Residues Phe342-Asn346 of Activated Coagulation Factor IX Contribute to the Interaction with Low Density Lipoprotein Receptor-related Protein*

Jakub RohlenaDagger , Joost A. KolkmanDagger , Ria C. BoertjesDagger , Koen MertensDagger §, and Peter J. Lenting||

From the Dagger  Department of Plasma Proteins, Sanquin Research at CLB, 1066 CX Amsterdam, The Netherlands, the || Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Centre, Utrecht, The Netherlands, and the § Utrecht Institute for Pharmaceutical Sciences, University Utrecht, 3584 CA Utrecht, The Netherlands

Received for publication, September 5, 2002, and in revised form, December 20, 2002

    ABSTRACT
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When blood coagulation factor IX is converted to activated factor IX (factor IXa), it develops enzymatic activity and exposes the binding sites for both activated factor VIII and the endocytic receptor low density lipoprotein receptor-related protein (LRP). In the present study we investigated the interaction between factor IXa and LRP in more detail, using an affinity-purified soluble form of LRP (sLRP). Purified sLRP and full-length LRP displayed similar binding to factor IXa. An anti-factor IX monoclonal antibody CLB-FIX 13 inhibited factor IXa·sLRP complex formation. Both the antibody and a soluble recombinant fragment of LRP (i.e. cluster IV) interfered with factor IXa amidolytic activity, suggesting that the antibody and LRP share similar binding regions near the active site of factor IXa. Next, a panel of recombinant factor IXa variants with amino acid replacements in the surface loops bordering the active site was tested for binding to antibody CLB-FIX 13 and sLRP in a solid phase binding assay. Factor IXa variants with mutations in the region Phe342-Asn346, located between the active site of factor IXa and factor VIII binding helix, showed reduced binding to both antibody CLB-FIX 13 and sLRP. Surface plasmon resonance analysis revealed that the variant with Asn346 replaced by Asp displayed slower association to sLRP, whereas the variant with residues Phe342-Tyr345 replaced by the corresponding residues of thrombin showed faster dissociation. Recombinant soluble LRP fragment cluster IV inhibited factor IXa-mediated activation of factor X with IC50 values of 5 and 40 nM in the presence and absence of factor VIII, respectively. This inhibition thus seems to occur via two mechanisms: by interference with factor IXa·factor VIIIa complex assembly and by direct inhibition of factor IXa enzymatic activity. Accordingly, we propose that LRP may function as a regulator of blood coagulation.

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Factor IX (FIX)1 is a vitamin K-dependent serine protease precursor that, upon activation, participates in the blood coagulation process (1). Its relevance for hemostasis is demonstrated by the fact that the absence of functional FIX is associated with severe hemophilia B. FIX circulates in plasma as a 56-kDa single-chain zymogen. Upon initiation of blood coagulation, FIX is converted into its active form FIXa by means of limited proteolysis mediated by factor VIIa·tissue factor complex or factor XIa (2). During this process, the activation peptide is cleaved off resulting in a two-chain molecule comprising covalently linked light and heavy chains. The NH2-terminal light chain (18 kDa) is composed of a gamma -carboxyglutamic acid (Gla)-containing domain and two epidermal growth factor-like domains (3, 4). The COOH-terminal heavy chain (28 kDa) contains a trypsin-like protease domain that carries the enzymatic activity of FIXa (5). Upon activation, FIXa forms a complex with activated factor VIII (FVIIIa). This complex then catalyzes FX activation in the presence of Ca2+ ions and phospholipid surface, which leads to further propagation of blood coagulation (6, 7). Various mechanisms have been proposed to control FIXa activity. First, FIXa may be inactivated via limited proteolysis mediated by elastase or plasmin (8-10). Second, FIXa may form a complex with the serine protease inhibitors antithrombin or protease nexin-2 (11-13). It has been reported that the FIXa·protease nexin-2 complex is recognized and internalized by the low density lipoprotein receptor-related protein (LRP) (14). Moreover, we have recently demonstrated that the FIXa enzyme itself is a ligand for LRP as well (15).

LRP, also known as alpha 2-macroglobulin receptor or CD91, is a member of the low density lipoprotein receptor family of transmembrane glycoproteins (16, 17). LRP consists of two noncovalently associated chains: the amino-terminal extracellular alpha -chain (515 kDa) and the carboxyl-terminal transmembrane beta -chain (85 kDa). The cytoplasmic part of the beta -chain contains the endocytosis-specific signal motif, whereas the alpha -chain contains 4 regions enriched in complement-type repeats, also called LRP clusters I, II, III, and IV (18). LRP clusters II and IV have been identified to be predominant in terms of ligand binding (19). LRP exhibits a remarkable ability to bind a broad range of structurally and functionally unrelated ligands (16, 17). This implies that LRP may participate in a number of processes that include lipoprotein metabolism, cell growth and migration, neuronal regeneration, fibrinolysis, and blood coagulation. LRP is most prominently present in the brain, liver, lung, and placenta. The many cell types that express LRP are, among others, parenchymal cells, Kupffer cells, neurons, astrocytes, smooth muscle cells, monocytes, adipocytes, and fibroblasts (20). A truncated form of LRP, referred to as soluble LRP or sLRP, circulates in plasma (21). It comprises the complete ligand binding alpha -chain and the NH2-terminal portion of the beta -chain (22).

