©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mechanism for Inhibition of Factor VIII Binding to Phospholipid by von Willebrand Factor (*)

Evgueni L. Saenko , Dorothea Scandella (§)

From the (1) Holland Laboratory, American Red Cross, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

von Willebrand factor (vWf) acts as a carrier for blood coagulation factor VIII (fVIII) in the circulation. The amino-terminal 272 residues of mature vWf contain a high affinity fVIII binding site. Upon thrombin activation, fVIII is released from vWf, thereby allowing its binding to phospholipid which is required for its procoagulant activity. Although phospholipid and vWf compete for fVIII binding, it was previously suggested that their binding sites are not closely juxtaposed within the fVIII protein because only amino-terminal vWf proteolytic fragments larger than SPIII-T4(1-272) were able to block the binding of fVIII to phospholipid. We have demonstrated, however, that SPIII-T4 is able to inhibit fVIII binding to phosphatidylserine (PS) in a dose-dependent fashion, but only at concentrations higher than those used in previous experiments. Our demonstration that the K values for vWf and SPIII-T4 for fVIII are 0.52 nM and 48 nM, respectively, explain this discrepancy.

Inhibition (>95%) of SPIII-T4 binding to fVIII by a purified recombinant fVIII C2 domain polypeptide demonstrated that SPIII-T4 binds directly to C2, as we had previously shown for vWf. The similarity of the C2 binding sites for vWf and SPIII-T4 was further confirmed by the identical inhibitory effects of synthetic peptides and monoclonal antibodies (mAbs) on vWffVIII or SPIII-T4fVIII binding. In both cases, binding was inhibited by synthetic peptide 2303-2332, containing a PS binding site, and by mAb NMC-VIII/5 Fab` (epitope within C2 residues 2170-2327). We propose that vWf, via residues 1-272, and PS compete for fVIII binding because they recognize overlapping sites within fVIII C2 domain residues 2303-2332.


INTRODUCTION

Factor VIII (fVIII)() and von Willebrand factor (vWf) are distinct glycoproteins that both have important but different roles in hemostasis. fVIII functions as a cofactor for factor IXa in the intrinsic activation of factor X on a membrane surface (1) , and vWf promotes the adhesion of platelets to the injured vessel wall. fVIII and vWf circulate in plasma as a noncovalently linked complex. Formation of a complex between fVIII and vWf is crucial to the stability of fVIII, since patients with severe von Willebrand's disease, who have a complete deletion of the vWf gene or mutations which reduce binding between fVIII and vWf, have a secondary deficiency of fVIII (2) .

fVIII internal protein sequence homology has led to the designation of six domains arranged in the order A1-A2-B-A3-C1-C2 (3) , Fig. 1. The light chain of fVIII corresponding to the A3-C1-C2 sequence contains sites for binding of vWf (4, 5) , activated protein C (6) , factor IXa (7), and phosphatidylserine (PS) (8) . A PS binding site was localized to fVIII amino acid residues 2303-2332 within the C2 domain based on the ability of overlapping peptides from this region to inhibit the binding of fVIII to immobilized PS (9) . Observations that the fVIIIvWf complex is dissociated by phospholipid vesicles (10) and that vWf prevents fVIII from binding to such vesicles (10, 11) and to platelets(12) demonstrated antagonism between phospholipid and vWf for binding to fVIII. Thrombin cleavage of the light chain of fVIII at amino acid residue 1689 separates an acidic region (amino acid residues 1649-1689) from the rest of the light chain (13) . This leads to release of fVIII from vWf (5) and allows its subsequent binding to phospholipid.


Figure 1: Domain structure of fVIII and binding sites for mAbs and PS. The domain structure of fVIII (top line) was previously published (3). Epitopes of mAbs were determined using immunoblotting assays (16, 25). The PS binding site was determined based on the ability of overlapping fVIII synthetic peptides to inhibit the binding of fVIII to immobilized PS (9).



The importance of the fVIII light chain acidic region for binding to vWf was also supported by the observation that several monoclonal antibodies with epitopes within residues 1670-1689 (14, 15, 16) inhibit fVIII binding to vWf. The presence of post-translationally sulfated Tyr was shown to be essential for vWf binding (17) . However, synthetic peptide 1673-1689 failed to inhibit fVIII binding to vWf, regardless of whether Tyr was sulfated(17). It was suggested (17) that residues 1673-1689 and sulfated Tyr may be required indirectly to maintain the necessary tertiary structure for high affinity vWf binding to the light chain, but the exact function of this region is not clear.

Proteolytic fragments of mature vWf amino acids 1-272 (SPIII-T4) (18) or 1-298 (P34) (19) , but not fragments lacking these regions, bind to fVIII. Monoclonal antibodies with epitopes localized to vWf residues 78-96 (20) and 51-60 (21) prevent binding of vWf or SPIII-T4 to fVIII, and naturally occurring human point mutations of vWf (residues 19, 28, 53, 54, and 91) (22) abolish fVIII binding. Thus, a major high affinity fVIII binding domain is believed to correspond to amino acid residues 1-272 of the mature vWf protein. A binding site for vWf was localized to the C2 domain of fVIII (residues 2173-2332) based on the ability of a fusion protein, glutathione S-transferase-C2, to bind to vWf. It was shown that this site has some overlap with a PS binding site (residues 2303-2332) since a peptide corresponding to this sequence and a monoclonal antibody with an epitope within amino acid residues 2170-2327 prevented fVIII binding to vWf (23) . Two antibodies with C2 epitopes 2170-2218 or 2248-2285, which do not overlap the PS binding site, did not have any inhibitory effect (23) . These data suggest that the antagonistic binding of vWf and phospholipid to fVIII is due to the involvement of some C2 domain amino acids in both processes.

