©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Residues 484508 Contain a Major Determinant of the Inhibitory Epitope in the A2 Domain of Human Factor VIII (*)

John F. Healey , Ira M. Lubin , Hiroaki Nakai , Evgueni L. Saenko , Leon W. Hoyer , Dorothea Scandella , Pete Lollar (§)

From the (1)From Emory University, Atlanta, Georgia 30322 and the Holland Laboratory, American Red Cross, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484-508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitor, mAb 413, whereas hybrids containing substitutions at 387-403, 387-444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg and Ile contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser-Arg bond within the Arg-Ile segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population.


INTRODUCTION

Inhibitory antibodies (inhibitors) to factor VIII (fVIII)()develop either in about 25% of hemophilia A patients following fVIII infusions or, rarely, as autoantibodies in nonhemophiliacs. Most inhibitors are directed against epitopes located in the A2 domain (residues 373-740) or the C2 domain (residues 2173-2332) of fVIII(1, 2, 3) . Additionally, an epitope of unknown significance has been identified within residues 1690-2172 in the A3 or C1 domain(3, 4, 5) . C2 inhibitors act by blocking the binding of fVIII to phospholipid(6) . Additionally, they inhibit the binding of fVIII to von Willebrand factor(7) , although the clinical significance of this is not known. A2 inhibitors bind noncompetitively to the intrinsic pathway factor X activation complex (fVIIIa/factor IXa/phospholipid) and block factor X activation(8) . Competition antibody binding assays indicate that the A2 antigenic region is either a single epitope or a narrowly clustered set of epitopes(9) .

Deletion mapping studies by immunoblotting have limited the A2 epitope to residues 373-536 in the NH-terminal region of the A2 domain and the C2 epitope to residues 2170-2327(2, 10) . Deletion mapping studies are informative when immunoreactivity is retained in the deletion mutant. However, it is difficult to interpret results from nonreactive mutants because of the possibility of misfolding and loss of antigenic structure. Recently, we eliminated the A2 epitope in a recombinant fVIII molecule by substituting porcine residues 387-604 for the homologous human sequence(11) . Because this hybrid was active, the substituted region is likely to have a native conformation. This finding was consistent with the deletion mapping studies and demonstrated the feasibility of using hybrid human/porcine fVIII molecules for detailed structural analysis of fVIII inhibitor epitopes. In this study, we constructed a series of active hybrids that contained progressively smaller segments of porcine fVIII to localize a major determinant of the A2 epitope to residues Arg-Ile.


EXPERIMENTAL PROCEDURES

Materials

Citrated hemophilia A and normal pooled human plasmas were purchased from George King Biomedical, Inc. Heparin-Sepharose was purchased from Sigma. Fetal bovine serum, G418, penicillin, and streptomycin were purchased from Life Technologies, Inc. Unilamellar phosphatidylcholine/phosphatidylserine (75:25) (w/w) vesicles were prepared as described previously(12) .

Proteins

Published procedures were used to isolate porcine factors IXa, X, and Xa and thrombin(13, 14) . Inhibitor IgG was prepared as described previously(9) . Recombinant desulfatohirudin was a gift from Ciby-Geigy. Albumin-free pure recombinant human fVIII was a gift from Baxter Biotech. FVIII heavy chains (A1, A2, and B domains covalently linked) were purified by dissociating heterodimeric fVIII chains with EDTA, followed by chromatographic isolation. EDTA was added to a final concentration of 50 mM to fVIII in 0.6 M NaCl, 50 mM HEPES, 0.01% Tween 80, pH 6.0. After 18 h, the preparation was subjected to cation exchange chromatography on Resource S (Pharmacia Biotech Inc.), which binds light chains but not heavy chains. Prior to application to the column, the fVIII solution was diluted with 20 mM MES, 10 mM EDTA, 0.01% Tween 80, pH 6.0, and applied to the column equilibrated in 0.25 M NaCl, 20 mM histidine chloride, 5 mM CaCl, 0.01% Tween 80, pH 7.4. The flow through containing heavy chains was adjusted to pH 7.4 with 1 M Tris-Cl, pH 8.5, and applied to a Resource Q column equilibrated in 0.15 M NaCl, 20 mM HEPES, 5 mM CaCl, 0.01% Tween 80, pH 7.4. Heavy chains were eluted with a NaCl gradient (0.15-1 M). Typical recovery of heavy chains was 60-70%. Purity was confirmed by SDS-polyacrylamide gel electrophoresis.