It has been shown that low molecular weight heparin effectively interferes with the binding of FIXa to LRP (15). Because FIXa binds heparin with high affinity (23), this suggests that heparin and LRP might require the same structural determinants for their interaction with FIXa. Nevertheless, FIX zymogen also exhibits high affinity heparin binding (24), yet it fails to bind to LRP. Clearly, additional regions that become available upon FIX activation must participate in FIXa interaction with LRP.

The aim of the present study was to identify regions within the FIXa molecule that contribute to the interaction with LRP. By using monoclonal anti-FIX antibodies and recombinant FIXa chimeric molecules, we have found that the FIXa surface region Phe342-Asn346 contributes to the interaction between FIXa and LRP. Moreover, we show that LRP binding affects the enzymatic function of FIXa.

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Materials-- CNBr-Sepharose 4B, Q-Sepharose FF, glutathione-Sepharose, and Protein A-Sepharose were from Amersham Biosciences. Heparin (grade A-1), vitamin K1, and benzamidine were obtained from Sigma. Low molecular weight (LMW) heparin was from Pharmacia and Upjohn (Woerden, The Netherlands). 4-(2-Aminoethyl)-benzenesulfonyl fluoride (Pefabloc) was from Roche Molecular Diagnostics. Pfu-polymerase was from Stratagene (Cambridge, United Kingdom). Oligonucleotide primers, restriction enzymes, DNA modifying enzymes, Dulbecco's modified Eagle's medium, geneticin, and Fungizone were purchased from Invitrogen. Penicillin/streptomycin and fetal calf serum were from BioWhittaker (Verviers, Belgium). CH3-SO2-D-leucyl-L-glycyl-L-arginyl-p-nitroanilide (CH3-SO2-LGR-pNA), product name CBS 31.39 was from Diagnostica Stago (Asnières, France). Pefachrome Xa was from Pentapharm AG, Basel, Switzerland. Microtiter plates were from Dynatech (Plockingen, Germany) or, for FIXa amidolytic activity tests, FX activation assay and antithrombin titration, from Corning (Badhoevedorp, The Netherlands). Cell factories (6000 cm2) were from Nunc A/S (Roskile, Denmark). BIAcoreTM-2000 and -3000 biosensor system and reagents (amino-coupling kit and CM-5 biosensor chips) were from Biacore AB (Uppsala, Sweden).

sLRP Purification-- sLRP was purified from human plasma by affinity chromatography employing receptor-associated protein fused to glutathione S-transferase (GST-RAP) being coupled to Sepharose, with 1 mg of GST-RAP (see the following section) coupled per ml of CNBr-Sepharose 4B according to the manufacturer's instructions. Plasma was centrifuged and the supernatant was filtrated through a 0.4-µm filter. The filtrate was supplemented with 10 mM CaCl2, 10 mM benzamidine, 50 units/ml heparin, 25 mM Hepes (pH 7.4) by the addition of 10 times concentrated buffer. 400 ml of filtrate was then loaded onto a RAP-Sepharose column (20 ml). The column was extensively washed with buffer containing 150 mM NaCl, 10 mM CaCl2, 10 mM benzamidine, 5% (v/v) glycerol, 25 mM Hepes (pH 7.4), and bound sLRP was subsequently eluted using the same buffer, but then with CaCl2 replaced by 20 mM EDTA. Remaining contaminants were removed by ion-exchange chromatography on a Q-Sepharose column employing a gradient from 0.025 to 1 M NaCl in 5% (v/v) glycerol, 25 mM Hepes (pH 8.0). Purified sLRP was subsequently concentrated on the same column by elution with 1 M NaCl in the presence of 10 mM Pefabloc. Concentrated sLRP was extensively dialyzed against the buffer containing 100 mM NaCl, 5 mM CaCl2, 50 mM Tris (pH 7.4).

Other Proteins-- GST-RAP fusion protein was expressed in Escherichia coli DH5alpha as described previously (25). GST-RAP was purified employing glutathione-Sepharose according to the manufacturer's instructions. Because the GST tag does not interfere with binding properties of RAP (25), GST-RAP was used in the present study. Full-length human LRP was generously provided by Dr. S. Moestrup, University of Aarhus, Aarhus, Denmark. Transfected baby hamster kidney cell lines (a kind gift of Dr. H. Pannekoek, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands) were used to produce recombinant LRP clusters II and IV. Both LRP clusters were purified by affinity chromatography employing Sepharose-coupled GST-RAP (19). Monoclonal antibodies CLB-FIX 11 (26) and CLB-FIX 14 (27) have been described previously. Mouse monoclonal antibodies CLB-FIX 12 and CLB-FIX 13 were prepared as described (28). Polyclonal anti-FIX antibodies were obtained by immunizing rabbits and were immunopurified by Sepharose-coupled human FIX (28). All monoclonal antibodies were purified employing Protein A-Sepharose as recommended by the manufacturer. Horseradish-conjugated antibodies were prepared as described (29). FX was obtained by conventional chromatography techniques as described (30). Normal human plasma FIX was purified by immunoaffinity chromatography using monoclonal antibody CLB-FIX D4 (26). FXIa was purchased from Enzyme Research Laboratories (South Bend, IN). FVIII light chain was obtained from human plasma FVIII by EDTA dissociation followed by immunoaffinity chromatography (28). Antithrombin and human serum albumin (HSA) were from the Sanquin Plasma Products Division.