It was previously shown that the binding of fVIII to phospholipid was inhibited by vWf but not by SPIII-T4 when each was used at a 10-fold molar excess over fVIII (11) , suggesting that the effect of vWf may be due to steric hindrance. If SPIII-T4 is believed to contain the major fVIII binding site, it would be expected to interfere with fVIIIPS binding as vWf does. Alternatively, the binding of vWf to the fVIII C2 domain may be mediated by a different region of vWf. In this study, we have examined the interaction of vWf and various vWf proteolytic fragments with the fVIII C2 domain in order to determine the mechanism by which vWf prevents phospholipid binding of fVIII.


EXPERIMENTAL PROCEDURES

Materials

Purified IgG from monoclonal antibody (mAb) ESH8 was obtained from American Diagnostica. Highly purified, human recombinant fVIII (3809 units/mg) and gelatin-Sepharose were generously provided by Baxter/Hyland (Glendale, CA) and Shelesa Brew (American Red Cross), respectively.

Monoclonal Antibodies

mAbs 413 (epitope within fVIII A2 domain amino acid residues 373-606) and 37 were purified as described previously (23) to 90-95%, as estimated by electrophoresis in 10% polyacrylamide-SDS gels and staining with Fast Stain (Zoion Research Inc.). The preparations of mAbs NMC-VIII/5 and NMC-VIII/10 IgG and their Fab` or F(ab)` and biotinylation of mAb 413 were described previously (16, 24, 25) .

Quantification of Proteins

Concentrations of proteins were determined by the method of Bradford (26) . The molar concentrations of vWf, SPIII, and SPIII-T4 were calculated using molecular masses of 270 kDa (27) , 170 kDa (28) , and 34 kDa (29) , respectively. The protein concentration of recombinant fVIII was determined by absorbance at 280 nm, using the extinction coefficient previously reported for porcine fVIII (30) . The molar concentration of fVIII was calculated using the molecular mass of the deduced fVIII amino acid sequence, equal to 265 kDa. fVIII and vWf antigen concentrations were determined using immunoradiometric (31) assays with a normal plasma standard.

C2 Expression

The C2 domain cDNA preceded by the prepro polypeptide cDNA of tissue plasminogen activator was constructed as described (32) . It was subcloned into the baculovirus transfer vector pVL941, kindly provided by Dr. Max Summers, by a polymerase chain reaction procedure which introduced new restriction sites at the 5` and 3` ends. The resulting plasmid, pDS188, and baculovirus Autographa californica DNA were used to cotransfect Spodoptera frugiperda Sf9 insect cells. A recombinant baculovirus encoding C2 was purified as described (25, 33), and expression of C2 with the expected molecular mass of 18.5 kDa was verified by immunoblotting with anti-C2 mAb ESH8. The cDNA sequence of the entire C2 domain was verified by sequencing using the dideoxy chain termination method. The C2 domain polypeptide was produced in Sf9 cells as described (25) .

Expression of C2 was measured by enzyme-linked immunosorbent assay, as follows. Immulon I plates were coated with 5 µg/ml mAb ESH4, blocked with 200 µl/well TBS, 1% bovine serum albumin, and recombinant fVIII (41-524 ng/ml), or dilutions of a C2 sample with unknown concentration were added. All incubation steps were at 37 °C for 2 h with shaking, and wells were washed three times with 200 µl/well TBS, 0.05% Tween 20 (Bio-Rad). Binding of C2 and fVIII was detected with biotinylated mAb ESH8 followed by streptavidin-alkaline phosphatase. The fVIII standard curve was analyzed by linear regression, and it was used for calculation of the C2 concentration. Molecular masses of 18.5 kDa and 265 kDa were used for C2 and fVIII, respectively, to determine their molar concentration. Wells coated with ESH4 and containing all components except fVIII were used as negative controls. The average values of all the negative controls were 10% of the maximal signal. The color developed by alkaline phosphatase cleavage of p-nitrophenyl phosphate was read at 410 nm on an MR5000 microtiter plate reader (Dynatech Laboratories). All samples were assayed in duplicate. The average values of negative controls were subtracted from the average values for all other samples.

Purification of C2

To precipitate C2, ammonium sulfate was added to the growth medium of the infected cells at a final concentration of 50%. The precipitate containing C2 was dissolved in 20 mM Tris, 0.15 M NaCl, pH 7.4 (TBS) and subjected to FPLC on a Superose-12HR (Pharmacia) gel filtration column (1.6 60 cm) equilibrated in TBS. Pooled fractions containing C2 were further purified by FPLC using a Superdex G-75 gel filtration column (Pharmacia) equilibrated in TBS. The C2 peak (V /V= 0.54) was diluted 1:2 with 50 mM histidine, pH 6.0, 0.01% Tween 80, loaded on a Resource S column equilibrated with the same buffer, and eluted with a linear gradient of NaCl (0-1.2 M). C2 was eluted as a single peak at 0.8 M NaCl. Recovery of purified C2 from the initial extract was about 25% as measured by enzyme-linked immunosorbent assay and the Quantigold protein assay (Diversified Biotech). The two assays gave similar results.

