3 domain region Gln-Asp(24) . In the present study, we addressed the possibility that this region is involved in the assembly of the enzyme-cofactor complex. Therefore, we first located the binding site of antibody CLB-CAg A in more detail. Subsequently, a series of synthetic peptides was employed in order to define the FVIII region involved in FIXa binding. This approach allowed us to identify the FVIII light chain region Glu-Lys as being involved in FIXa binding and in the assembly of the FX-activating FIXabulletFVIIIa complex.


EXPERIMENTAL PROCEDURES

Materials

Protein A-Sepharose CL-4B was from Pharmacia Biotech Inc. Microtiter plates (Immulon) were from Dynatech (Plockingen, Germany). The in vitro transcription and translation kits employing the SP6-expression system as well as the plasmid pSP64 were from Promega. Restriction enzymes were obtained from Life Technologies, Inc. Goat anti-mouse antibodies, rabbit anti-mouse antibodies, and human serum albumin (HSA) were from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, The Netherlands).

Antibodies and Other Proteins

The monoclonal anti-FVIII antibodies CLB-CAg 12, CLB-CAg 69, and CLB-CAg A have been described previously(24, 25) . The murine anti-FIX antibody CLB-FIX 14 (isotype IgG) was obtained as outlined previously, employing a screening strategy based on binding to immobilized FIX in the absence of calcium ions(21) . Monoclonal antibodies were purified from culture medium employing Protein A-Sepharose CL-4B as recommended by the manufacturer. Polyclonal antibodies against human FIX were obtained as described(21) . Antibodies were conjugated with horseradish peroxidase as described(26) . Human FVIII light chain was purified as outlined previously(21) . Human FIXa was prepared from immunopurified FIX as described(27) .

Synthetic Peptides

Peptides encompassing residues Tyr-Glu, Gly-Lys, Lys-Lys, Glu-Gln, Tyr-Ala, and His-Ala from human FVIII were synthesized using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry by the manual ``T-bag'' method(28, 29) , or employing a 430A Applied Biosystems instrument (Pharmacia). Purity of the peptides was checked by HPLC reversed phase chromatography(29) . Peptides Gly-Lys, Lys-Lys, and Glu-Gln were more than 90% pure. Peptides Tyr-Glu, Tyr-Ala and His-Ala were further purified by reversed phase HPLC to the same degree of purity. The identities of peptides Gly-Lys, Lys-Lys, and Glu-Lys were confirmed by mass spectrometry analysis (Eurosequence B.V., Groningen, The Netherlands). Mass spectrometry further confirmed that Cys-containing peptides did exist as monomers. Peptide Lys-Lys was used from two separate preparations and produced identical results.

Construction of Recombinant FVIII Fragments

Plasmid pSP/F8-80K1 (24) was used as template for the construction of truncated FVIII fragments employing the polymerase chain reaction. The DNA fragments were made by using the sense primer 5`-ACG ATT TAG GTG ACA CTA TAG-3` (containing a part of the SP-6 promotor) in combination with the respective antisense primers 5`-TTA GGA TCC TCA CAT ATG ATG TTG CAC TTT-3`, 5`-TTA GGA TCC TCA TTC ATT AGG CTT GAC AAA-3`, 5`-TTA GGA TCC TCA TTC TGC TCC TTG CCT CTG-3`, and 5`-TTA GGA TCC TCA AGA AAT AAG GCT AGA ATA-3`. Polymerase chain reaction amplifications of these primer combinations yielded the FVIII DNA sequences between base pairs 4893 and 5676, 5640, 5610, and 5580, respectively. The antisense primers enclosed a BamHI restriction site (underlined) and a stop-codon (boldface type). The sense primer was designed to contain a HindIII restriction site at the 5` terminus after polymerase chain reaction. After digestion of the polymerase chain reaction products and plasmid pSP64 with HindIII and BamHI, the products were ligated into the plasmid. The identities of the constructs were verified by restriction mapping and dideoxy termination sequencing. The constructs encode for the FVIII fragments Asp-Met, Asp-Glu, Asp-Glu, and Asp-Ser.

