©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Amino Acid Residues Essential for von Willebrand Factor Binding to Platelet Glycoprotein Ib
CHARGED-TO-ALANINE SCANNING MUTAGENESIS OF THE A1 DOMAIN OF HUMAN VON WILLEBRAND FACTOR (*)

Tadashi Matsushita , J. Evan Sadler (§)

From the (1) Howard Hughes Medical Institute, Departments of Medicine and Biochemistry & Molecular Biophysics, The Jewish Hospital of St. Louis, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). The VWF-GPIb interaction was investigated by clustered charged-to-alanine scanning mutagenesis of VWF domain A1 between His-473 and Gly-716. Recombinant variants of VWF were assayed for binding to conformation-dependent monoclonal antibody NMC-4, for ristocetin-induced and botrocetin-induced binding to platelets, and for direct binding to botrocetin. Substitutions at 32 amino acids had no effect on VWF function. The epitope of NMC-4 depended on charged residues between Asp-514 and Arg-632 and not on segments previously implicated by peptide inhibition studies, Cys-474-Pro-488 and Leu-694-Pro-708. Substitutions at Glu-626 and in the segment Asp-520-Lys-534 abolished ristocetin-induced binding of VWF to GPIb but did not affect botrocetin-induced binding, suggesting that these regions are required for modulation by ristocetin but not for binding of VWF to GPIb. Mutations at Glu-596 and Lys-599 decreased binding of VWF to GPIb without affecting its binding to botrocetin, suggesting that this segment interacts directly with GPIb. Alanine substitutions at Arg-545 and in the segments Glu-497-Arg-511 and Arg-687-Glu-689 caused increased binding of VWF to GPIb. These results, and the locations of von Willebrand disease type 2B mutations, suggest that two acidic regions containing the Cys-509-Cys-695 disulfide (Glu-497-Arg-511, Arg-687-Val-698) and one predominantly basic region (Met-540-Arg-578) cooperate to inhibit a distinct GPIb binding site in the VWF A1 domain. This inhibition is relieved by specific mutations, by the modulators ristocetin and botrocetin, or by binding to subendothelial connective tissue.


INTRODUCTION

von Willebrand factor (VWF)() is a multimeric glycoprotein that plays an important role in primary hemostasis. It circulates in the blood as disulfide-linked multimers that are assembled from subunits of 250 kDa. The VWF multimers range in size from dimers of 500 kDa to >10,000 kDa. VWF does not bind spontaneously to platelets in blood but promotes thrombus formation by mediating platelet adhesion at sites of vascular injury. This activation of adhesive properties is induced in vivo upon the binding of VWF to subendothelial connective tissue, particularly under the conditions of high shear stress that occur in the microcirculation. Activated VWF binds to the chain of platelet glycoprotein Ib (GPIb) (1, 2, 3) . This interaction results in platelet adhesion, followed by platelet activation and aggregation.

Binding of VWF to GPIb in vitro can be induced by the antibiotic ristocetin or by the snake venom protein botrocetin. Ristocetin apparently can bind both to platelets and to VWF (4) , whereas botrocetin binds to VWF but not to GPIb (5) . The precise mechanism by which these agents promote VWF-GPIb interaction is not known.

The binding site on VWF for platelet GPIb corresponds approximately to the first of three repeated A domains in the VWF subunit. Domain A1 extends from Glu-497 to Gly-716 and contains an intrachain disulfide loop that is defined by the disulfide bond Cys-509-Cys-695 (6, 7) . A proteolytic fragment of VWF that contains residues Leu-480/Val-481 to Gly-718 (7) binds directly to platelet GPIb. This binding is stimulated by ristocetin or botrocetin (8) , suggesting that at least some structures required to regulate the VWF-GPIb binding interaction are contained in this proteolytic fragment.

The VWF domain that binds to GPIb has been studied by using peptide inhibitors of VWF function (9, 10, 11) , by mapping the epitopes of inhibitory monoclonal antibodies (9, 12) , and by deletion mutagenesis (13). The results suggest that several discontinuous segments of VWF domain A1 interact with GPIb, ristocetin, and botrocetin. However, none of these approaches has sufficient resolution to identify single amino acid residues that are necessary for VWF function. The low potency of most synthetic peptide inhibitors also raises concerns about their specificity and mechanism of action.

Scanning mutagenesis has proved to be a powerful method for the accurate high resolution mapping of protein interaction sites (14, 15) . In this study, we have employed charged-to-alanine scanning mutagenesis to define functional amino acid residues within the A1 domain of human VWF. The results implicate residues of the A1 domain not previously known to participate in the positive and negative regulation of VWF binding to platelet GPIb.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes were obtained from New England BioLabs (Beverly, MA). Taq DNA polymerase was from Perkin Elmer Corp. Highly purified two-chain botrocetin was provided by Dr. Yoshihiro Fujimura (Nara Medical College, Japan). Monoclonal antibody 6D1 against human platelet GPIb (16) was provided by Dr. Barry Coller (Mt. Sinai Medical Center, NY). Monoclonal antibody NMC-4 against the GPIb binding domain of human VWF (9, 12, 17) was provided as ascites fluid by Dr. Midori Shima (Nara Medical College, Japan). Anti-VWF monoclonal antibody 33E12 (18) was provided by Dr. Claudine Mazurier (CRTS, Lille, France).