Purification and Characterization of vWf and Its Proteolytic Fragments

vWf was prepared from cryoprecipitate (Cutter Biological) as described (5) with the following modifications: 1) after precipitation with 1.55 M NaCl, the precipitate containing vWf was dissolved in 20 ml of TBS, 0.3 M CaCl, and vWf was further purified on a 2.6 100 cm gel filtration column of Sepharose 4B-CL equilibrated in the same buffer; 2) fibronectin (4% of total protein) was removed from the resulting preparation by passage of vWf in TBS over a gelatin-Sepharose column. The final vWf preparation (1 mg/ml) contained <0.2 µg/ml fVIII, as determined by the immunoradiometric fVIII antigen assay (31) . The purity of vWf was determined by SDS-7.5% polyacrylamide gel electrophoresis of reduced protein and staining with Coomassie Blue. Protein distribution among the bands was determined by laser densitometry.

Fragments SPII and SPIII were obtained by limited proteolysis of vWf with Staphylococcus aureus V8 protease (ICN Biomedicals Inc.) and purified by fast protein liquid chromatography (FPLC) using a Resource Q column (Pharmacia) as described (34) . The SPIII-T4 fragment was prepared by digestion of SPIII with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated bovine pancreatic trypsin (Sigma) (29) . The digested mixture was treated with the serine protease inhibitor Pefabloc (Boehringer Mannheim) at 1 mM and subjected to FPLC on a Superose 12HR (Pharmacia) column under denaturing conditions (8 M urea, 0.1 M Tris, pH 7.2) as described (35) . Fractions containing SPIII-T4 were identified by SDS-PAGE and staining with silver nitrate. SPIII-T4 was further purified by FPLC on a Superdex G-75 (Pharmacia) column in TBS and by affinity chromatography on a column of 1.9 mg of purified plasma fVIII (36) immobilized on Affi-Prep 10 gel (Bio-Rad) at 1.4 mg/ml. Eighty µg of SPIII-T4 was loaded in TBS, and the bound material was eluted with a linear, 20-ml gradient of CaCl from 0 to 0.5 M in TBS. SPIII-T4 was eluted at 0.2 M CaCl as a single peak containing 85% of the loaded protein, and no other protein peaks were eluted at higher CaCl concentrations.

Synthetic Peptides

Peptides were synthesized, purified, and characterized as described (23) . Peptide solutions of 0.7-0.9 mM in 10% acetonitrile were diluted for use to 0.2 mM with 50 mM NHHCO, and the pH was adjusted to 7.4 with 0.1 M NaOH. The final concentration of NHHCO used had no effect on assays in which peptides were used.

Protein Iodination

Ten micrograms of vWf, SPIII, or SPIII-T4 in 50 µl of 0.5 M Tris, pH 7.4, were radiolabeled by incubation with 0.5 mCi of NaI (Amersham), 50 µl of Enzymobead reagent (Bio-Rad), and 25 µl of 1% -D-glucose for 15 min. Free NaI was removed on a PD10 column (Pharmacia). Recombinant fVIII was iodinated with the Bolton-Hunter reagent I kit (ICN Radiochemicals). fVIII (30 µg) in 60 µl of 100 mM sodium borate buffer, pH 8.5, 5 mM CaCl, was incubated with 1 mCi of the iodinating reagent for 6 h at 4 °C. The reaction was terminated by addition of 100 µl of 0.5 M ethanolamine, 10% glycerol in sodium borate buffer, pH 8.5. The specific radioactivity of the proteins labeled by either method was 4-10 µCi/µg.

Solid Phase Radioimmunoassays

Anti-fVIII mAbs 413 or ESH8 (3.5 µg/ml) in 0.1 M NaHCO, pH 9.6, were incubated in Immulon 1 (Dynatech) microtiter plates for 16 h at 4 °C. Each well contained 100 µl, except at the TBS, bovine serum albumin (BSA) blocking step (200 µl). All incubation steps were at 37 °C for 1 h with shaking, and the plates were washed 4 times after each step with 200 µl/well of TBS, 0.05% Tween 20 (Bio-Rad). After blocking with TBS, 1% bovine serum albumin (Sigma, fatty acid-free), anti-fVIII antibodies were saturated with fVIII by addition of 1.6 µg/ml fVIII, followed by the addition of increasing concentrations of I-vWf or I-SPIII-T4. Negative controls were mAb 413 or mAb ESH8 coated wells containing all components except fVIII or uncoated blocked wells with all other components, and these values were subtracted from average values of duplicates for all other samples. The values of these controls were similar to values of nonspecific binding of I-labeled SPIII-T4, SPIII, or vWf in the presence of a 200-fold excess of unlabeled SPIII-T4 or vWf, respectively, i.e. 20% and 10% of the maximal signal for mAb 413 or ESH8 fVIII immobilization, respectively. In competition studies, fixed concentrations below those achieving saturation of I-vWf or I-SPIII-T4 were added together with increasing concentrations of antibodies or synthetic peptides.