Immunoprecipitation Studies

The FVIII fragments were in vitro transcribed and translated employing the SP6 expression system as described previously(24) . Translation was performed in the presence of [S]methionine to obtain radiolabeled FVIII fragments, which were analyzed by 12% (w/v) SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. All constructs appeared to encode for single polypeptides (not shown). The fragments were diluted 100-fold in 25 mM Tris (pH 8.0), 5 mM NaCl, 2 mM EDTA, 0.5% (w/v) dideoxycholate, 0.5% (v/v) Triton X-100 (immunoprecipitation buffer). The fragments were then incubated with monoclonal antibody CLB-CAg A or CLB-CAg 69 (5 µg/ml) for 1 h at room temperature. Subsequently, rabbit anti-mouse antibodies immobilized onto Protein A-Sepharose were added, and the incubation was continued for another hour at room temperature. After washing twice with immunoprecipitation buffer and once with 0.1 M NaCl, 0.1% (v/v) Triton X-100, 50 mM Tris (pH 8.0), the beads were boiled for 5 min in 8% (w/v) SDS, 40% (v/v) glycerol, 0.04% (w/v) bromphenol blue, 20 mM dithiothreitol, 0.25 M Tris (pH 6.8). Finally, the supernatants were subjected to 12% (w/v) SDS-polyacrylamide gel electrophoresis, and polypeptides were visualized by autoradiography.

Binding Assays

Synthetic peptides were immobilized (0.8 nmol/well added) to microtiter wells in a volume of 100 µl, and remaining binding sites were blocked with 2% (w/v) HSA in 0.1% (v/v) Tween 20, 0.1 M NaCl, 25 mM Tris (pH 7.4). After washing, the immobilized peptides were incubated with antibody CLB-CAg A (625 nM) or FIXa (50 nM) in the same buffer for 1 h at 37 °C in a 100-µl volume. After washing, bound antibody CLB-CAg A was probed by incubating with peroxidase-conjugated goat anti-mouse antibodies for 15 min at room temperature and detected by peroxidase hydrolysis of the substrate 3,3`,5,5`-tetramethylbenzidine (Sigma). Bound FIXa was probed by employing the peroxidase-conjugated anti-FIX antibody CLB-FIX 14 (5 µg/ml). Binding of FIXa to immobilized FVIII light chain and calculation of binding parameters were performed as described(21) . The affinities of FIX and FIXa for peptides in solution were determined employing a previously described method(30) . Briefly, FIX or FIXa (30 nM) was incubated with various concentrations of peptide (0.2-0.6 mM) in a buffer containing 2% (w/v) HSA, 0.1% (v/v) Tween 20, 0.1 M NaCl, 25 mM Tris (pH 7.4). The mixtures were incubated for 16 h at room temperature in order to reach equilibrium. Samples were subsequently incubated with immobilized peptide to allow noncomplexed FIX or FIXa to bind to the immobilized peptide. FIX or FIXa bound to the immobilized peptide was quantified employing the peroxidase-conjugated antibody CLB-FIX 14. The dissociation constants for the interaction with peptides in solution could be calculated as described(30) .

FX Activation

FXa formation was determined as described (31) . FX (0.2 µM) was activated in 3 mM CaCl(2), 0.1 M NaCl, 0.2 mg/ml HSA, 0.05 M Tris (pH 7.4) at 37 °C by FIXa (0.7 nM) in the presence of phospholipids (0.1 mM) and FVIIIa (0.4 nM). FVIII was preactivated in the same buffer for 5 min by thrombin prior to the addition. FX activation experiments in the absence of FVIIIa were performed employing a FIXa concentration of 30 nM. FXa formation was quantified employing the chromogenic substrate S-2222 (Chromogenix AB, Mölndal, Sweden). An active site titrated FXa preparation was used as a reference to convert absorbance values into molar FXa concentrations.


