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INTRODUCTION |
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
-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
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
-chain (515 kDa)
and the carboxyl-terminal transmembrane
-chain (85 kDa). The
cytoplasmic part of the
-chain contains the endocytosis-specific signal motif, whereas the
-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
-chain and the NH2-terminal
portion of the
-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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EXPERIMENTAL PROCEDURES |
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 DH5
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.
 |
RESULTS |
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.
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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.
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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.
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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.
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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).
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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.
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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.
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DISCUSSION |
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.