Plasmid Constructs

The strategy for mutagenesis was similar to that previously described (19) . Expression plasmid pSVHVWF1 (20, 21) contains a unique NgoMI site at nucleotide 3608 and two KpnI sites at nucleotides 298 and 4748 (relative to the initiation codon) in the full-length coding sequence of human VWF. The first KpnI site was mutated by changing the codon for G100 from GGT to GGA, generating the plasmid pSVHVWF1.1. Plasmid pGEM-4ZNK (19) contains the 1140-base pair NgoMI-KpnI human VWF cDNA fragment that encodes amino acid residues 442-821 of the mature subunit; this fragment was cloned into the NgoMI and KpnI sites of plasmid pGEM-4ZNae (19) (NgoMI is an isoschizomer of NaeI but leaves a 3`-overhang). DNA fragments containing each of 33 mutations were produced by oligonucleotide-directed mutagenesis using a polymerase chain reaction method essentially as previously described (19) except for the choice of mutagenic primers. Mutated NgoMI-KpnI fragments were recloned into pGEM-4ZNae, and the entire sequence was confirmed by dideoxy sequencing (Sequenase 2.0, U. S. Biochemical Corp.). The mutated NgoMI-KpnI fragments then were cloned into pSVHVWF1.1.

Expression and Characterization of Recombinant VWF

Human 293T cells (22) were kindly provided by Dr. D. Ginsburg (University of Michigan, Ann Arbor, MI) and were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Cells were transfected by a calcium-phosphate method (23) . In mock transfections, vector pSV7D DNA (24) was used. 24 h after transfection, cells were washed once with phosphated-buffered saline (PBS) and then incubated with serum-free medium (Optimem-1, Life Technologies, Inc.). Recombinant VWF (rVWF) secreted in the medium was harvested 48 h later and concentrated using Centriprep-30 and Centricon-100 devices (Amicon, Beverly, MA). The VWF antigen was measured by an ELISA assay using polyclonal rabbit anti-human VWF antibody 082 and peroxidase-conjugated rabbit anti-human VWF antibody P226 (DAKO, Carpinteria, CA) (25) . Multimer analysis was performed as described (26) . Briefly, samples (<40 µl) of concentrated medium containing 70 ng of VWF were subjected to SDS-1.5% agarose gel electrophoresis and blotted onto polyvinylidene difluoride membranes (Millipore). The VWF was detected with polyclonal rabbit anti-human VWF 082 (DAKO) and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA).

Epitope Mapping of Antibody NMC-4

Monoclonal antibody NMC-4 was purified from ascites fluid by chromatography on recombinant protein A-agarose (RepliGen, Cambridge, MA) (27) . The binding of NMC-4 to rVWF was assayed by ELISA as described above (25) except that microtiter ELISA plates with U-shaped bottoms (Coster, Cambridge, MA) were coated for 24 h at 4 °C with 25 µl of NMC-4, 7.5 µg/ml, in 0.1 M sodium carbonate, pH 9.6. The wells were washed with PBS containing 0.1% Tween 20 and then incubated for 105 min at room temperature with 15 µl of various concentrations of wild type or mutant rVWF diluted in PBS containing 3% BSA. The wells were washed again and incubated for 105 min at room temperature with 20 µl of peroxidase-conjugated rabbit polyclonal anti-human VWF P226 (DAKO) diluted 1:5000 in PBS containing 3% BSA. The plates were washed and incubated for 7 min with 100 µl of o-phenylenediamine solution (Sigma). The reaction was stopped by adding 3 M sulfuric acid, and absorbance at 490 nm was determined. Control assays performed with concentrated conditioned media from mock-transfected 293T cells gave absorbance values of zero.

Platelet Binding Assays

Ristocetin-induced binding of VWF to platelets was assayed as described (20) . Briefly, the total reaction volume (100 µl) contained 500 ng/ml rVWF, 2 10/ml human lyophilized platelets (Biodata, Hatboro, PA), 4% BSA (Sigma), and various concentrations (0-1.5 mg/ml) of ristocetin (Helena Laboratories, Beaumont, TX) in Tris-buffered saline (TBS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl). The reaction mixtures were incubated for 30 min at room temperature and centrifuged for 10 min at 10,000 g. The VWF antigen present in the supernatant was measured by ELISA. Reaction mixtures without platelets were tested simultaneously to verify the absence of nonspecific VWF flocculation and sedimentation in the presence of ristocetin.