RESULTS

Interaction between Recombinant FVIII Fragments and Antibody CLB-CAg A

Binding of FIXa to FVIII light chain is inhibited by the monoclonal anti-FVIII antibody CLB-CAg A(21) . Since this antibody is known to bind to the FVIII region Gln-Asp(24) , the same region may be involved in the interaction with FIXa. To address this possibility, we first located the binding site for this antibody in more detail. Constructs comprising DNA sequences encoding for the FVIII fragments Asp-Met and carboxyl-terminal truncations thereof (Fig. 1) were in vitro transcribed and translated, the latter of which in the presence of [S]methionine. As determined by SDS-polyacrylamide gel electrophoresis, each radiolabeled polypeptide migrated as a single band with the expected M(r) between 32,000 and 36,000 (not shown). These polypeptides were examined for their binding to antibodies CLB-CAg A and CLB-CAg 69 in immunoprecipitation studies. In these studies, antibody CLB-CAg 69 served as a control that should bind to all four polypeptides, as its epitope is known to encompass residues Lys-Arg(24) . Indeed, the antibody effectively bound the various polypeptides to the same extent (Fig. 1, lanes 1-4). With respect to antibody CLB-CAg A, the largest polypeptide (Asp-Met) was readily recognized by this antibody, whereas only a faint band was observed for the polypeptide Asp-Glu (Fig. 1, lanes 7 and 8). In contrast, the two smaller polypeptides comprising the sequences Asp-Glu and Asp-Ser did not bind to antibody CLB-CAg A (Fig. 1, lanes 5 and 6). From these results it appears that residues between Glu and Met are of particular importance for binding of antibody CLB-CAg A to the FVIII polypeptide Asp-Met.


Figure 1: Immunoprecipitation of FVIII fragments with monoclonal antibodies. Recombinant FVIII fragments were obtained as outlined under ``Experimental Procedures.'' The fragments were labeled by in vitro translation employing [S]methionine(24) . Immunocomplexes were analyzed by 12% (w/v) SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. Lanes represent immunocomplexes of the radiolabeled FVIII fragments from Asp to Ser (lanes 1 and 5), Glu (lanes 2 and 6), Glu (lanes 3 and 7) and Met (lanes 4 and 8) with antibodies CLB-CAg 69 and CLB-CAg A, respectively. The residues Lys-Arg encompass the previously defined epitope for antibody CLB-CAg 69(24) .



Interaction between Synthetic Peptides and Antibody CLB-CAg A or FIXa

A hydropathy analysis of the FVIII A3 domain primary structure was performed in order to determine the hydrophilicity of the region Glu-Met. As can be seen in Fig. 2, this region is part of a markedly hydrophilic exosite encompassing the residues Arg-Asp, which indicates that the region Glu-Met may be exposed at the exterior of the FVIII light chain molecule. The possibility that the hydrophilic exosite comprises a FIXa binding region was addressed by employing a series of overlapping peptides that encompass the FVIII region between Tyr and Ala (Fig. 2). The interaction between these peptides and FIXa was assessed in binding studies employing immobilized peptides. FIXa displayed particular effective binding to peptide Lys-Lys and, to a lesser extent, to peptide Glu-Gln (Fig. 3). The same series of peptides were used to examine antibody CLB-CAg A binding (Fig. 3). Antibody CLB-CAg A displayed a similar pattern of specificity for these peptides as FIXa (Fig. 3), as most effective binding was observed for peptides Lys-Lys and Glu-Gln. Collectively, these data indicate that the A3 domain comprises a hydrophilic exosite that contributes to FIXa binding.


Figure 2: Synthetic peptides of the FVIII A3 domain. Kyte-Doolittle hydropathy analysis (32) of the FVIII A3 domain sequence using a sliding window of 19 amino acids is shown on top. The most hydrophilic region comprises the residues Arg-Asp. Peptides overlapping the FVIII region Tyr-Ala that are used in this study are shown below. The primary sequence of the FVIII region Tyr-Ala is represented using the single-letter code. The lines below the amino acid sequence denote the inclusive amino acid residues of the peptides synthesized.