Affinity Purification of Inhibitor Antibodies

Affinity columns were prepared by coupling purified fVIII heavy chains to Affi-Prep 10 (Bio-Rad). Coupling was done at 0.82 mg/ml resin with 93% efficiency. The column was equilibrated in 0.5 M NaCl, 50 mM Tris-Cl, pH 7.4. Purified inhibitor IgG was dialyzed into the equilibration buffer and loaded onto the column, the flow through was collected in loading buffer and concentrated, and the column was washed further with 50-100 volumes of the same buffer. The elution buffer was 50 mM Tris, 0.5 M MgCl, 50% ethylene glycol, pH 7.5. Yields of inhibitor activity ranged from 20 to 75%. Bovine serum albumin was added to 0.5 mg/ml as a stabilizing agent to the pooled, eluted IgG.

Construction and Expression of Hybrid Human/Porcine FVIII Molecules

The expression vector ReNeo, which contains a neomycin resistance gene and B-domainless human fVIII cDNA, designated HB, were gifts from Biogen, Inc. HB lacks the entire B domain, which is defined as the Ser-Arg cleavage fragment produced by thrombin. All DNA constructs containing modified sequences were prepared using Bluescript II K/S- (Stratagene) as the cloning vector. Modified sequences were transferred into the ReNeo expression vector using SpeI/ApaI fragment replacement (see Fig. 1). ReNeo plasmid DNA was purified using a Qiagen Plasmid Maxi kit (Qiagen, Inc.) and was used for subsequent transfections into baby hamster kidney cells. All novel sequences of DNA generated by the polymerase chain reaction were confirmed by dideoxy sequencing(15) .


Figure 1: Hybrid B-domainless human/porcine fVIII constructs and their inhibition by mAb 413. Black regions correspond to areas of porcine substitution whose boundaries are defined by residues of human/porcine nonidentity. Hatched regions correspond to regions of identity between human and porcine fVIII that are contained within splice sites. The 5` and 3` arrows show positions of a and d primers used for SOE mutagenesis. BU, Bethesda units of anti-fVIII inhibitory activity.



Construction of fVIII Hybrids HP4 and HP5 by Restriction Fragment Insertion

HP4 and HP5 were prepared by restriction fragment insertions using HB and HP1, a fVIII cDNA containing a complete substitution of the human A2 domain by the porcine A2 domain (11) (see Fig. 1). A SpeI/BglII fragment from HP1 was inserted into SpeI/BglII-cut HB to produce the HP4 cDNA, which encodes a porcine substitution of human residues 387-541. Similarly, an AlwN1/AlwN1 fragment from HP1 was inserted into AlwN1/AlwN1-cut HB to produce the HP5 cDNA, which encodes a porcine substitution of human residues 387-508. Prior to this, the single AlwN1 site in Bluescript II K/S- was removed, and HB was inserted into the NotI site in the plasmid polylinker region.

Construction of fVIII Hybrids HP6 through HP13 by Splicing-by-overlap Extension (SOE) Mutagenesis