Botrocetin-induced binding of VWF to platelets was assayed essentially as previously described (19) . Each reaction mixture (25 µl) contained 570 ng/ml rVWF, 0.2% BSA and various concentrations (0-20 µg/ml) of botrocetin in TBS. After incubation for 30 min at room temperature, the reaction mixtures were centrifuged, and VWF in the supernatant was measured by ELISA. Control assays were performed in the absence of platelets.

With two exceptions, the mutant rVWF proteins did not bind spontaneously to platelets, and the results of platelet binding assays are expressed as the percentage of unbound VWF antigen relative to the value obtained with platelets but without ristocetin or botrocetin. In the case of the variant R545A in both assays and the variant (687-689)3A in the botrocetin-induced platelet binding assay, spontaneous binding to platelets occurred, and the values obtained in the presence of modulators were compared with those obtained without platelets or modulators.

To summarize binding data in graphical form, values obtained for each mutant at specific concentrations of ristocetin or botrocetin were normalized to the corresponding values obtained for wild type rVWF as follows.

On-line formulae not verified for accuracy

Radiolabeled Botrocetin Binding Assay

Botrocetin binding to VWF was assayed according to Fujimura et al.(28) with some modifications. Botrocetin (60 µg) was radioiodinated with I using a chloramine-T method (29) to a specific radioactivity of 2.5 10 cpm/µg, and the concentration of botrocetin was adjusted to 3 µg/ml in TBS containing 1% BSA. Anti-VWF monoclonal antibody 33E12 (18) binds to the carboxyl-terminal region of the VWF subunit and has no effect on VWF binding to platelets in the presence of either ristocetin or botrocetin. The concentration of each mutant rVWF was determined by ELISA, with antibody 33E12 as the first antibody, and adjusted to 5 µg/ml with TBS. Polystyrene microtiter plates with eight strips (Coster) were coated for 16 h at 4 °C with 100 µl of antibody 33E12 (10 µg/ml). After coating, the wells were washed three times with TBS, blocked with 3% BSA in TBS for 2 h at room temperature, and incubated with 25 µl of rVWF (5 µg/ml) for 3 h at room temperature. The wells were washed four times with TBS containing 0.1% Tween 20 (TBS-Tween), and radiolabeled botrocetin solution (5 µl, 35,000 cpm) was added for 30 min at room temperature. The wells were washed rapidly five times with TBS-Tween; the total time of washing wells was <1 min. After drying, each well was cut out, and the bound radioactivity was measured by spectroscopy. Nonspecific binding was obtained by testing concentrated supernatant from mock-transfected cells in the same assay system and was 1,400 cpm. Binding to wild type rVWF was 7,500 cpm. Specific binding for each mutant rVWF was calculated by subtracting nonspecific from total binding and normalized to the value obtained for wild type rVWF.


RESULTS

Design, Construction, and Expression of Human Recombinant VWF Variants

The segment of VWF that was targeted for mutagenesis consists of 254 amino acid residues between His-463 and Gly-716. This segment contains domain A1 and part of VWF domain D3 (Fig. 1) and encompasses a fragment produced by digestion of VWF with dispase (Leu-480/Val-481-Gly-718); this fragment binds GPIb in a ristocetin-dependent or botrocetin-dependent manner (7, 8) . To simplify the production and analysis of mutant constructs, a clustered charged-to-alanine scanning strategy was chosen (14, 15) . All 68 charged amino acids including arginine, lysine, aspartate, glutamate, and histidine were changed singly or in small clusters to alanine. The 68 charged residues in the target sequence were covered in a total of 33 constructs (Fig. 1). For convenience the variant proteins were named according to the residue number of the mutated amino acid in the mature VWF subunit. If more than one charged amino acid was mutated, the range of residue numbers and the number of alanine substitutions is indicated. For example, in construct R545A, one arginine at position 545 was changed to alanine; in construct(557-563)4A, the four residues Glu-557, His-559, Asp-560, and His-563 were changed to alanine. To facilitate the mutagenesis procedure, the expression construct pSVHVWF1 was modified by eliminating the KpnI site at codon 99 of the VWF cDNA sequence. The resultant plasmid pSVHVWF1.1 has unique KpnI and NgoMI sites that facilitated the mutagenesis of amino acids 442-821 by a cassette replacement method.