Figure 3: Binding of CLB-CAg A and FIXa to immobilized synthetic peptides. Antibody CLB-CAg A (625 nM) or FIXa (50 nM) were incubated with immobilized synthetic peptides (0.8 nmol/well added) in 1% (w/v) HSA, 0.1% (v/v) Tween 20, 10 mM CaCl(2), 0.1 M NaCl, 25 mM Tris (pH 7.4) for 1 h at 37 °C. After washing the microtiter plate with the same buffer without HSA, bound CLB-CAg A (solid bars) and FIXa (shaded bars) were quantified by incubating for 15 min at room temperature with peroxidase-labeled goat anti-mouse antibodies and peroxidase-labeled anti-FIX antibody CLB-FIX 14, respectively, as described under ``Experimental Procedures.'' Binding is expressed as percentage of the maximum response. Data represent the mean ± S.D. of at least three experiments. Peptides are indicated by the positions of the amino- and carboxyl-terminal amino acid in the corresponding FVIII sequence (cf.Fig. 2).



Effect of Synthetic Peptides on FIXa Binding to FVIII Light Chain

In order to investigate the interaction between FIXa and various synthetic peptides in solution, the effect of synthetic peptides on binding of FIXa to immobilized FVIII light chain was determined. In these experiments not all of the peptides of Fig. 2could be evaluated, as the solubility of peptides Tyr-Glu, Tyr-Ala, and His-Ala was limited to concentrations that are below the concentrations required for competition studies in solution. As shown in Fig. 4A, the peptide comprising the sequence Gly-Lys was incapable of interfering with binding of FIXa to immobilized FVIII light chain. In contrast, peptides Lys-Lys and Glu-Gln were found to inhibit binding of FIXa to immobilized FVIII light chain in a dose-dependent manner (Fig. 4A). By analyzing these data in a model for competitive inhibition(21) , the inhibition constants were calculated to be 0.19 ± 0.01 mM (mean ± S.D.) and 0.27 ± 0.02 mM for peptide Lys-Lys and Glu-Gln, respectively. Thus, both peptides effectively compete with binding of FVIII light chain to FIXa. Therefore, these peptides should also interfere with FVIII-dependent activation of FX by FIXa.


Figure 4: Effect of synthetic peptides on FIXa-FVIII light chain interaction and FX activation. A, Glu-Gly-Arg chloromethyl ketone-treated FIXa (30 nM) was incubated with immobilized FVIII light chain (0.7 pmol/well) in 0.15 M NaCl, 1% (w/v) HSA, 0.1% (v/v) Tween 20, 5 mM CaCl(2), 25 mM histidine (pH 6.2) for 4 h at 37 °C in the presence of various concentrations of peptide Gly-Lys (bullet), Lys-Lys (circle), or Glu-Gln (box). FIXa binding and calculation of binding parameters was performed as described previously (21) . Binding parameters revealed a K of 0.19 ± 0.01 mM and 0.27 ± 0.02 mM for peptides Lys-Lys and Glu-Gln, respectively. B, activation of FX (0.2 µM) by FIXa (0.7 nM) in the presence of phospholipids (0.1 µM), FVIIIa (0.4 nM), and various concentrations of peptide Gly-Lys (bullet), Lys-Lys (circle), or Glu-Gln (box) in 3 mM CaCl(2), 0.1 M NaCl, 0.2 mg/ml HSA, 0.05 M Tris (pH 7.4) was assayed as described(31) . FXa formation was quantified as described under ``Experimental Procedures.'' The data represent the mean ± S.D. of three experiments.