To study the effect of C2 on vWf or SPIII-T4 binding to immobilized fVIII, I-SPIII-T4 or I-vWf were added to the wells together with increasing concentrations of unlabeled purified C2 or fVIII. All the determinations were done in triplicate, and inhibitory data were analyzed as described below. After washing, radioactivity bound to the wells was counted in a Pharmacia LKB 1274 counter.

Affinity Measurements

Step a: Binding of SPIII-T4, SPIII, and vWf to fVIII

Binding affinities were determined by both homologous and heterologous ligand displacement assays. I-labeled vWf, SPIII, or SPIII-T4 were added together with varying concentrations of the homologous unlabeled protein and incubated at 37 °C for 2 h in microtiter wells containing fVIII immobilized on mAb ESH8, followed by washing and determination of bound radioactivity. Preliminary kinetic studies of the binding of I-labeled ligands to immobilized fVIII in the presence of unlabeled competitors demonstrated that for all ligands binding approaches equilibrium within 2 h at 37 °C. Data from homologous ligand competitions were analyzed using the computer program LIGAND (37) . For heterologous ligand displacement studies, in which the displacing ligand differs from the labeled ligand, the data were analyzed assuming two distinct equilibria, R + L RL and R + L RL, where R is immobilized fVIII, L is I-SPIII-T4, and L is vWf or SPIII. These equilibria are described by the equilibrium constants K and K. Since there is insufficient information in a single heterologous displacement curve to solve for all of the unknown parameters (K, K, and maximal number of binding sites, B), the values for K and B were determined in a parallel homologous ligand displacement experiment measuring the binding of I-SPIII-T4 to immobilized fVIII.

Step b: Binding of fVIII to Immobilized PS

PS was immobilized on a microtiter plate, and wells were blocked as described (23). To determine K and the maximal number of fVIII binding sites (B) for PS, I-fVIII was homologously displaced by unlabeled fVIII and the data were fitted to a model with a single class of binding sites as in Step a. In competition studies, vWf, SPIII, SPIII-T4, or SPII were added to the PS-coated, blocked wells together with 0.18 nMI-fVIII. The binding step was at 37 °C for 2 h with shaking, which is sufficient time to achieve equilibrium for fVIII binding to PS (data not shown) and fVIII binding to vWf and its fragments. Negative controls were uncoated, blocked wells containing all other components. The values of the negative controls were <5% of the positive signal, representing I-fVIII binding to PS in the absence of competitor. All samples were assayed in triplicate for Steps a and b.

Analysis of Competition Data

Binding data obtained in the presence of varying concentrations of competitor were analyzed using the following equation (7, 38) :

On-line formulae not verified for accuracy

Herein, represents the ratio of bound ligand (I-fVIII, I-SPIII-T4, or I-vWf, respectively) in the presence and absence of competitor, [L] is the concentration of added labeled ligand, and [I] is the concentration of competitor added. K, the apparent inhibition constant, was derived from nonlinear regression analysis fitting versus [I] to Equation 1 using the Sigmaplot 1.02 program (Jandel Scientific). Correcting the value of K for contribution of the direct interaction between labeled ligand and immobilized PS or fVIII led to determination of the K as follows:

On-line formulae not verified for accuracy

where dissociation constant K and maximal number of binding sites B are parameters describing fVIII binding to immobilized PS or SPIII-T4 and vWf binding to immobilized fVIII obtained from independent homologous displacement experiments as described above.


RESULTS

Purification and Characterization of vWf and Its Proteolytic Fragments

Our previous finding that vWf binds to the fVIII C2 domain and that this binding is inhibited by peptide 2303-2332, a PS binding site (Fig. 1), indicated that vWf and PS have overlapping fVIII binding sites. Since the major fVIII binding site of mature vWf has been localized to residues 1-272, this region should also prevent fVIIIPS binding. In order to confirm this possibility, we prepared and characterized proteolytic vWf fragment SPIII-T4, a monomer of 1-272, by limited proteolysis of vWf with S. aureus V8 protease and trypsin. The larger vWf fragments SPIII, a dimer of 1-1365 which binds to fVIII, and SPII, a dimer of 1366-2050 which does not (18) , were also purified for use as controls.

Purified, reduced vWf migrated on an SDS-polyacrylamide gel as four vWf-specific bands of 270, 215, 140, and 120 kDa, Fig. 2, lane 1, identified by immunoblotting with a polyclonal monospecific anti-vWf antibody, which were present at 80%, 2%, 4%, and 6.3% of the total protein, respectively. There was also a faint unidentified band at 80 kDa (<3% total loaded protein), as observed in other studies (23, 27) , and two faint bands at 40-50 kDa (<4.5%), which may be fibrinogen (27) . Purified, reduced SPIII and SPII fragments are shown in Fig. 2(lanes 2 and 3) where they migrate as major bands containing >85% of total protein at 170 kDa and 110 kDa, respectively, as previously reported (27, 28) . The identity of the fragments was additionally confirmed by determination of the amino-terminal amino acid sequences, which were identical with those reported (28) .


Figure 2: SDS-polyacrylamide gel electrophoresis of vWf and its proteolytic fragments. Lane 1, 7.5% gel of 10 µg of purified, reduced vWf, 5 µg of SPIII (lane 2), or SPII (lane 3) fragments stained with Coomassie Blue. Left panel, 8-25% gel of SPIII-T4 fragment reduced (lane 4) and unreduced (lane 5) stained with silver nitrate. The positions of molecular mass markers in kilodaltons are indicated to the left.