Construction of Expression Vectors Encoding Recombinant FIX Variants-- FIXdes(N264,K265), FIX-K265A, and FIX199-204/FII were described previously (31, 32). FIX258-267/FX, FIX-N346D, and FIX342-345/FII were constructed using the mammalian expression plasmid pKG5 containing human FIX cDNA (33) as a template for the PCR-based mutagenesis (34), employing partially overlapping oligonucleotide primers: 5'-TTCACAAAGGAGACCTATGACCATGACATTGCCCTTCTG-3' (sense) and 5'-ATAGGTCTCCTTTGTGAACCGGTGGTGAGGAATAATTGC-3' (antisense) for FIX258-267/FX; 5'-TTCTGTGCCGGCTTCCATGAAGGA-3' (sense), 5'-ATGGAAGCCGGCACAGAACATGTTGTTAGTGATTCTGATCTTTGTAGATCGAAG-3' (antisense) for FIX342-345/FII and 5'-ATGGAAGCCGGCACAGAACATGTTGTCATAGATGGT-5' (antisense) for FIX-N346D. The mutated full-length cDNA was digested with restriction enzymes BamHI and HindIII and subsequently ligated into the corresponding restriction sites in pKG5. The final FIX constructs were verified by DNA sequence analyses. Table II shows changes in the amino acid sequence for all recombinant FIX variants as opposed to FIX wild type.

Recombinant FIX-- Stable cell lines producing FIX variants were obtained by the calcium phosphate co-precipitation method and selection with geneticin (32). As reported previously, the expression system used in this study yields recombinant FIX molecules with normal calcium-dependent properties and similar activities for recombinant wild type and plasma-derived FIXa (26, 33, 35) with an average Gla content of 11 mol of Gla/mol of protein (36). Recombinant FIX was purified by affinity chromatography employing anti-FIX monoclonal antibody CLB-FIX 14 from concentrated medium obtained by culturing cell lines producing FIX in 1-liter cell factories (35). FIX was converted into FIXa by limited FXIa-mediated proteolysis as described (26). FIXa was purified from the activation mixture employing anion exchange chromatography (26).

Protein Concentrations-- Protein concentrations were determined by the method of Bradford (37), using HSA as a standard. FIX antigen levels were determined by enzyme-linked immunosorbent assay employing a previously described method (26). FIXa concentrations were determined by active-site titration with antithrombin in the presence of heparin (26).

Amidolytic Activity of FIXa-- FIXa amidolytic activity was measured as described previously (26). Shortly, the conversion of 2.5 mM CH3-SO2-LGR-pNA by FIXa was determined in a 96-well microtiter plate (Costar) in 100 mM NaCl, 10 mM CaCl2, 0.2% (w/v) HSA, 50 mM Tris (pH 7.4 or 8.4) by measuring the absorbance at 405 nm.

FX Activation Assay-- FX activation was performed in the presence of phospholipid vesicles (phosphatidylserine and phosphatidylcholine in 1 to 1 M ratio), essentially as previously described (30). Phospholipid vesicles (100 µM) and calcium ions were preincubated in siliconized glass tubes for 10 min in a buffer containing 100 mM NaCl, 10 mM CaCl2, 0.5% (w/v) ovalbumin, 50 mM Tris (pH 7.4). After preincubation, FIXa (15 nM) with or without the LRP clusters II or IV (also preincubated for 10 min) was added. The reaction was started by addition of FX (200 nM). When FX activation was measured in the presence of FVIII, FVIII (0.3 nM) and thrombin (5 nM) were added 1 min before the reaction was started. FIXa concentration was then 0.3 nM. Subsamples were drawn in time and the reaction was terminated in buffer containing EDTA and 1 unit/ml hirudin. The amount of FXa generated was determined spectrophotometrically at 405 nm, employing Pefachrome Xa substrate as previously described (30).

Surface Plasmon Resonance Analysis-- Studies were performed using the BIAcoreTM biosensor system, based on surface plasmon resonance (SPR) technology. SPR analysis was performed essentially as described previously (38). Proteins were immobilized onto CM5 sensorchips using the amine coupling method according to the manufacturer's instructions. Routinely, a control channel was activated and blocked in the absence of protein. Binding to coated channels was corrected for binding to noncoated channel (<5% of binding to coated channel). SPR analysis was performed in 150 mM NaCl, 2 mM CaCl2, 0.005% (v/v) Tween 20, 20 mM Hepes (pH 7.4) at 25 °C with a flow of 20 µl/min. The sensorchips were regenerated by incubating with 100 mM H3PO4 for 30 s at a flow of 20 µl/min. For quantitative measurements of FIXa binding to LRP or sLRP, experiments were performed using 7 different concentrations (6-110 nM) of FIXa. BIAevaluation 3.1 software (Biacore AB) was used to analyze the association and dissociation curves of the sensorgrams. Interaction constants were determined by performing nonlinear global fitting of data corrected for bulk refractive index changes. Data were fitted according to various models available within the software. A model describing the interaction between FIXa and two independent binding sites (heterologous ligand, parallel reactions) was found to provide the best fit of the experimental data for both sLRP and full-length LRP. This same model was previously used by us to describe the interaction between FIXa and full-length LRP (15).