Effect of Synthetic Peptides on FX Activation

The effect of peptides Glu-Gln, Lys-Lys, and Gly-Lys on FX activation by FIXa in the presence of phospholipids, calcium ions, and FVIII was determined. In the presence of peptide Gly-Lys little, if any, inhibition was observed (Fig. 4B). In contrast, FX activation was effectively inhibited in the presence of peptides Lys-Lys and Glu-Gln (Fig. 4B). Neither of the peptides inhibited FX activation in the absence of FVIII (not shown). Thus, peptides Lys-Lys and Glu-Gln indeed interfere with a FVIII-dependent step within the process of FX activation. The mechanism of inhibition was addressed by studying the effect of the inhibitory peptides on the kinetic parameters K(m) and V(max). In experiments employing peptide Lys-Lys, the apparent K(m) remained unchanged, whereas the apparent V(max) was dependent on the concentration of peptide (Fig. 5). Thus, peptide Lys-Lys inhibits FVIII-dependent FX activation by FIXa by a noncompetitive mechanism. The same results were obtained employing the peptide Glu-Gln (not shown). Using the normal model of noncompetitive inhibition, the inhibition constant was found to be 0.23 ± 0.05 mM and 0.25 ± 0.02 mM for peptides Lys-Lys and Glu-Gln, respectively. These values are similar to the inhibition constant found for the inhibition of the FIXa-FVIII light chain interaction (see Fig. 4A). Apparently, both peptide Lys-Lys and Glu-Gln inhibit FVIII-dependent FX activation by FIXa by interfering in the association between FIXa and FVIII light chain.


Figure 5: Kinetic analysis of FX activation in the presence of peptide Lys-Lys. FXa generation experiments were performed in the absence (bullet) or presence of 0.2 mM (), 0.4 mM (), or 0.6 mM () of the peptide Lys-Lys as described under ``Experimental Procedures,'' except that variable FX concentrations (2.5-50 nM) were used. Initial rates of FXa formation are plotted as a function of the substrate concentration. Data represent the mean ± S.D. of three experiments. The curves were obtained by fitting the data employing the Michaelis-Menten equation. The calculated apparent V(max) values in the absence or presence of 0.2 mM, 0.4 mM, or 0.6 mM peptide were 6.0 ± 0.3, 4.5 ± 0.2, 3.8 ± 0.2, and 2.8 ± 0.1 nM FXa/min, respectively (mean ± S.D.). The apparent K values were 5.3 ± 1.0, 5.0 ± 0.7, 6.1 ± 0.9, and 4.6 ± 0.8 nM FX, respectively.



Interaction of FVIII Light Chain or Synthetic Peptides with Uncleaved and Cleaved Forms of FIX

The FIX zymogen is dissimilar to the fully activated FIXa in that it does not activate FX(13, 27) . To examine whether FIX and FIXa also differ in cofactor binding, the interaction with FVIII light chain and, more specifically, with peptide Lys-Lys was addressed. In line with our previous finding(27) , FIX was less efficient than FIXa in binding FVIII light chain (Fig. 6). In contrast, when FIX and FIXa were compared for binding to the immobilized peptide Lys-Lys, this peptide bound the FIX zymogen and the FIXa enzyme to the same extent (Fig. 6, inset). The same observation was made employing peptide Glu-Gln (not shown). However, studies employing immobilized peptides do not provide quantitative binding parameters. Therefore, the interaction between peptide Lys-Lys and FIX or FIXa was further studied in solution under equilibrium conditions (see ``Experimental Procedures''). These experiments revealed an apparent dissociation constant of 0.20 ± 0.02 mM (mean ± S.D.) for the peptide-FIXa interaction. This value is similar to the inhibition constants found for the inhibition of the FIXa-FVIII light chain interaction and FVIII-dependent activation of FX (see Fig. 4). The same value (0.23 ± 0.02 mM) was also found for the peptide-FIX interaction, demonstrating that indeed peptide Lys-Lys is equally effective in binding FIX or FIXa. Thus, peptide Lys-Lys does not distinguish between uncleaved and cleaved forms of FIX, whereas FVIII light chain does. This finding was confirmed by using FVIII light chain as competitor for the interaction between immobilized peptide Lys-Lys and FIX or FIXa. Binding of FIXa to the immobilized peptide was readily inhibited in the presence of FVIII light chain (Fig. 7). In contrast, FVIII light chain proved to be inefficient in interfering with binding of FIX to the immobilized peptide. These data demonstrate that peptide Lys-Lys and FVIII light chain compete for binding to FIXa but not to the FIX zymogen.