Since gel filtration of SPIII-T4 using buffer containing 8 M urea can lead to its partial denaturation, affinity purification of SPIII-T4 on an immobilized fVIII column was the final step of SPIII-T4 purification (see ``Experimental Procedures''). Purified SPIII-T4 migrated on an 8-25% SDS-polyacrylamide gel as a band containing >98% of the total protein at approximately 31 kDa (reduced) and 34 kDa (unreduced), Fig. 2 (lanes 4 and 5), as previously reported (29) . A faint unidentified band (1.1%) at 40 kDa is seen on the unreduced gel (lane 5). During affinity purification of SPIII-T4, the amount of the 40-kDa fragment decreased by 8-fold, which indicates that it does not to bind to fVIII with high affinity. SPIII-T4 was previously shown to contain 4 disulfide-linked peptides, vWf residues 1-10, 11-19, 264-272, and 21-263, due to internal proteolytic cleavages between residues 10-11, 19-20, and 263-264 (29) . The data shown in the demonstrate that our SPIII-T4 preparation contained the 4 expected amino-terminal sequences in equal amounts. In sequencing cycle numbers 11-15, only residues 30-34 of the von Willebrand peptide 20-263 were detected, which indicates that >90% of the SPIII-T4 molecules contained internal cleavages at positions 10-11, 19-20, and 263-264. Since the peptides with sequences 1-10, 11-19, and 264-272 are attached to the 20-263 peptide through disulfide bonds (29) , upon reduction only the single band corresponding to sequence 20-263, which migrates at the expected mass of 31 kDa, is seen on the gel (Fig. 2, lane 4). The broader band of reduced SPIII-T4 (lane 4) is likely to be due to the ability of -mercaptoethanol to intensify silver stains, as described (39) , by reduction of the seven disulfide bonds within SPIII-T4 (29) and generation of thiol groups. The higher intensity of silver staining of reduced proteins could also explain some additional minor contaminants visible on the reduced gel which are not seen on the unreduced gel. The amino-terminal amino acid sequences determined from affinity-purified SPIII-T4 () demonstrate that it also contains these cleavages as does SPIII-T4 which is not affinity-purified (not shown). These results rule out the possibility that the original 34-kDa material contained a fraction of a different structure which selectively binds to an immobilized fVIII column.

Inhibition of Human fVIII Binding to PS by SPIII-T4

To test our hypothesis that SPIII-T4 should inhibit fVIII binding to PS, the effect of SPIII-T4 on I-fVIII binding to immobilized PS was investigated, and the results are shown in Fig. 3. In order to preserve the post-translationally sulfated Tyr which is essential for fVIII binding to vWf (17) , radioactive iodine was introduced into fVIII lysine residues using the Bolton-Hunter reagent (40) . Binding of I-fVIII to PS was inhibited by increasing concentrations of vWf, SPIII, and SPIII-T4, but not by SPII. The concentration of I-fVIII (0.18 nM) in this experiment was 6.5 times below K for fVIIIPS binding. The molar concentration of SPIII-T4 required to achieve an inhibitory effect was about 100-fold higher than the molar concentration of vWf or SPIII. The inhibition constants (K ) were calculated using Equations 1 and 2 by nonlinear regression analysis as described under ``Experimental Procedures.'' The K= 1.18 ± 0.21 nM and B = 0.155 ± 0.17 nM for I-fVIII binding to immobilized PS used in Equation 2 were determined from an independent homologous diplacement experiment (data not shown). The calculated K values for vWf, SPIII, and SPIII-T4 were 0.76 ± 0.18 nM, 1.02 ± 0.18 nM, and 68 ± 16 nM, respectively.


Figure 3: Effect of vWf and its proteolytic fragments on fVIII binding to PS. I-fVIII (0.18 nM) with increasing concentrations of unlabeled vWf (--) or vWf fragments SPIII (--), SPIII-T4 (--), or SPII (- - -) was added to wells with immobilized PS (see ``Experimental Procedures''). fVIII binding in the presence of competitor is expressed as the percentage of fVIII binding when no competitor was added. Each point represents the mean value ± S.D. of triplicates. The curves show a best fit of the data to a model describing competitive inhibition (see ``Experimental Procedures,'' Equation 1).



Determination of SPIII-T4 Affinity for fVIII

The greater molar concentration of SPIII-T4 required for inhibition of fVIIIPS binding compared to SPIII or vWf may reflect a lower affinity of SPIII-T4 for fVIII. Thus, quantitative measurements of the affinities of SPIII-T4, SPIII, and vWf for fVIII were obtained in homologous ligand displacement assays. fVIII was captured to microtiter wells by mAb ESH8, and I-labeled SPIII-T4, SPIII, or vWf binding to fVIII was determined in the presence of increasing concentrations of the corresponding unlabeled ligands. The data shown in Fig. 4A were used to calculate the K by computer analysis from the best fit of the data to a single class of binding sites by the LIGAND program (37) . The K values derived for the binding of vWf, SPIII, and SPIII-T4 to fVIII were 0.52 ± 0.11 nM, 0.82 ± 0.2 nM, and 48.5 ± 9.7 nM, respectively. These results indicate that the SPIII-T4 fragment binds to fVIII with 60 or 93 times lower affinity than SPIII or vWf. The value of K obtained for the fVIIIvWf interaction is similar to the previously reported K values of 0.44 nM and 0.21 nM(12, 15) .