Figure 1: Amino acid residues of human VWF targeted for clustered charged-to-alanine scanning mutagenesis. The target VWF segment contains a part of domain D3 (463-496) and entire A1 domain (497-716). Segments that contain mutations in each variant protein are indicated by shading, and the mutated charged residues are shown in boldcharacters. Note that the segment mutated in variant (527-531)3A is divided between two lines. The positions of two single residue mutant constructs, E527A and R632A, are indicated by underlinesbelowshadedsegments. Above the sequence, solidarrows indicate the two segments (474-488 and 694-708) reported to mediate ristocetin-induced platelet binding (9) and three segments (539-553, 569-583, 629-643) reported to bind botrocetin (10). Below the sequence, a dashedarrow marks another segment (514-542) proposed to interact with GPIb (11), and mutations in VWD type 2B are shown. The mutation V551F was identified originally as a VWD type 2A mutation, but a recent study indicates that it causes a type 2B phenotype (42).



Human kidney 293T cells were transfected with each construct, and serum-free media were analyzed for the expression of mutant human rVWF. Initially, a construct containing the two mutations R511A and D514A was not detectably secreted; therefore, the independent mutants R511A and D514A were constructed and analyzed instead. All other mutant constructs were expressed and secreted efficiently. By ELISA, the level of expression of the variant rVWF proteins was 66 ± 23% (S.D.) of wild type rVWF. The rVWF was concentrated and subjected to multimer analysis using SDS-agarose gel electrophoresis without protein reduction. The multimer distribution of all the mutant proteins was similar to that of wild type rVWF and plasma VWF (Fig. 2). In every case, at least 12 multimer bands could be detected.


Figure 2: Multimer analysis of secreted recombinant VWF variants. Samples (70 ng) of rVWF were analyzed by SDS, 1.5% agarose gel electrophoresis as described under ``Experimental Procedures.'' The abbreviated name of each variant is indicated above the corresponding lane.



The functional properties of the mutant VWF proteins were studied in four assays that depend on the GPIb binding domain. Antibody NMC-4 recognizes a conformation-dependent epitope within the A1 domain of VWF (9, 12, 17) . Reactivity with NMC-4 therefore was determined to provide an index of the structural integrity of the A1 domain of rVWF, and decreases in reactivity were used to localize the epitope of NMC-4. The ability to bind to platelet GPIb was assessed in the presence of ristocetin or botrocetin. In addition, the direct binding of radiolabeled botrocetin to each mutant rVWF protein was determined.

Epitope Mapping of Monoclonal Antibody NMC-4

Plasma VWF and wild type rVWF bound to NMC-4 with similar concentration dependence (Fig. 3A). Wild type rVWF appeared to bind slightly more avidly than plasma VWF; the cause of this apparent minor difference is not known but might be due to subtle differences in glycosylation or multimer structure or to proteolytic degradation of plasma VWF. The binding data for the mutant rVWF proteins are summarized in Fig. 3B. Compared to wild type rVWF, 26 mutant rVWF proteins bound normally to NMC-4. Seven mutant rVWF proteins exhibited 50% of normal binding to NMC-4: D514A,(520-524)2A,(549-552)2A,(608-611)3A,(613-616)2A, (629-632)2A, and R632A. These results suggest that amino acids in at least three discontinuous segments of domain A1 contribute directly to the epitope for NMC-4 or are required indirectly for its conformation.


Figure 3: Binding of VWF to anti-human VWF monoclonal antibody NMC-4. Antibody NMC-4 was immobilized in microtiter plates, and binding of VWF variants was determined as described under ``Experimental Procedures.'' Panel A, dose response to plasma VWF (open circles) and wild type rVWF (closed circles). Nonspecific binding (open squares) was determined with concentrated conditioned medium from mock-transfected 293T cells and was undetectable. Panel B, binding of rVWF variants to NMC-4 was determined at a fixed concentration of rVWF (500 ng/ml) and normalized to the value obtained for wild type rVWF.



Ristocetin-induced Binding of rVWF to Platelets

The conditions for ristocetin-induced binding of rVWF to platelets were optimized for wild type rVWF. In the absence of platelets, addition of ristocetin (1.5 mg/ml) directly to wild type rVWF (500 ng/ml) in conditioned medium was associated with precipitation and sedimentation of the VWF during centrifugation. This nonspecific aggregation was prevented by addition of 4% BSA (data not shown). Under these conditions, the binding of wild type rVWF to formalin-fixed human platelets increased as a function of ristocetin concentration, reaching a maximum of 40% binding at 1.5 mg/ml ristocetin (Fig. 4). Binding was blocked by monoclonal antibody 6D1 to platelet GPIb (16) (data not shown) as previously reported (20) .


Figure 4: Binding of VWF to platelets with increasing concentrations of ristocetin. Each rVWF variant (rVWF) was incubated at a concentration of 500 ng/ml with 2 10/ml human lyophilized platelets and ristocetin as described under ``Experimental Procedures.'' After 30 min at room temperature, the samples were centrifuged, and VWF antigen present in the supernatant was measured by ELISA. Platelet binding is expressed as the percentage of unbound VWF antigen compared with the values obtained with no ristocetin (A-C)or with neither ristocetin nor platelets (D). In each panel, VWF binding to platelets is shown for one mutant rVWF (closed circles) and for wild type rVWF (open circles) assayed at the same time. The mutant proteins are (671-673)2A (panel A), (629-632)2A (panel B), (687-689)3A (panel C), and R545A (panel D). Each data point represents the mean ± S.D. of values obtained in at least two independent sets of duplicate assays.