Binding of FIXa to Immobilized sLRP in a Solid-phase Binding Assay-- Purified sLRP was adsorbed onto microtiter wells (88 fmol/well) in 50 mM NaHCO3 (pH 9.5) for 16 h at 4 °C in a volume of 50 µl/well. The wells were then blocked with 2% (w/v) HSA in 150 mM NaCl, 5 mM CaCl2, 25 mM Hepes (pH 7.4) for 2 h at 37 °C in a volume of 100 µl. After washing, 50 µl of FIXa was added in 150 mM NaCl, 5 mM CaCl2, 0.1% (v/v) Tween 20, 0.1% (w/v) HSA, 25 mM Hepes (pH 7.4). Bound FIXa was detected by incubating with peroxidase-labeled monoclonal antibody CLB-FIX 11 for 15 min at 37 °C in a volume of 50 µl.

Binding of Recombinant FIXa Variants to Immobilized Monoclonal Antibodies-- Monoclonal antibodies CLB-FIX 13 or CLB-FIX 14 were adsorbed onto the microtiter wells (3.3 pmol/well and 0.33 pmol/well respectively) in 50 mM NaHCO3 (pH 9.5) for 16 h at 4 °C in a volume of 50 µl/well. The wells were blocked with 2% (w/v) HSA in 150 mM NaCl, 5 mM CaCl2, 25 mM Hepes (pH 7.4) for 2 h at 37 °C in a volume of 100 µl. After washing, 50 µl of FIXa in concentrations between 0 and 10 nM was added in 150 mM NaCl, 5 mM CaCl2, 0.1% (v/v) Tween 20, 0.1% (w/v) HSA, 25 mM Hepes (pH 7.4). Bound FIXa was detected by incubating with 50 µl of peroxidase-labeled monoclonal antibody CLB-FIX 11 for 1 h at 37 °C.

Inhibition of FIXa Activity by Antithrombin in the Presence of LMW Heparin-- Experiments were performed under pseudo first-order rate conditions essentially as previously described (39). Briefly, 150 nM FIXa was incubated with 1.5 µM antithrombin and 400 nM LMW heparin at 37 °C in 100 mM NaCl, 10 mM CaCl2, 0.2% HSA, 50 mM Tris, pH 7.4. Subsamples of 50 µl were drawn in regular intervals between 10 s and 5 min and pipetted into a 96-well microtiter plate containing Polybrene (1 mg/ml final concentration) to stop the action of heparin immediately. Chromogenic substrate CBS 31.39 was added at a final concentration of 1.5 mM and residual FIXa activity was measured at 405 nm. The time-dependent inhibition of FIXa activity was fitted to a first-order rate equation to obtain values for the apparent pseudo first-order rate constant.

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Soluble and Full-length LRP Display Similar Ligand Binding-- To study FIXa-LRP interaction we made use of the truncated soluble form of LRP (sLRP) (21). sLRP was purified from human plasma employing RAP-GST based affinity chromatography. The product obtained was homogeneous as determined by SDS-polyacrylamide gel electrophoresis. In addition, sLRP efficiently bound GST-RAP, FVIII light chain, and FIXa (data not shown), all of which are established ligands for LRP. The interaction between FIXa and both full-length LRP and sLRP was analyzed in more detail by assessing the association and dissociation rate constants. For both LRP species, the experimental data displayed a faster and a slower phase in FIXa binding and could be adequately described employing a heterologous two-site binding model, indicating the presence of (i) an interaction site with fast binding and somewhat lower affinity, and (ii) an interaction site with slower binding but high affinity for FIXa (class 1 and class 2 binding sites, respectively). The calculated association (kon) and dissociation (koff) rate constants that followed from this model were similar for full-length LRP and sLRP (Table I), indicating that sLRP and full-length LRP are indistinguishable in terms of FIXa binding.

                              
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Table I
Kinetics of pd-FIXa binding to immobilized LRP and sLRP in SPR
Association and dissociation between pd-FIXa and both LRP and sLRP immobilized onto CM5-sensorchip (surface density 13 fmol/mm2 for both LRP species) was investigated by passing 7 different concentrations (6-110 nM) over the LRP- or sLRP-coated channels for 120 s. Data were analyzed by performing nonlinear global fitting of data corrected for bulk refractive index changes to calculate association (kon) and dissociation (koff) rate constants employing a two-site binding model. Each binding site is referred to as 1 and 2, respectively. Data represent average values (± S.D.) of multiple experiments.