Figure 4: Determination of affinity of vWf and its fragments for fVIII. A, homologous displacement by unlabeled analogs. Binding of I-labeled vWf (0.23 nM), SPIII (0.33 nM), or SPIII-T4 (7.7 nM) to fVIII immobilized on mAb ESH8 in the presence of increasing concentrations of the corresponding unlabeled ligands is shown. Binding conditions are described under ``Experimental Procedures.'' The data represent mean values ± S.D. from three independent experiments. The curves show a best fit of the data to a single class of sites using the computer program LIGAND. B, heterologous displacement of I-SPIII-T4 by vWf or its fragments. I-SPIII-T4 (5.9 nM) and increasing concentrations of competitors SPIII-T4, vWf, SPIII, or SPII were added to wells with fVIII immobilized on mAb ESH8, as described under ``Experimental Procedures.'' Symbols used in A and B are: --, SPIII-T4; --, vWf; --, SPIII; and - - - - -, SPII.



To further confirm that SPIII-T4 affinity for fVIII is much lower than that of vWf or SPIII, inhibition of SPIII-T4 binding to immobilized fVIII by vWf and SPIII was studied (Fig. 4B). The concentration of I-SPIII-T4 was 8.3 times below K for SPIII-T4fVIII binding. The inhibitory effects of vWf and SPIII on I-SPIII-T4 binding to immobilized fVIII were similar, as would be predicted from their similar K for binding to fVIII. However, it required a molar concentration of unlabeled SPIII-T4 almost 100 times higher than that of vWf or SPIII to achieve the same inhibition of I-SPIII-T4fVIII binding. The SPII fragment was not inhibitory in this assay. The inhibition constants for vWf and SPIII calculated from heterologous displacement data using the LIGAND program were 0.5 ± 0.15 nM and 1.03 ± 0.25 nM, respectively. Values of the inhibition constants for both vWf and SPIII are close to the corresponding K calculated from homologous displacement data. Thus, both homologous and heterologous displacement data demonstrate that the affinity of SPIII-T4 for fVIII is substantially lower than that of vWf and SPIII.

Localization of the SPIII-T4 Binding Site to the fVIII C2 Domain

In order to determine if inhibition of fVIII binding to PS resulted from SPIII-T4 binding to the C2 domain of fVIII as reported for vWf (23) , the effect of a recombinant C2 domain polypeptide on SPIII-T4 binding to fVIII was tested. The C2 domain was produced as a soluble, secreted polypeptide by Sf9 insect cells infected with a recombinant baculovirus encoding C2. The growth medium from a baculovirus infection of Sf9 cells contained 8-10 µg/ml C2, as measured by enzyme-linked immunosorbent assay using two different anti-C2 antibodies, mAbs ESH8 and ESH4, which do not compete with each other for binding to fVIII (data not shown). C2, purified as described under ``Experimental Procedures,'' was characterized by amino-terminal amino acid sequencing which revealed that cleavage of the prepro polypeptide was complete in 60% of the C2 molecules. Twenty percent of the expressed C2 contained 3 residues of the prepro sequence and 20% contained 9 residues. SDS-PAGE of purified, reduced C2 is shown in Fig. 5. Amino-terminal sequencing of each band of the doublet was carried out. The bottom band contained predominantly C2 without prepro residues, whereas the upper band contained C2 with 3 or 9 residues of the prepro sequence.


Figure 5: SDS-polyacrylamide gel electrophoresis of the purified recombinant C2 domain polypeptide. The reduced sample (2.5 µg) was analyzed by electrophoresis in a 12% gel stained with Coomassie Blue. The positions of molecular mass markers in kilodaltons are indicated to the left.



Inhibition of I-SPIII-T4 binding to fVIII by C2 was tested in an assay using fVIII immobilized in microtiter wells by mAb ESH8, which does not interfere with vWf binding (23) . A saturating concentration of fVIII was used to prevent subsequent binding of fluid phase C2 or fVIII to mAb ESH8. Dose-dependent inhibition of I-SPIII-T4 binding to immobilized fVIII by fluid phase C2 or fVIII is shown in Fig. 6A. Inhibition constants for C2 and fVIII were calculated using the model describing competitive inhibition (see ``Experimental Procedures,'' Equations 1 and 2) were 960 ± 203 nM and 47.7 ± 10.3 nM, respectively. The C2 polypeptide and fVIII also inhibited I-vWf (0.1 nM or 0.2 K ) binding to immobilized fVIII, Fig. 6B. The calculated K values for C2 and fVIII were 105.3 ± 17.3 nM and 0.50 ± 0.15 nM, respectively.


Figure 6: Effect of recombinant C2 domain on SPIII-T4 or vWf binding to immobilized fVIII. A, I-SPIII-T4 (5.9 nM) and increasing concentrations of unlabeled C2 or fVIII were added to the wells with fVIII immobilized on mAb ESH8. Binding of I-SPIII-T4 is expressed as a percentage of its binding in the absence of competitor. Each point represents the mean value ± S.D. of triplicates. The curves show a best fit of the data to a model describing competitive inhibition (Equation 1 under ``Experimental Procedures''). B, I-vWf (0.1 nM) and increasing concentrations of C2 or fVIII were added to wells with immobilized fVIII as in A. Symbols used in A and B: --, C2; --, fVIII.