Ristocetin-induced binding to platelets was determined for each mutant rVWF at several concentrations of ristocetin.() The dose response for wild type rVWF was determined simultaneously to control for variations among platelet preparations. Normal and loss-of-function phenotypes were distinguished clearly at the higher ristocetin concentrations. A total of 16 mutant constructs, representing 36 charged amino acid residues, had essentially normal ristocetin-induced binding to platelets. For another 12 constructs, containing mutations at 23 charged residues, little or no ristocetin-induced binding was observed. Representative results for several mutant rVWF proteins with these phenotypes are shown in Fig. 4B. The results for variants with normal and decreased binding can be summarized satisfactorily by histograms of the values obtained at 1.0 and 1.5 mg/ml ristocetin, normalized to wild type rVWF (Fig. 5).


Figure 5: Histogram of ristocetin-induced binding of VWF to platelets. The binding assay was performed as described in the legend to Fig. 4. The value for each mutant rVWF is expressed relative to that for wild type rVWF performed at the same time. Binding to platelets was determined in the presence of 1.5 mg/ml ristocetin (closed column) and 1.0 mg/ml ristocetin (hatched column). Each column represents the mean ± S.D. of values obtained in at least two independent sets of duplicate assays.



For 5 constructs, representing 10 charged residues, a gain-of-function phenotype was demonstrated most clearly at a low ristocetin concentration of 0.5 mg/ml (Fig. 4, C and D). These constructs were(497-501)3A,(505-506)2A, R511A, R545A, and (687-689)3A. One variant, R545A, bound spontaneously to platelets in the absence of ristocetin (Fig. 4D). For this group of mutant proteins, the increase in binding also was evident at higher ristocetin concentrations, although it was less dramatic (Fig. 5).

Botrocetin-induced Binding of rVWF to Platelets

The conditions selected for botrocetin-induced binding of rVWF to platelets are similar to those for ristocetin-induced binding, except that high concentrations of BSA were not required to prevent platelet-independent precipitation of VWF. Representative dose responses to botrocetin are shown in Fig. 6 for wild type rVWF and for the mutant rVWF proteins that were assayed for ristocetin-induced binding in Fig. 4. Two variants exhibited spontaneous binding. Approximately 30% of mutant R545A sedimented with platelets in the absence of botrocetin (Fig. 6D); this variant also exhibited spontaneous binding to platelets in the ristocetin-dependent assay (Fig. 4D). Approximately 27% of mutant(687-689)3A bound spontaneously to platelets under the botrocetin-dependent assay conditions, but none bound spontaneously under the ristocetin-dependent assay conditions. This difference appears to depend on the BSA concentration in the respective assays (data not shown).


Figure 6: Binding of VWF to platelets with increasing concentrations of botrocetin. Highly purified two-chain botrocetin was incubated with 570 ng/ml rVWF and 2 10/ml lyophilized human platelets as described under ``Experimental Procedures.'' After 30 min at room temperature, the samples were centrifuged, and VWF antigen in the supernatant was measured by ELISA. Binding is expressed as the percentage of unbound VWF compared with the values obtained with no botrocetin (A, B) or with neither botrocetin nor platelets (C, D). In each panel, VWF binding to platelets is shown for one mutant rVWF (closed circles) and for wild type rVWF (open circles) assayed at the same time. The mutant proteins are (671-673)2A (panel A), (629-632)2A (panel B), (687-689)3A (panel C), and R545A (panel D). Each data point represents the mean ± S.D. of values obtained in at least two independent sets of duplicate assays.



Half maximal binding of wild type rVWF occurred at 3 µg/ml botrocetin (Fig. 6). The binding of the mutant proteins at this fixed concentration of botrocetin is summarized in Fig. 7. With few exceptions, mutant rVWF proteins behaved similarly in both the ristocetin-dependent and botrocetin-dependent binding assays. A total of 18 constructs, encompassing 39 charged residues, exhibited normal botrocetin-induced binding. Interestingly, four constructs had normal botrocetin-induced binding but no ristocetin-induced binding:(520-524)2A,(527-531)3A, K534A, and E626A. Ten constructs, including 20 amino acids, exhibited decreased botrocetin-induced binding. Only two of these variants with decreased botrocetin-induced binding had normal ristocetin-induced binding: R636A and(663-667)3A. Two constructs with normal botrocetin-induced binding exhibited increased ristocetin-induced binding to platelets: (505-506)2A and R511A.