SOE mutagenesis of fVIII was performed as described by Horton et al.(16) . This method consists of two rounds of polymerase chain reactions using outer primer pairs (``a'' and ``d''), which are common to reactions with all constructs, and inner primer pairs (``b'' and ``c''), which define the splice site. HP6, HP7, HP8, HP9, HP10, and HP11 were prepared using HB and HP5 as the templates for the two first round polymerase chain reactions. HP12 and HP13 were prepared using HB and HP9 as the templates for the two first round polymerase chain reactions. The 5` outer primer, a, 5`-GGCATGGAAGCTTATGTCAAAGTAN-3`, contains the AccI restriction site in the A1 domain (see Fig. 1). The 3` outer primer, d, 5`-CCTCTTTGGTCTACAGATTCTTTG-3`, lies outside the BglII restriction site in the A2 domain. The inner primers were i) 5`-ATAAAGTAAAGGTCCCA-3` and 5`-TGGGACCTTTACTTTAT-3` for both HP6 (porcine substitution 387-444) and HP7 (porcine substitution 468-508), ii) 5`-TGAGGGTAGATGTTATATGGTC-3` and 5`-GACCATATAACATCTACCCTCA-3` for HP8 (porcine substitution 387-468) and HP9 (porcine substitution 484-508), iii) 5`-GACTTTTATAACTTCTGTCA-3` and 5`-TGACAGAAGTTATAAAAGTC-3` for both HP10 (porcine substitution 387-403) and HP11 (porcine substitution 410-508), and iv) 5`-TGAATACAAAGGACGGACATC-3` and 5`-GATGTCCGTCCTTTGTATTCAGGGAGACTTCTAAAAGG-3` for HP12 (porcine substitution 489-508) and 5`-TGGGTGCAAAGCGCT-3` and 5`-AGCGCTTTGCACCCAAGGAGATTACCAAAAGGTG-3` for HP13 (porcine substitution 484-488). HP6 and HP7, HP8 and HP9, HP10 and HP11, and HP12 and HP13, represent complementary pairs (see Fig. 1). Complementary pairs are generated by reversing the templates in the first round polymerase chain reactions. Inner primers are identical between pairs if sequences are identical at the splice site (HP6/HP7 and HP10/HP11). In all constructs, AccI/BglII fragments were inserted into HB in Bluescript that had been cut with AccI and BglII to generate the final Bluescript cDNA products.

Expression of HB and Hybrid Proteins

All transfected cell lines were maintained in Dulbecco's modified Eagle's medium-F12 containing 10% fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin. Fetal bovine serum was heat-inactivated at 56 °C for 1 h prior to use. HB and hybrid proteins initially were transfected into COS-7 cells to confirm that active protein could be expressed. Then they were stably transfected into baby hamster kidney cells and selected for G418 resistance as described previously (11) except that expressing cells were maintained in growth medium containing 400 µg/ml G418. Maximum expression was obtained using Nunc triple flasks. Cells from Corning T-75 flasks grown to 80-90% confluence were transferred to Nunc triple flasks in medium containing 100 µg/ml G418 and grown to approximately 90% confluence. The medium was removed and replaced with fresh medium without G418. FVIII expression was monitored by coagulation assay and 100-150 ml of medium was collected twice daily for 4-5 days. Typical expression levels were 60 µg (300 units) of fVIII/200 ml of medium/24 h. Pooled medium was stored at 4 °C in 0.05% (w/v) sodium azide.

FVIII and fVIII hybrids were partially purified and concentrated from the growth medium by heparin-Sepharose chromatography. A 10-ml column of heparin-Sepharose was equilibrated with 0.075 M NaCl, 10 mM HEPES, 2.5 mM CaCl, 0.005% Tween 80, 0.02% sodium azide, pH 7.40. Medium (100-200 ml) from expressing cells was applied to the column, which then was washed with 30 ml of equilibration buffer without sodium azide. FVIII was eluted with 0.65 M NaCl, 20 mM HEPES, 0.01% Tween 80, pH 7.40, and was stored at -80 °C. The yield of fVIII coagulant activity was typically 50-75%.

FVIII Assays

The activity of recombinant fVIII proteins was measured by one-stage clotting assay (17) and by a plasma-free assay(11) . FVIII concentrations were determined by enzyme-linked immunoassay(11) . One unit of fVIII is defined as the activity in 1 ml of normal citrated human plasma. The standard was recombinant heterodimeric fVIII that contained variable amounts of B domain. An extinction coefficient at 280 nm of 1.0 (mg/ml/cm) was used to calculate the concentration of fVIII and to determine a specific coagulant activity of 5000 units/mg. Inhibitor titers were measured by the Bethesda assay (18), in which recombinant fVIII was added to hemophilia A plasma to a final concentration of 1 unit/ml. In the plasma-free assay, the activity of the intrinsic pathway factor X activation complex (intrinsic fXase), consisting of factor IXa, activated fVIII, and phospholipid, was measured under conditions in which the intitial velocity of factor X activation was directly proportional to the concentration of activated fVIII. fVIII preparations (0.025-0.2 nM) were activated by 40 nM porcine thrombin in the presence of 0.5 nM factor IXa, 425 nM factor X, 50 µM phosphatidylcholine/phosphatidylserine vesicles in 0.15 M NaCl, 20 mM HEPES, 5 mM CaCl, 0.01% Tween 80, pH 7.40. After 5 min at room temperature, the reaction was stopped with 0.05 M EDTA and 100 nM desulfatohirudin. Factor Xa generated by the reaction was determined using a chromogenic substrate assay(14) .