The binding site of SPIII-T4 within C2 was further localized with an fVIII synthetic peptide, 2303-2332, which contains PS and vWf binding sites (Fig. 1). Peptide 2303-2332 but not its randomized version (TLHQAEIWIRLGAMDPSYERQLTYHEVCVR) inhibited I-SPIII-T4 binding to fVIII in a dose-dependent fashion, Fig. 7. Peptides with the amino acid sequences 2218-2233 from the C2 domain of fVIII or 351-365 from the fVIII heavy chain were not inhibitory, as was previously observed for fVIII binding to immobilized vWf (23) . The dose of peptide 2303-2332 that reduced the binding of SPIII-T4 to fVIII to 50% was approximately 6 µM. In a control experiment, incubation of immobilized fVIII with peptide 2303-2332 did not lead to dissociation of fVIII from mAb ESH8 (not shown); therefore, peptide 2303-2332 directly interfered with SPIII-T4 or vWf binding to fVIII.


Figure 7: Effect of synthetic peptides on SPIII-T4 binding to fVIII. Increasing concentrations of fVIII peptides were preincubated with I-SPIII-T4 (6.7 nM) for 1 h at 37 °C, and the mixture was added to wells containing fVIII immobilized on mAb ESH8. Binding of I-SPIII-T4 in the presence of peptide is expressed as a percentage of fVIII binding when no peptide was added. Symbols represent peptides with the following fVIII amino acid sequences: --, 2303-2332; --, randomized version of 2303-2332; --, 2218-2233; and --, 351-365.



Inhibition of fVIIIvWf binding by mAbs with epitopes within the C2 domain (23, 24) and within the acidic region of the fVIII light chain (1649-1689) (14, 15, 16) (Fig. 1) suggested that both regions of fVIII are critical for its binding to vWf. In order to determine if these two regions are also important for binding of fVIII to SPIII-T4, the effect of anti-C2 domain mAbs NMC-VIII/5, ESH8, 37, and NMC-VIII/10, with the C2 epitopes shown in Fig. 1, was tested. Anti-A2 domain mAb 413 was used to capture fVIII onto microtiter wells. Each of the other mAbs was added in increasing concentrations at the I-SPIII-T4 binding step. As shown in Fig. 8A, binding was inhibited in a dose-dependent fashion by mAb NMC-VIII/5 IgG and Fab`, but not by IgG of mAbs ESH8 or 37. Similar concentrations of the mAb NMC-VIII/5 IgG and Fab` reduced binding by 50% (7.5 nM and 8 nM, respectively), suggesting that whole IgG is not required for inhibition. mAb NMC-VIII/10 IgG and F(ab)` also demonstrated dose-dependent inhibition of SPIII-T4fVIII binding (Fig. 8B), and the IgG and F(ab)` concentrations for 50% inhibition were approximately 5 nM and 130 nM. Neither mAb NMC-VIII/5 nor mAb NMC-VIII/10 led to dissociation of fVIII from mAb 413 (not shown); therefore, they directly inhibited SPIII-T4fVIII binding.


Figure 8: Effect of monoclonal antibodies on the binding of SPIII-T4 to fVIII. I-SPIII-T4 (5.9 nM) and increasing concentrations of antibodies with epitopes in the C2 domain of fVIII (A) or with epitopes in the acidic region of fVIII light chain (B) were added to wells with fVIII immobilized on mAb 413, as described under ``Experimental Procedures.'' The following symbols are used for antibodies in A: --, NMC-VIII/5 IgG; --, NMC-VIII/5 Fab`; --, mAb 37 IgG; --, mAb ESH8 IgG. Symbols used in B: --, NMC-VIII/10 IgG; --, NMC-VIII/10 F(ab)`.




DISCUSSION

It was previously proposed that the binding sites in fVIII for phospholipid and vWf are not closely juxtaposed, because only amino-terminal vWf fragments larger than SPIII-T4(1-272), which is known to contain a major fVIII binding site (18) , are able to inhibit binding of phospholipid to fVIII (11, 19) , possibly by steric hindrance. We have, however, demonstrated that SPIII-T4 is able to block fVIII access to PS, but the molar concentration of SPIII-T4 required for effective inhibition is almost 100 times greater than that for vWf or SPIII. In our experiments, the K values for the respective binding of vWf, SPIII, and SPIII-T4 to fVIII demonstrated that the higher concentration of SPIII-T4 than SPIII or vWf required for inhibition of fVIIIPS binding is due to the lower affinity of SPIII-T4 for fVIII binding. We propose that this explains why the inhibition of fVIII binding to phospholipid vesicles by SPIII-T4 was not observed in the previous study (11) , in which equimolar concentrations of SPIII-T4, vWf, and SPIII were used to compete for fVIIIPS binding.