Figure 7: Histogram of botrocetin-induced binding of VWF to platelets and direct binding of radiolabeled botrocetin to VWF. Botrocetin-induced platelet binding (closed columns) was measured at 3 µg/ml botrocetin as described in the legend to Fig. 6. Direct binding of radiolabeled botrocetin to rVWF (hatched columns) was measured as described under ``Experimental Procedures.'' Each column represents the mean ± S.D. of values obtained in at least two independent duplicated assays.



Binding of Botrocetin to rVWF

Decreased botrocetin-induced platelet binding could be the result of mutations that affect the interaction of VWF with either botrocetin or GPIb; these two binding properties also could be linked conformationally (30). To address this point, the binding of radiolabeled botrocetin to rVWF mutants was measured directly as described by Fujimura et al.(28) with minor modifications. In this assay, I-botrocetin (two chain) bound to wild type rVWF with an EC of 3.0 µg/ml (data not shown). For comparison, an EC of 1.7 µg/ml was determined for botrocetin binding to purified human plasma VWF in a similar assay (28) .

The binding of botrocetin to rVWF variants was determined at a fixed concentration of 3 µg/ml I-botrocetin (Fig. 7). With one exception, decreased binding of botrocetin to rVWF correlated with decreased botrocetin-induced binding of rVWF to platelets. For mutant (596-599)2A, direct botrocetin binding was normal, but botrocetin-induced binding to platelets was decreased.


DISCUSSION

The contact sites between proteins can be shown directly by structural methods such as x-ray crystallography, although structure alone cannot indicate the functional importance of a specific contact. At this time, no three-dimensional structures are available for VWF or GPIb. Mutagenesis provides an indirect approach to define contacts and also can define the energetic contribution of individual amino acids to protein-protein interactions (31) .

A clustered charged-to-alanine scanning strategy (14, 15) was chosen for the production and analysis of mutant VWF. The rationale underlying this approach has been reviewed (15) . In particular, alanine was selected as the replacement residue because it is the most common amino acid in proteins, is compatible with all types of secondary structures, and does not impose new structural effects related to hydrogen bonding, unusual hydrophobicity, or steric bulk (15) . Charged amino acids usually are at or near the protein surface. For this reason, charged-to-alanine substitution generally does not interfere with the packing of buried residues and disrupt the structural integrity or expression of the protein (15) . Each of these critical points has been confirmed during the successful application of this strategy to study the binding interactions of many proteins (15) .

Recombinant human VWF proved to be quite tolerant to mutations within domain A1. Among 34 different constructs covering 68 charged residues, only one was not secreted. Deletion of the entire A1 domain also is compatible with the secretion of VWF multimers that retain the ability to bind collagen and factor VIII (32) . These results suggest that domain A1, containing the GPIb binding site, is structurally independent of the remainder of the VWF subunit.

Localization of the NMC-4 Epitope

Recognition of VWF by monoclonal antibody NMC-4 is decreased markedly by reduction and alkylation of the Cys-509-Cys-695 disulfide bond or by proteolytic excision of the sequences between Leu-512 and Lys-674 (9, 12, 13, 33) , so that normal reactivity with NMC-4 indicates the normal folding of a complex epitope in VWF domain A1. In the present study, all of the rVWF proteins with decreased binding to NMC-4 had mutations between Leu-512 and Lys-674. Three mutant rVWF proteins had 60% of normal NMC-4 binding; another two had 35% of normal NMC-4 binding (Fig. 3). These results suggest that the functional epitope of NMC-4 contains amino acid residues in the segments Asp-514-Arg-524, Lys-549-Arg-552, Lys-608-Arg-616, and Arg-629-Arg-632. Several of these segments are widely separated in the primary sequence of VWF and may be held in proximity by the Cys-509-Cys-695 disulfide bond; if so, this would explain the effect of reduction on the recognition of VWF by NMC-4.

Peptide inhibition studies appear to implicate segments Cys-474-Pro-488 and Leu-694-Pro-708 of VWF in the interaction with antibody NMC-4 (9) , but recent deletion mutagenesis experiments do not support this proposal. A rVWF fragment that lacked all residues amino-terminal to Tyr-508 still reacted strongly with NMC-4; another fragment that lacked all residues carboxyl-terminal to Glu-700 also retained strong reactivity with NMC-4 (13) . These results suggest that the NMC-4 epitope does not contain most of the residues in the synthetic peptides Cys-474-Pro-488 and Lys-694-Pro-708. Further study will be required to define the mechanism by which these peptides inhibit NMC-4 binding to VWF.