Figure 6: Binding of FIX or FIXa to immobilized peptide Lys-Lys or FVIII light chain. Various concentrations of Glu-Gly-Arg chloromethyl ketone-treated FIXa (bullet) or FIX (circle) were incubated with immobilized FVIII light chain (0.7 pmol/well) as described under ``Experimental Procedures.'' Association between FVIII light chain and FIX or FIXa was assessed as described(21) . Inset, various concentrations of Glu-Gly-Arg chloromethyl ketone-treated FIXa (bullet) or FIX (circle) were incubated with immobilized peptide Lys-Lys (0.8 nmol/well added) as described under ``Experimental Procedures.'' Binding was detected employing the peroxidase-labeled anti-FIX antibody CLB-FIX 14. Absorbance was measured at 450 nm using 540 nm as reference. Plotted is the absorbance versus the concentration of FIX or FIXa. Data represent mean values ± S.D. of three to six experiments.




Figure 7: Effect of FVIII light chain on binding of FIX or FIXa to peptide Lys-Lys. FIX (circle) or FIXa (bullet) (both 50 nM) were incubated with immobilized peptide Lys-Lys (0.8 nmol/well added) in the presence of various concentrations of FVIII light chain as described under ``Experimental Procedures.'' Binding is presented as percentage of binding in the absence of FVIII light chain. The data represent the mean ± S.D. of three experiments.




DISCUSSION

During the process of FX activation, the enzyme FIXa assembles with the nonenzymatic cofactor FVIIIa into a lipid-bound complex. In previous studies we have shown that FVIII light chain contains a site that binds FIXa with high affinity (K(d) approx 15 nM) and that FIXa binding is inhibited by the FVIII light chain-directed antibody CLB-CAg A(21) . Here we show that this antibody is directed against an extensive hydrophilic exosite within the A3 domain ( Fig. 2and Fig. 3). Because such hydrophilic regions are likely to be exposed at the exterior of the protein(32) , we addressed the possibility that this exosite comprises a FIXa binding site. Indeed, FIXa binds to synthetic peptides that consist of FVIII sequences that are part of the hydrophilic exosite Arg-Asp (Fig. 3). Competition studies demonstrated that peptides corresponding to the exosite regions Lys-Lys and Glu-Gln effectively inhibit binding of FIXa to immobilized FVIII light chain (Fig. 4A). The same peptides also interfere with FVIII-dependent activation of FX by FIXa (Fig. 4B). Inhibition of FX activation is noncompetitive (Fig. 5), which strongly suggests that the peptides inhibit the enzyme FIXa by binding at a site distinct from the substrate binding pocket. Collectively, our data demonstrate that peptides consisting of the FVIII amino acid residues Lys-Lys and Glu-Gln represent a FIXa binding site. It is of importance to note that the K(i) for the binary FIXa-FVIII light chain interaction is similar to the K(i) found for FX activation in the complete FX activating complex, thus including the entire FVIIIa heterotrimer ( Fig. 4and Fig. 5). Assembly of the functional FIXabulletFVIIIa complex apparently is directly related to binding of FIXa to the FVIII A3 domain exosite. In this respect it should be mentioned that FIXa binding is not an exclusive property of the FVIII A3 domain. FIXa recognition sites have been identified within the FVIII A2 domain regions Ser-Gln(22) and Arg-Ser(23) . As synthetic peptides corresponding to these A2 domain regions also interfere with FVIIIa cofactor function, it seems reasonable to assume that both FVIII heavy chain and light chain regions participate in FIXa-FVIIIa complex formation.