FIXa but Not FIX Zymogen Binds to Immobilized sLRP-- Binding of FIXa to sLRP was further analyzed in an immunosorbent assay by incubating immobilized sLRP with FIXa in various concentrations. Bound FIXa was detected using monoclonal anti-FIX antibody CLB-FIX 11, which is directed against the Gla domain of FIXa (40). As shown in Fig. 1A, FIXa bound to immobilized sLRP in a dose-dependent and saturable manner with half-maximal binding at 45 nM FIXa. In contrast, no binding of FIX zymogen to immobilized sLRP could be detected. This is consistent with our previous observation in which no binding of FIX zymogen to immobilized full-length LRP was observed using SPR analysis (15). The specificity of the interaction between FIXa and sLRP was further characterized in competition experiments using sLRP in solution. Whereas FIXa bound efficiently to immobilized sLRP in the absence of sLRP in solution, binding was inhibited in a dose-dependent manner in its presence (Fig. 1B). Half-maximum inhibition was observed at 8 nM sLRP. We further tested the ability of recombinant LRP fragments clusters II and IV to interfere with binding of FIXa to immobilized sLRP. Both recombinant fragments inhibited binding of FIXa to sLRP, albeit with different efficiencies (Fig. 1C). Half-maximum inhibition was obtained at 18 and 4 nM for clusters II and IV, respectively. In addition, binding of FIXa to cluster II was blocked in the presence of cluster IV (Fig. 1D). These results demonstrate that sLRP, like full-length LRP, interacts exclusively with the activated form of FIX and that LRP clusters II and IV share overlapping binding sites in FIXa.


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Fig. 1.   Analysis of FIX and FIXa binding to immobilized sLRP and LRP cluster II. A, binding of FIX(a) to sLRP. FIX or FIXa in various concentrations were incubated with immobilized sLRP (88 fmol/well) in the solid-phase binding assay as described under "Experimental Procedures." Factor IXa is expressed as the percentage of maximal binding. B, competition of sLRP in solution for FIXa binding to sLRP. FIXa (25 nM) was incubated with immobilized sLRP as described above in the presence of various concentrations of sLRP in solution. C, competition of LRP clusters II and IV for FIXa binding to sLRP. FIXa (25 nM) was incubated with immobilized sLRP as described above in the presence of various concentrations of LRP clusters II (open circles) or IV (closed circles). D, competition of LRP cluster IV for FIXa binding to cluster II. FIXa (25 nM) was incubated with immobilized LRP cluster II (890 fmol/well) in the presence of increasing concentrations of LRP cluster IV. The bound FIXa was detected as described under "Experimental Procedures." For panels B-D, binding is expressed as the percentage of binding in the absence of competitors. Data represent mean (±S.D.) of three independent experiments.

Monoclonal Anti-FIX Antibody CLB-FIX 13 Interferes with FIXa·sLRP Complex Formation-- To gain insight into the location of LRP interactive sites within the FIXa molecule, several monoclonal antibodies directed against FIX were analyzed for their ability to inhibit the FIXa-sLRP interaction. Binding of FIXa to immobilized sLRP was assessed in the presence of monoclonal anti-FIX antibodies in various concentrations. As shown in Fig. 2, antibodies CLB-FIX 12 and CLB-FIX 14 did not inhibit the binding of FIXa to sLRP. In contrast, antibody CLB-FIX 13 inhibited binding of FIXa to sLRP in a dose-dependent manner, although the inhibition was incomplete (Fig. 2). Apparently, antibody CLB-FIX 13 impairs the formation and/or the stability of the FIXa·sLRP complex. This suggests that this particular antibody and sLRP share overlapping binding regions within the FIXa molecule.


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Fig. 2.   Effect of anti-FIX monoclonal antibodies on FIXa binding to immobilized sLRP. FIXa (25 nM) was incubated with immobilized sLRP as described under "Experimental Procedures" in the presence of various concentrations (0-100 nM) of monoclonal anti-FIX antibodies CLB-FIX 14 (squares), CLB-FIX 12 (triangles), and CLB-FIX 13 (circles). Binding is expressed as the percentage of binding in the absence of monoclonal antibodies. Data represent the mean (± S.D.) of three independent experiments.

Both Antibody CLB-FIX 13 and LRP Interfere with FIXa Amidolytic Activity-- If sLRP and antibody CLB-FIX 13 bind to the same region in FIXa, they should have a similar effect on FIXa enzymatic function. As for antibody CLB-FIX 13, it interferes with FIXa-mediated FX activation in both the presence and absence of FVIII (results not shown). Furthermore, the effect of this antibody on FIXa-mediated hydrolysis of the synthetic substrate CH3-SO2-LGR-pNA was examined. Whereas in the presence of monoclonal anti-FIX antibody CLB-FIX 14 the rate of hydrolysis remained unchanged, the addition of CLB-FIX 13 resulted in a dose-dependent inhibition of substrate hydrolysis (Fig. 3A). Next, the effect of recombinant LRP clusters II and IV on FIXa-mediated hydrolysis of the same substrate was investigated. The addition of LRP cluster II did not affect FIXa activity. In contrast, LRP cluster IV inhibited substrate hydrolysis in a dose-dependent manner (Fig. 3B) indicating that LRP clusters II and IV bind FIXa in a different manner. Collectively, these data suggest that CLB-FIX 13 and LRP interact with the same region, which is located close to the active site of FIXa. Identification of the epitope of antibody CLB-FIX 13 should therefore help to locate the interactive region for LRP.


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Fig. 3.   Effect of LRP and anti-FIXa monoclonal antibody CLB-FIX 13 on FIXa amidolytic activity. A, hydrolysis of the substrate CH3-SO2-LGR-pNA (2.5 mM) by FIXa (150 nM) in 100 mM NaCl, 10 mM CaCl2, 0.2% (w/v) HSA, 50 mM Tris (pH 8.4) was assessed in the presence of increasing concentrations (0-500 nM) of anti-FIXa antibodies CLB-FIX 13 (closed circles) and CLB-FIX 14 (open circles). B, hydrolysis of the substrate CH3-SO2-LGR-pNA (2.5 mM) by FIXa (75 nM) in 100 mM NaCl, 10 mM CaCl2, 0.2% (w/v) HSA, 50 mM Tris (pH 7.4) was assessed in the presence of increasing concentrations (0-250 nM) of recombinant LRP clusters II (open circles) and IV (closed circles). Data represent the mean (± S.D.) of three independent experiments.