Synthetic Peptides

Two COOH-terminal carboxamide peptides corresponding to residues 484-509 in human and porcine fVIII were synthesized and purified using published methods(19) . The identity of both peptides was confirmed by sequence analysis.


RESULTS

Mapping the Human A2 Epitope for Monoclonal Antibody 413

Previously, we eliminated the A2 epitope in fVIII by making a recombinant hybrid human/porcine molecule, HP2, that contains a substitution of a porcine sequence corresponding to residues 387-604 (11) (Fig. 1). This region had previously been shown to contain an inhibitor epitope(2, 10) . In the present study, we made a series of hybrid fVIII proteins containing smaller substitutions of porcine fVIII in the NH-terminal region of the A2 domain (Fig. 1). After initial screening by transient protein expression in COS-7 cells, these constructs were stably expressed in baby hamster kidney cells and partially purified for assay with A2 inhibitors. The specific coagulant activity of the hybrid proteins was indistinguishable from that of B-domainless and B domain-containing fVIII.

cDNAs for HP4 and HP5 were constructed by using convenient restriction sites as described under ``Experimental Procedures.'' The expressed hybrids were tested in a coagulation assay for inhibition by mAb 413, a murine monoclonal antibody that competes with human A2 inhibitors for binding to A2(9) . mAb 413 inhibited neither HP4 nor HP5, indicating that it does not bind these hybrids and that a major determinant of the A2 epitope is in the sequence bounded by residues 387-508 (Fig. 1).

To further localize the epitope, constructs HP6 through HP11 were made by the SOE method(16) . The SOE constructs are complementary pairs defined by a splice site within the 387-508 substitution in HP5. Because of sequence identity between human and porcine fVIII, we define boundaries of porcine substitutions by the first amino acids that differ between human and porcine fVIII at the NH-terminal and COOH-terminal ends of the insertions. The regions of identity are shown in Fig. 1as hatched regions. Thus, the spice sites for the HP6-HP7, HP8-HP9, HP10-HP11, and HP12-HP13 pairs are at residues 444-445, 483-484, 403-404, and 488-489, respectively. mAb 413 did not inhibit HP7, HP9, and HP11, but it inhibited their complementary pairs, HP6, HP8, and HP10, as well as wild-type HB (Fig. 1). The lack of inhibition of HP9 indicates that amino acid side chains in the 484-508 region contribute significantly to the binding of human fVIII to mAb 413. Additionally, the inhibition of HP8 by mAb 413 suggests that the 373-483 region is not part of the A2 epitope.

There are nine differences between human and porcine fVIII in the 484-508 segment (). We made two more complementary constructs, HP12 and HP13, which contain porcine substitutions at residues 489-508 and 484-488 and which differ from human fVIII by four and five residues, respectively. Neither HP12 nor HP13 was inhibited by mAb 413 (Fig. 1). This indicates that reactive side chains of the A2 epitope for mAb 413 are on both sides of the Ser-Arg bond.

We also measured the inhibition of the recombinant fVIII proteins by mAb 413 using a plasma-free assay, which measures the ability of fVIIIa to support factor X activation by factor IXa. The IC of mAb 413 for HP9, HP12, and HP13 was greater than 0.8 µM (Fig. 2), whereas the IC for HB and HP8 was 3 nM. These results are consistent with the Bethesda assay and they indicate that mAb 413 binds HP9, HP12, and HP13 more than 3 orders of magnitude less tightly than HB and HP8.


Figure 2: Inhibition of control, B-domainless fVIII, and hybrid human/porcine fVIII molecules by mAb 413. fVIII activity was measured in a plasma-free intrinsic fXase assay as a function of mAb 413 concentration as described under ``Experimental Procedures'' and expressed as the percentage of activity observed in the absence of mAb 413. FVIII constructs: , HB; , HP8; , HP9; ▾, HP12; , HP13.