Normal and Nonselective Loss-of-Function Phenotypes

Mutations of 32 different charged amino acid residues did not significantly change the properties of VWF in any of the assays tested, suggesting that the corresponding amino acid side chains do not participate in the regulation of VWF binding to platelets. Mutations of an additional 14 residues markedly reduced modulator-induced binding to platelets and direct binding to botrocetin. Five loss-of-function constructs contain mutations between Lys-608 and Lys-645; two other constructs, D514A and(549-552)2A, also exhibited this phenotype. These results implicate three discontinuous segments of domain A1 in binding to botrocetin and in ristocetin-induced binding to GPIb.

Since GPIb and modulators (ristocetin or botrocetin) can bind simultaneously to VWF, a strong correlation between loss of binding to GPIb and to modulators was not expected. These results suggest several explanations. Mutations that cause nonselective loss-of-function could grossly disrupt the folding of domain A1 and impair the binding of all of these macromolecules. However, reduced and alkylated fragments of VWF will bind normally to GPIb in the presence of botrocetin at concentrations (5 µg/ml) that were employed in these studies (13, 30) , indicating that little secondary structure is required. Alternatively, botrocetin binding and GPIb binding may be conformationally linked, as suggested by quantitative binding studies (30). In that case, mutations at either the GPIb or botrocetin binding site could impair binding to both ligands. More than one residue was altered in five of these loss-of-function constructs. Therefore, further mutagenesis will be required to determine which amino acids are necessary for normal function and whether modification of single amino acids can dissociate the effects on botrocetin binding and GPIb binding.

Selective Loss of Modulation by Ristocetin or Botrocetin

Four constructs with mutations at a total of seven charged residues exhibited a selective loss of ristocetin-induced binding to platelets:(520-524)2A,(527-531)3A, K534A, and E626A. The single mutant E527A had normal ristocetin-induced binding, so the abnormal phenotype of(527-531)3A may be due to mutation of Glu-529 or Glu-531, or both. Botrocetin-induced binding to GPIb was normal for all four of these constructs, so the mutated amino acid residues probably are not part of the GPIb binding site. Instead, these mutations may identify a specific ristocetin modulator site. This phenotype is similar to that of the naturally occurring VWF mutation G561S, which was identified in a patient with VWD and severe bleeding (34) .

Peptide inhibition studies have suggested that botrocetin binds to VWF through discontinuous sites that are represented in three synthetic peptides: D539-V553, K569-Q583, and R629-K643 (10) . The results of scanning mutagenesis are consistent with the participation of residues in the segments Asp-539-Val-553 and Arg-629-Lys-643 but do not support a role for the segment Lys-569-Gln-583.

The strongest support for the participation of specific residues in botrocetin binding is provided by the two constructs, R636A and (663-667)3A, that exhibited normal ristocetin-induced binding but defective botrocetin-induced binding to GPIb. The loss of botrocetin-induced GPIb binding correlated with decreased direct binding to botrocetin. The preservation of ristocetin-induced binding indicates that the GPIb binding site is intact. Therefore, these mutations identify amino acid residues that are required specifically for botrocetin binding and may contribute to the binding site.

GPIb Binding Sites

Construct(596-599)2A exhibited normal binding to botrocetin but defective modulator-induced binding to GPIb. Thus, Glu-596 or Lys-599 may bind directly to GPIb. Mutations at nearby residues also impaired VWF binding to GPIb (Fig. 5), suggesting that the GPIb binding site may require several amino acid side chains in this predominantly basic segment of domain A1. All mutations that decreased the binding of VWF to GPIb are located within the Cys-509-Cys-695 disulfide loop, and they overlap with the sets of mutations that decreased binding to botrocetin or to antibody NMC-4.

Peptide inhibition experiments suggested that segments of domain A1 outside the Cys-509-Cys-695 loop interact directly with GPIb (9) , but subsequent studies do not support this interpretation. Synthetic peptides corresponding to VWF residues Cys-474-Pro-488 and Leu-694-Pro-708 blocked ristocetin-induced binding of VWF to platelets; they also inhibited the binding of asialo-VWF to platelets in the absence of modulators (9) . These peptides did not inhibit botrocetin-induced binding of VWF to GPIb (12, 35) , however, suggesting that they do not interact with GPIb but may instead interact with ristocetin. Proteins that bind ristocetin often are rich in proline, and a proline-containing motif in the Cys-474-Pro-488 peptide was proposed to be a ristocetin binding site (4) . This conclusion was supported by ultraviolet difference spectroscopy; peptides Cys-474-Pro-488 and Leu-694-Pro-708 and other unrelated proline-rich peptides were shown to bind directly to ristocetin (11) . A role for proline residues in the binding of ristocetin also is supported by mutagenesis of VWF. Substitution of proline residues 702-704 either by aspartate or by arginine abolished ristocetin-induced binding to GPIb but had no significant effect on the spontaneous binding of the reduced recombinant proteins (36) . Finally, recombinant VWF fragments that lack the Cys-474-Pro-488 and Leu-694-Pro-708 peptide segments bind to GPIb with high affinity (13) . Thus, the VWF sequences represented in peptides Cys-474-Pro-488 and Leu-694-Pro-708 are dispensable for binding to platelet GPIb but may associate with ristocetin.