FIXa Regions Asn258-Asn267 and Phe342-Asn346 Are Involved in Binding of Monoclonal Antibody CLB-FIX 13-- Surface loops Asn199-Ala204, His256-His268, and Thr340-Asn347, bordering the active site of FIXa, are implicated in the recognition of macromolecular substrates (31, 32, 41). To locate its interactive site in the FIXa molecule, the binding of antibody CLB-FIX 13 to a panel of recombinant FIXa variants with alterations in these three loops was investigated in a solid phase binding assay (see Table II and Fig. 4). All FIXa variants bound the control antibody CLB-FIX 14 in a manner similar to recombinant wild type FIXa (Table II). With regard to CLB-FIX 13, however, only variants FIXa199-204/FII and FIXa-K265A were similar to wild type FIXa in binding to this antibody. In contrast, deletion of residues Asn264-Lys265 resulted in a 10-fold decrease in half-maximal binding (Table II). Moreover, replacement of the sequence Asn258-Asn267 by the corresponding residues of FX was associated with a complete loss of binding to antibody CLB-FIX 13 (Table II). A severe interactive defect was also observed for FIXa variants FIXa342-345/FII and FIXa-N346D (Table II). Thus, surface regions Asn258-Asn267 and Phe342-Asn346 both contribute to the binding of antibody CLB-FIX 13. 

                              
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Table II
Amino acid substitutions and binding of antibodies CLB-FIX 13 and CLB-FIX 14 for individual recombinant FIX variants
Changes introduced in the amino acid sequence are shown in bold. FIX amino acid numbering is used. Values of half-maximal binding (± S.D.) for interaction of FIXa variants with antibodies CLB-FIX 13 and CLB-FIX 14 were derived from the solid phase binding assay. Experiments were performed in triplicate as indicated under "Experimental Procedures."


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Fig. 4.   Representation of the protease domain of porcine FIXa. FIXa regions 199-204, 258-267, and 342-346 are shown in black. Positions of the mutations are indicated. The covalently bound inhibitor D-Phe-Pro-Arg-chloromethyl ketone is also shown (61).

FIXa Region Thr340-Asn347 Contributes to FIXa·sLRP Complex Formation-- The observations that (i) antibody CLB-FIX 13 can inhibit FIXa-sLRP complex formation and (ii) surface regions His256-His268 and Thr340-Asn347 are involved in the interaction of FIXa with antibody CLB-FIX 13 suggest that the same regions contribute to LRP binding as well. Therefore, the set of recombinant FIXa variants described in the previous section were investigated for sLRP binding. To this end, FIXa variants in various concentrations were incubated with sLRP immobilized onto a microtiter plate (Fig. 5A). FIXa variants FIXa199-204/FII, FIXa258-267/FX, FIXa-K265A, and FIXades(N264,K265) bound sLRP similarly to wild type recombinant FIXa. In contrast, FIXa variants FIXa-N346D and FIXa342-345/FII displayed markedly reduced binding. To distinguish between defects in association and dissociation, the two FIXa variants with reduced LRP binding were further analyzed by surface plasmon resonance (Fig. 5B). For these mutants, reduced sLRP binding was particularly apparent in the second, slower phase of association and dissociation. Under the conditions of Fig. 5B, the FIXa-N346D variants displayed 5-fold slower association than wild type FIXa, whereas FIXa342-345/FII displayed 2-fold faster dissociation. Full kinetic analysis employing multiple FIXa concentrations (40-100 nM) revealed that KD2 (mean ± S.D.) was 19 ± 5 nM for wild type FIXa, while KD2 was 120 ± 10 nM for FIXa-N346D and 44 ± 9 nM for FIXa342-345/FII. Thus, the surface loop Thr340-Asn346 contributes to the affinity of FIXa for sLRP.


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Fig. 5.   Analysis of binding of recombinant FIXa variants to immobilized sLRP. A, binding of recombinant FIXa variants (0-100 nM) to sLRP immobilized on a microtiter plate (88 fmol/well) was assessed as described under "Experimental Procedures." Data represent the mean of three independent experiments. Standard deviation was 15% on average. Wild type recombinant FIXa (closed circles), FIXa258-267/FX (open circles), FIXa-K265A (open squares), FIXades(N264,K265) (closed triangles), FIXa-N346D (open triangles), FIXa342-345/FII (closed squares), and FIXa199-204/FII (inverted open triangles) are shown. B, SPR analysis of binding of recombinant FIXa variants to immobilized sLRP. 80 nM wild type FIXa (I), FIXa-342-345/FII (II), and FIXaN346D (III) were passed over sLRP immobilized on a CM5 sensor chip at a density of 12 fmol/mm2 as described under "Experimental Procedures." Association and dissociation were monitored for 975 s. Data represent a typical experiment.