Since vWf was previously shown to bind to fVIII residues 2303-2332, a PS binding site, we tested SPIII-T4 for binding to the same region. SPIII-T4 binding to fVIII was inhibited by a recombinant C2 domain polypeptide and by synthetic peptide 2303-2332. The epitope of mAb NMC-VIII/5 overlaps amino acids 2303-2332, but those of mAbs ESH8 and 37 do not. Only mAb NMC-VIII/5 inhibited SPIII-T4 binding to fVIII. Since the peptide and mAb competition results for SPIII-T4 were identical with those we reported for vWf (23) , we conclude that SPIII-T4 and vWf bind to the same, or very closely spaced, sites within amino acid residues 2303-2332 of the C2 domain. Thus, we propose that the ability of SPIII-T4 to inhibit fVIIIPS binding can also be explained by its direct prevention of fVIII access to phospholipid.

The diminished fVIII binding capacity of SPIII-T4 compared to SPIII and vWf is similar to that previously observed for the P34 fragment of vWf (residues 1-298) (19) , and it may be due to the presence of the three internal proteolytic cleavages in all SPIII-T4 molecules (29) . Our experiments, however, do not completely exclude the possibility that the low affinity of SPIII-T4(1-272) for fVIII binding may be due to the presence of another fVIII binding region within residues 272-1365 of vWf, both of which are required for high affinity binding to fVIII. We believe that this is unlikely since we have demonstrated that the properties of the sites for SPIII-T4 and vWf binding to fVIII are similar or identical. In addition, it was previously shown that proteolytic fragments SPIII-T2 (heterodimer of 273-511 and 674-728), SPI (monomer of 911-1365), and 39/34 (monomer of 480-718) did not bind fVIII (18, 19) .

The presence of PS in membranes is required for mediation of fVIII binding (41) . An fVIII PS binding site was localized to C2 domain residues 2303-2332 (9) , and a recombinant glutathione S-transferase-C2 domain fusion protein binds to 100% immobilized PS (23) . Deletion analysis of coagulation factor V (fV), an fVIII homolog, demonstrated that removal of the C2 domain resulted in a complete loss of PS binding by fV (42) , whereas both fV proteolytic fragments containing A3 and C1-C2 bound to pure phosphatidylcholine (PC) vesicles but only C1-C2 bound to 100% PS (43) . A model for fV interaction with phospholipid was proposed to consist of a first step mediated by C2 interaction with PS. This would be followed by fV penetration into a PS/PC membrane, mediated by A3 (43, 44) . Since fV does not bind to pure PC (43, 45) both steps would be required for fV high affinity interaction with PS/PC membrane. A multistep character of fVIII interaction with PS/PC membranes (46) and possible parallels to fV membrane binding suggest that regions of fVIII other than the C2 domain sequence 2303-2332 may also be involved in membrane binding. The 100% PS binding assay we have used would represent the first step in fVIII binding to phospholipid.

Our finding that SPIII-T4 binding to fVIII is inhibited by mAb NMC-VIII/10 (epitope within residues 1675-1684) is consistent with the suggestion from previous studies that the acidic region of the light chain(1649-1689) either directly or indirectly participates in the binding of fVIII to vWf. If both the acidic region of the fVIII light chain and the carboxyl-terminal region of C2 are in close proximity and form one high affinity binding site for vWf, removal of either region may reduce binding to vWf. This could explain why thrombin cleavage at residue 1689, which removes the acidic region of the light chain, leads to dissociation of the fVIIIvWf complex (5) . This hypothesis predicts that the affinities of the C2 domain and thrombin-cleaved light chain for vWf are lower than that of the uncleaved light chain. Since we showed that the K value (0.5 nM) for inhibition of vWf binding to immobilized fVIII by fluid phase fVIII is similar to the K for fVIIIvWf interaction (0.52 nM), the K for C2 binding to vWf is expected to be similar to the K (105 nM), which we measured for inhibition of vWffVIII binding by C2. This value is about 200 times lower than that for fVIII. Similar affinities for mAb ESH8 binding to C2 and fVIII, determined by homologous displacement of I-ESH8 by the unlabeled analog,() suggest that the recombinant C2 is likely to be correctly folded. Further characterization of the C2 structure and determination of its affinities for binding to vWf and PS are in progress.

  
Table: Amino-terminal sequencing of the affinity purified SPIII-T4 fragment

The numbers in parentheses indicate picomoles of amino acid at the given cycle; - means that the expected C residue cannot be determined by the method of sequencing used.



FOOTNOTES

*
This work was supported in part by United States Public Health Service Grants P50-HL44336 and R01-HL36094. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0750; Fax: 301-738-0794.

The abbreviations used are: fVIII, factor VIII; vWf, von Willebrand factor; mAb, monoclonal antibody; PS, phosphatidylserine; PC, phosphatidylcholine; TBS, Tris-buffered saline; FPLC, fast protein liquid chromatography; SPIII and SPII, homodimeric fragments of vWf, containing vWf residues 1-1365 and 1366-2050, respectively; SPIII-T4, monomeric fragment of vWf, residues 1-272.

E. Saenko, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Midori Shima for the generous gift of mAbs NMC-VIII/5 and NMC-VIII/10. We also thank Drs. Yury Matsuka and Sergey Litvinovich for their helpful advice regarding purification of proteolytic fragments of von Willebrand factor and Dr. Dudley Strickland for advice on the data analysis. We also thank Matthew Felch for his help in the preparation of the manuscript.


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