As peptides Lys-Lys and Glu-Gln proved more efficient in their interaction with FIXa than the other peptides tested ( Fig. 3and Fig. 4), we propose that the minimal requirements for FIXa binding are met by the overlapping residues Glu-Lys. This region, including its direct environment (residues Gly-Gln), is strikingly rich in basic Lys residues, which are located at positions 1804, 1808, 1813, and 1818 (Fig. 8). These Lys residues appear to be unique for the FVIII A3 domain, as they are not only lacking in the FVIII A1- and A2 domains but also in the A3 domains of the structurally related proteins FV and ceruloplasmin (Fig. 8). The same Lys residues are conserved in the FVIII A3 domain of a rodent species (Fig. 8), which would be compatible with the involvement of these residues in a FVIII A3 domain-specific event such as FIXa binding. However, peptide Gly-Lys with Lys residues at 1804, 1808, and 1813 proved considerably less efficient in its interaction with FIXa than peptide Glu-Gln with Lys residues at 1813 and 1818 ( Fig. 3and Fig. 4). Apparently, the presence of the Lys residues alone is not sufficient for FIXa binding. It should be mentioned that peptide Glu-Gln contains a triplet of aromatic residues (Tyr-Phe-Trp), which is also present in the inhibitory peptide Lys-Lys but lacking in the noninhibitory peptide Gly-Lys. Because part of this sequence is conserved in other A domains (Fig. 8), it is unclear how these residues may be involved in a FVIII A3 domain-specific function. To what extent individual amino acids in the FIXa binding region contribute to FIXabulletFVIII light chain complex formation, therefore, remains to be investigated. It seems of interest to note that mutations at positions Ser, Leu, Met, Pro, and Thr have been determined to be associated with moderately severe hemophilia A(33) . As these mutations are in close proximity to the FIXa binding region, it is tempting to speculate that the bleeding tendency that is associated with these mutations is due to a suboptimal assembly of the FIXabulletFVIII light chain complex.


Figure 8: Comparison of the FVIII A3 domain region Gly-Gln with corresponding portions of other A domains. The primary structure of the A3 domain of human FVIII (2, 3) is compared with the corresponding regions of the A3 domains of murine FVIII (mFVIII)(36) , human FV (hFV)(4, 5) , and ceruloplasmin (hCer.) (6) and with both A1 and A2 domains of human FVIII (hFVIII)(2, 3) . The sequences are aligned as described(36, 37) . Amino acids that are identical to the corresponding residues in the FVIII A3 domain are boxed.



Recently, we demonstrated that uncleaved FVIII light chain is similar to FVIII light chain derivatives that have been cleaved by the activators thrombin or FXa in that they display similar affinity for FIXa(31) . Apparently, the FIXa recognition site is fully exposed in the intact FVIII light chain. In agreement with previous observations, we have found that FVIII light chain is more efficient in binding the fully activated FIXa than the uncleaved FIX zymogen ( Fig. 6and Fig. 7; (27) ). These data indicate that FVIII light chain displays preferential binding to the enzyme FIXa rather than to the nonactivated FIX zymogen. In this regard, FVIII light chain seems similar to FVa and thrombomodulin, because these cofactors are more efficient in binding to their respective enzymes than to the uncleaved proenzymes(34, 35) . Surprisingly, this seems to be untrue for the FIXa-binding peptides Lys-Lys and Glu-Gln, because these peptides do not distinguish between the enzyme FIXa and the FIX zymogen ( Fig. 6and 7). Several possibilities may be considered that may explain these observations. First, it is possible that the relative size of the FVIII light chain prevents binding to the intact FIX zymogen, while this restriction is overcome by limited proteolysis of the zymogen at its activation sites Arg or Arg(27) . It should be noted here that cleavage at Arg is sufficient for full exposure of the FVIII light chain binding site, whereas cleavage at Arg results in a suboptimal exposure(27) . Alternatively, the conformation of the FIXa-binding motif in synthetic peptides may differ from its conformation in the complete FVIII light chain. This may be due to other portions of the light chain that provide the region Glu-Lys its specificity for binding to the activated form of FIX. In this respect it is of importance to note that the Asn residue at position 1810 is a potential site for N-linked glycosylation in FVIII(2, 3) . As this site is located adjacent to the FIX-binding motif Glu-Lys, it seems conceivable that glycosylation of this site contributes to the specificity for binding of FIXa to its binding sequence Glu-Lys.


FOOTNOTES

*
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§
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(^1)
The abbreviations used are: FVIII, factor VIII; FV, factor V; FX, factor X; FIX, factor IX; FIXa, factor IXabeta; HSA, human serum albumin; HPLC, high pressure liquid chromatography.


ACKNOWLEDGEMENTS

We express our gratitude to Prof. Dr. W. G. van Aken, Dr. O. D. Christophe, and Dr. J. Voorberg for helpful discussions and critical reading of the manuscript. We thank M. van den Berg for the preparation of synthetic peptides.


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