We have previously reported that LMW heparin inhibits the binding of FIXa to LRP, suggesting that part of the LRP-interactive site overlaps that of heparin. It was of interest therefore to compare FIXa to FIXa-N346D and FIXa342-345/FII for their interaction with LMW heparin. This interaction was examined by the ability of LMW heparin to enhance inhibition of amidolytic activity of these proteases by antithrombin (see also "Experimental Procedures"). As expected, inhibition of FIXa activity by antithrombin was enhanced by LMW heparin, and the pseudo first-rate constant of this reaction was (1.1 ± 0.2) × 10-2 s-1. A similar rate constant was obtained for FIXa342-345/FII (e.g. (1.1 ± 0.2) × 10-2 s-1), whereas it was reduced for FIXa-N346D (4.6 ± 0.2) × 10-2 s-1. This suggests that FIXa-N346D but not FIXa342-345/FII displays impaired interaction with LMW heparin. It seems conceivable therefore that the interactive regions for LMW heparin and LRP partially overlap.

Recombinant LRP Clusters II and IV Interfere with FIXa-mediated FX Activation Both in the Absence and Presence of FVIII-- Surface loop Thr340-Asn346 is located in a crucial position between the FVIIIa interactive helix (residues 333-339) and the entry to the substrate binding cleft of FIXa. The possibility was therefore considered that LRP interferes with the enzymatic activity of FIXa. To address this issue, FIXa-mediated activation of FX was examined in the presence of phospholipids, calcium ions, and recombinant LRP clusters II and IV in various concentrations. In the absence of protein cofactor FVIIIa, the addition of LRP clusters resulted in a decrease in the rate of FXa generation by FIXa in a dose-dependent fashion, with half-maximal inhibition at 1000 and 40 nM for clusters II and IV, respectively (Fig. 6A). The effect of both LRP clusters II and IV was also examined under conditions where FIXa activity was enhanced by the presence of FVIIIa. In this situation, addition of both LRP clusters II and IV also led to reduced FXa formation, with half-maximal inhibition at 200 and 5 nM, respectively (Fig. 6B). It appears that LRP clusters II and IV are able to inhibit FIXa-mediated activation of FX both in the absence and presence of FVIII with cluster IV being a more potent inhibitor under both conditions.


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Fig. 6.   Effect of LRP clusters II and IV on FIXa-mediated FXa generation. A, FIXa (15 nM) was incubated with increasing concentrations of LRP cluster II (open circles), concentration range 0-1 µM, or LRP cluster IV (closed circles), concentration range 0-100 nM. Subsequently, the FX activation assay was performed in the presence of phospholipid (100 µM) calcium ions and FX (200 nM), as described under "Experimental Procedures." B, FIXa (0.3 nM) was incubated with LRP clusters II (open circles) or IV (closed circles). FX activation assay was performed in the presence of phospholipids (100 µM), calcium ions, FVIII (0.3 nM), thrombin (5 nM), and FX (200 nM), as described under "Experimental Procedures." Data represent the mean (± S.D.) of multiple independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Most serine proteases of the hemostatic system bind LRP only when they are complexed with serpins or other macromolecular protease inhibitors (reviewed in Ref. 17). FIXa is one of the few serine proteases, along with tissue-type plasminogen activator and urokinase, that have the potential to associate with LRP as free enzymes (15, 42, 43). However, FIXa is unique in that only the activated form, but not the FIX zymogen, interacts with LRP (15). This suggests that binding involves structural elements that become exposed upon conversion of FIX into its enzymatically active derivative. During this process, the activation peptide is released, which leads to apparent rearrangement of the surface loops that conceal the active site and limit the accessibility thereof in the proenzyme (44). These loops have previously been shown to modulate interactions with macromolecular substrates and inhibitors (31, 32, 41, 45). The present study therefore addresses the possibility that the same surface loops also contribute to LRP binding. Using anti-FIX monoclonal antibodies (Fig. 3) and a panel of recombinant FIXa variants (Table II) we show that one of these loops is indeed involved in LRP binding (Fig. 5). This interaction is inhibitory for FIXa enzymatic activity and the assembly of the FX activating complex (Figs. 3B and 6).