Peptide Inhibition of mAb 413

Synthetic COOH-terminal carboxamide peptides corresponding to HP9 region residues Arg-Phe in human fVIII and Ser-Phe in porcine fVIII were tested in the plasma-free assay for their ability to block the inhibition by mAb 413. Fig. 3shows the concentration dependence of inhibition of fVIIIa by mAb 413 in the presence and the absence of 25 µM peptide. The human peptide shifts the inhibition curve to the right (IC = 6 nM). The porcine peptide also reproducibly shifts the inhibition curve to the right, but its effect is significantly weaker (IC = 1 nM). There was no further inhibition by either peptide at concentrations up to 250 µM (data not shown). These results indicate that mAb 413 can specifically recognize the human peptide but that the peptide can only partially block mAb 413 inhibitory activity.


Figure 3: Inhibition of mAb 413 inhibition by human and porcine segment 484-509 peptides. Purified human fVIII was incubated with increasing concentrations of mAb 413 for 10 min at room temperature in the presence and the absence of 25 µM peptide prior to activation with thrombin and measurement of fVIII activity by plasma-free assay. , mAb 413 only; , mAb 413 plus porcine peptide; , mAb 413 plus human peptide.



Inhibition of Hybrid fVIII by Heavy Chain-specific Patient IgG

Usually inhibitor plasmas consist of a mixture of antibodies against both A2 and light chain epitopes(3) . We isolated heavy chain-specific (A1, A2, and B domains covalently linked) antibodies from four patients by chromatography on human heavy chain Affi-Gel. Patients SC, JM, and NS are nonhemophiliacs with anti-fVIII autoantibodies, and RC is a hemophiliac with alloantibodies. All four affinity-purified antibodies were shown to be more than 95% neutralized by recombinant A2 domain(9) , indicating that they contain only anti-A2 inhibitors. The concentration-dependent inhibition of HB, HP8, HP9, HP12, and HP13 by these antibodies was compared with mAb 413 by fVIII coagulation assay (Fig. 4). All antibody preparations inhibited HB and HP8 similarly, as expected. There was less than 50% inhibition of HP9, HP12, and HP13 by mAb 413 and antibodies from SC, JM, and NS at the highest antibody concentrations used. RC antibody inhibited HP12 and HP13 by 50 and 90%, respectively, at the highest concentrations of antibody used, but they were inhibited more weakly than HB at lower concentrations. These results indicate that mAb 413 and SC, JM, NS, and RC antibody A2 inhibitors recognize a very similar epitope. The inhibition of HP13 by RC antibody indicates that its major binding determinant is located within residues 489-508.


Figure 4: Inhibition of fVIII and hybrid human/porcine fVIII by A2 inhibitors. Residual fVIII coagulant activity was measured after incubation with antibody as defined by the Bethesda assay (18). Abscissa values refer to final antibody (Ab) concentrations in the assay. FVIII constructs: , HB; , HP8; , HP9; ▾, HP12; , HP13.




DISCUSSION

By systematic replacement of human fVIII sequences with homologous porcine fVIII sequences, we have shown that a major determinant of the A2 inhibitor epitope lies in a 25-residue segment bounded by Arg-Ile (Fig. 1). Although there is always concern when using recombinant proteins that the observed differences in binding may be due to misfolding of the protein, the hybrids used in this study had normal procoagulant activity, which minimizes this possibility.

Antibody competition binding experiments have indicated that A2 inhibitors bind to a common epitope or a narrowly clustered set of epitopes(9) . Comparison of inhibitor activity against porcine substitution mutants in the 484-508 segment (HP9, HP12, and HP13) offer a higher resolution analysis of inhibitor specificity than competition binding experiments. We observed decreased binding to HP9, HP12, and HP13 by all four inhibitors tested, although RC antibody showed significant reactivity with HP13 (Fig. 4). Thus, residues with the 489-508 segment are important for recognition by RC antibody, with a minor contribution due to residues with the 484-488 segment. Overall, the results with all four human inhibitors and with mAb 413 indicate that there is little variation of the A2 epitope in the human inhibitor response. However, further mutational analysis in this region may reveal fine specificity that is not yet apparent.