The data presented in this study indicate that the interaction of FIXa with LRP involves surface loop Thr340-Asp347, and region Phe342-Asn346 in particular. This is supported by multiple lines of evidence. First, region Phe342-Asn346 forms a part of the binding epitope for anti-FIXa monoclonal antibody CLB-FIX 13 (Table II), which efficiently interferes with FIXa·LRP complex assembly (Fig. 2). Second, recombinant FIXa variants with amino acid replacements within region Phe342-Asn346 have reduced interaction with LRP. This reduction is particularly manifest in the solid-phase assay system (Fig. 5A), presumably because the multiple washing steps in this system may amplify association or dissociation defects. Real time kinetic analysis revealed that effects of both mutations are more subtle. In comparison with wt-FIXa, variant FIXa-N346D shows slower association to LRP, whereas variant FIXa342-345/FII displays faster dissociation from LRP (Fig. 5B). The abnormality in LRP interaction is more pronounced for the FIXa-N346D variant (Fig. 5B). The difference between the two variants is interesting, because their substitutions are located within the same exposed structural element (46). Apparently, the NH2-terminal and COOH-terminal portions of this region play dissimilar roles in LRP interaction. How residues Phe342-Asn346 contribute to the interaction with LRP remains unclear. The FIXa residue Asn346 itself may be directly involved in LRP binding. Another possibility is that the introduction of a negatively charged Asp residue has an adverse effect on LRP interaction with other residues that are in the vicinity of position 346. Previously, positively charged residues have been shown to play a role in the interaction of LRP with some of its other ligands (47-52). In this respect it is interesting that certain positively charged residues, in particular Arg333 and Arg403, are located within 3.3 and 4.4 Å of Asn346 in the three-dimensional structure of the protease domain of human FIXa (46). Corresponding residues to those in the vicinity of FIXa position 346 (178 in chymotrypsin numbering) are known to contribute to heparin binding in the homologous serine proteases thrombin and FXa (39, 53-55). Moreover, the mutation at position 346 itself led to a decreased sensitivity of the FIXa-N346D variant for the inactivation by antithrombin in the presence of LMW heparin, whereas no such effect was observed for the FIXa342-345/FII variant. This suggests that LRP and heparin interactive regions in FIXa are not identical, but overlap to a limited extent. This is compatible with our previous observation that although both FIX and FIXa bind heparin, only FIXa comprises the full requirements for LRP binding (Fig. 1A, Ref. 15). It seems conceivable therefore that LRP binding involves positively charged residues that are located in the vicinity of FIXa position 346, and which are part of a more extended binding site. This view is in line with the observation that replacement of FIX residues Phe342-Thr343-Ile344-Tyr345 by residues Ile-Arg-Ile-Thr of thrombin or Asn346 by Asp (Fig. 5B) affects KD by only 6-fold, and as such has limited impact on the binding energy of the FIXa-LRP interaction.

FIXa surface region Phe342-Asn346 is located in a crucial position between the FVIIIa interactive helix and the active site cleft of FIXa (Fig. 4) (56-58). The region itself also appears to contribute to the interactions of FIXa with both FVIIIa and FX (41, 59). In particular, FIX-N346D is a known hemophilia B variant (59). Patients with this variant suffer from a mild form of the disease. If a region important for FIXa activity participates in LRP binding, one would expect that LRP could interfere with the enzymatic function of FIXa. Previously we showed that FIXa binds to two ligand binding regions in LRP, called clusters II and IV, the latter having five times higher affinity for FIXa (15). Indeed, cluster IV proved more effective than cluster II in inhibiting FIXa activity toward a tripeptide substrate (Fig. 3B). Because the contact area between such a small substrate and FIXa will be limited to the immediate vicinity of the active site (Fig. 4), it seems reasonable to assume that regions proximal to the active site are affected by LRP binding. In the situation when FIXa mediated the hydrolysis of its physiological substrate FX, both LRP clusters interfered with its activity, although cluster IV with higher efficiency than cluster II (Fig. 6A). This is again consistent with the fact that cluster IV displays higher affinity toward FIXa than cluster II. In the presence of the protein cofactor FVIIIa, we observed an increase in the rate of inhibition of FX activation for both clusters IV and II (Fig. 6B). With an IC50 value of 5 nM, cluster IV was the more efficient inhibitor. Accounting for the importance of residue 346 in the enhancement of FIXa activity by FVIIIa, we suggest that LRP prevents the proper assembly of the FIXa·FVIIIa complex by interfering with the binding of the A2 domain of FVIII to the protease domain of FIXa. This issue may be more complex, however. Not only FIXa, but also both the heavy and light chains of FVIIIa, contain binding sites for LRP (38, 52, 60). It is conceivable therefore that LRP or its recombinant derivatives clusters II and IV interfere with FX activation by binding to both protein components of the FX activation complex.

In view of these observations we propose that LRP serves a dual role in the down-regulation of FIXa/FVIIIa-dependent activation of FX at the sites of vascular injury. Membrane-bound LRP, and possibly other proteins containing complement-type repeats, could interfere with substrate binding and induce dissociation of FIXa from its complex with FVIIIa. The individual components then could be removed from the circulation via LRP-mediated endocytosis. In addition, not only membrane-bound LRP, but also its soluble form that circulates in plasma could participate in this process. The concentration of soluble LRP has been reported to be in the range of 2-10 nM (21, 22). This is close to the value of half-maximal inhibition that we found for isolated LRP cluster IV (Fig. 6B). Accordingly, sLRP may play a role as an inhibitor of FIXa activity in plasma. This would constitute a novel mechanism controlling the pro-thrombotic effects of FIXa.

    FOOTNOTES

* This work was supported by The Netherlands Organization for Scientific Research (NWO) Grant 902-26-214.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 Plasma Proteins, Sanquin Research at CLB, Sanquin Blood Supply Foundation, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. Tel.: 31-20-5123120; Fax: 31-20-5123680; E-mail: K_Mertens@clb.nl.

Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M209097200

    ABBREVIATIONS

The abbreviations used are: FIX, factor IX; GST, glutathione S-transferase; HSA, human serum albumin; FIXa, activated factor IX; FVIII, factor VIII; FVIIIa, activated factor VIII; FX, factor X; FXa, activated factor X; LMW, low molecular weight; LRP, low density lipoprotein receptor-related protein; pNA, para-nitroanilide; RAP, receptor-associated protein; sLRP soluble LRP, SPR, surface plasmon resonance.

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