Although the 484-508 segment contributes substantially to the binding of fVIII to A2 inhibitors, a synthetic 26-residue peptide corresponding to the human sequence in this region only weakly blocked inhibition by A2 antibodies (Fig. 3). For several reasons, synthetic peptides frequently are poor antigen mimics(20, 21) . In this case, incomplete antibody blocking was observed at saturating concentrations of peptide. This partial competitive behavior is consistent with partial formation of the native fold of the 484-508 segment by the peptide. Thus, at saturating concentrations, it would bind only part of the inhibitor idiotope, thereby reducing the affinity of the inhibitor for A2.

Protein antigens typically have 15-20 side chains in contact with antibody(22) . Side chain interactions, as opposed to main chain interactions, dominate protein-protein interactions. Recent studies indicate that only a few of these side chains contribute most of the binding energy(23, 24, 25, 26) . An extensive analysis of growth hormone epitopes for several murine monoclonal antibodies revealed the following hierarchy for side chain contributions to the binding energy: Arg > Pro > Glu Asp Phe Ile, with Trp, Ala, Gly, and Cys not tested(24) . Our results are consistent with this, because 12 of the 25 residues in the 484-508 A2 segment contain these side chains ().

It is clear from our study that a significant amount of binding energy for A2 inhibitors is present in the Arg-Ile sequence. The dissociation constant for the binding of mAb 413 to fVIIIa A2 is 0.1 nM(8) , which corresponds to standard free energy of binding of -13.6 kcal/mol (from G = RT ln K). HP12 and HP13 have nonoverlapping substitutions of porcine sequence and do not have a measurable affinity for mAb 413. From the inhibition curves in Fig. 2, we estimate that the dissociation constant is greater than 0.8 µM. This corresponds to a G of greater than 5.3 kcal/mol for each mutant. Thus, greater than 10 kcal/mol binding free energy is lost by substitution with porcine side chains in the 484-508 region. The sum of free energy changes due to individual side chain replacement is greater than the standard free energy of binding because of side chain interactions(27, 28) . Therefore, changes in standard free energy due to substitutions or mutations can only be used semiquantitatively to study the overall interaction. Additionally, it is possible that the substituted porcine side chains themselves adversely affect inhibitor binding. Nevertheless, the results with HP12 and HP13 suggest that most of the A2 epitope binding energy of mAb 413 and the four human inhibitors is in the 484-508 segment.

Because epitopes are usually made up of more than one fold because they consist of either discontinuous segments or a continuous segment in a loop region(20, 22, 29) , we cannot exclude the possibility that part of the A2 epitope lies outside the 484-508 region. Ware et al.(10) found that a triple mutation (Glu Gln, Glu Gln, and Glu Gln) of an A2 inhibitor reactive fVIII deletion polypeptide expressed as a fusion protein in Escherichia coli abolished A2 inhibitor reactivity. This suggests that the Glu-Glu segment, which is conserved in human and porcine fVIII(30) , contains part of the A2 epitope. Alternatively, loss of antigenic structure due to misfolding of this substitution mutant could also account for the loss of inhibitor binding.

We have shown that hybrid human/porcine fVIII molecules can be used to locate a major determinant of the A2 inhibitor epitope. The region we have identified is sufficiently small that site-directed mutagenesis may be used to characterize the contribution of individual side chains to the binding of fVIII to A2 inhibitors. Additionally, hybrid molecules may also be useful for characterizing the remaining inhibitor epitopes in fVIII. Further characterization of the inhibitory epitopes in fVIII may lead to the construction of recombinant fVIII molecules to deal with the inhibitor problem in hemophilia A.

  
Table: Sequence of human and porcine FVIII from residues 484-508

Identical residues are marked by asterisks.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants R01-HL46215 (to P. L.), P50-HL44336 (to D. S. and L. W. H.), and R01-HL36094 (L. W. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Drawer AJ, Emory University, Atlanta, GA 30322. Tel.: 404-727-5569; Fax: 404-727-3404; E-mail: jlollar@unix.cc.emory.edu.

The abbreviations used are: fVIII, factor VIII; fVIIIa, thrombin-activated fVIII; HB, B-domainless fVIII (lacking residues 741-1648); SOE, splicing-by-overlap extension; MES, 2-(N-morpholino)ethanesulfonic acid; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Renard Rawls, Chris Skoog, and Dr. Hiba Tamim and Dr. Kenneth Kaushansky (University of Washington) for advice on SOE mutagenesis.


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