A similar peptide inhibition study suggests that VWF residues Asp-514-Glu-542 contribute to the binding site for GPIb (11) . A synthetic peptide with this sequence inhibited ristocetin-induced and botrocetin-induced binding of VWF to GPIb, and it inhibited the agglutination of platelets by asialo-human VWF or bovine VWF. However, this peptide also inhibited the direct binding of botrocetin to VWF (11); whether it binds to botrocetin, VWF, or GPIb or to more than one site has not been determined.

Inhibition of Binding to GPIb

Type 2B VWD is a relatively rare subtype of VWD that is characterized by increased affinity of the mutant VWF for platelet GPIb (37) . The natural mutations that cause this phenotype all are in three major clusters within domain A1: amino-terminal to Cys-509 (e.g. P503L, H505D), between Met-540 and Arg-578, and carboxyl-terminal to Cys-695 (e.g. L697V, A698V) (38, 39) . These observations suggest that the segments affected by VWD type 2B mutations normally inhibit the binding of VWF to GPIb; type 2B mutations relieve this inhibition, causing VWF to bind GPIb constitutively.

The results of scanning mutagenesis reinforce this concept and identify additional residues that inhibit the binding of VWF to GPIb. Constructs (497-501)3A,(505-506)2A, R511A, R545A, and(687-689)3A had increased affinity for GPIb. Three of these constructs affect residues within segments that were identified previously by naturally occurring mutations in VWD type 2B, but constructs R511A and(687-689)3A mark sites in which no type 2B mutations have been observed. These sites are within the Cys-509-Cys-695 disulfide loop and adjacent to either cysteine residue.

Model for Binding of VWF to GPIb

The mutagenesis results and the interactions that have been reported among antibody NMC-4, botrocetin, VWF, and GPIb suggest a model for the location of ligand binding sites on VWF. Both botrocetin and GPIb can bind simultaneously to VWF (7) , whereas NMC-4 binding is not compatible with either botrocetin (40) or GPIb binding (9) . The scanning mutagenesis data suggest that the binding sites of NMC-4, GPIb, and botrocetin overlap but are not identical. In particular, construct(596-599)2A binds normally to botrocetin and NMC-4 but not to GPIb; constructs R636A and(663-667)3A bind normally to NMC-4 and GPIb (with ristocetin) but not to botrocetin; construct(520-524)2A binds normally to botrocetin and GPIb (with botrocetin) but has decreased binding to NMC-4; construct(642-645)4A binds normally to NMC-4 but not to GPIb or botrocetin. Seven other constructs are defective in binding to all three ligands. These data suggest that botrocetin and GPIb bind to adjacent sites and that the epitope of NMC-4 may overlap with both the botrocetin and GPIb sites.

These data also suggest a speculative but testable model for the regulation of the GPIb-VWF interaction (Fig. 8). As defined by mutagenesis and the location of natural VWD type 2B mutations, residues within three VWF segments cooperate to inhibit the binding of VWF to GPIb: Glu-497-Arg-511, Arg-687-Val-698, and Met-540-Arg-578. The first two segments are acidic, whereas the last contains many basic residues. The GPIb binding site itself may be located in a second region that contains amino acids from several discontinuous segments, including Glu-596-Arg-616, Arg-629-Arg-632, and Lys-642-Lys-645; these segments contain many basic amino acid residues. Inhibition of binding is relieved by certain mutations, by the modulators ristocetin and botrocetin, or by binding to subendothelial connective tissue. The striking distribution of positive and negative charges in distinct regions suggests that intramolecular electrostatic interactions among these sites play a major role in the regulation of VWF binding to GPIb. An acidic segment Asp-252-Asp-287 of the GPIb chain was identified as a binding site for VWF (2) , and botrocetin is notable for a striking preponderance of acidic residues, especially in the chain (41) . Thus, botrocetin and GPIb both may interact electrostatically with positively charged sites on VWF.


Figure 8: Model for the regulation of VWF binding to platelet GPIb. Panel A shows a schematic diagram of the VWF A1 domain. A disulfide bond links Cys-509 to Cys-695. Functionally important VWF segments are indicated that were identified by mutagenesis or VWD type 2B mutations. Mutations within three segments (grayshaded; 497-511, 540-578, and 687-798) cause mainly gain-of-function phenotypes and are labeled Inhibitor. Mutations within two acidic regions (hatched) abolish only ristocetin-induced binding to platelets; these are labeled Activator because the defect is bypassed by botrocetin. Three predominantly basic regions (black-filled; 596-616, 629-632, and 642-645) are labeled GPIb binding. Participation of segments 629-632 and 642-645 in GPIb binding is uncertain because mutations in them cause nonselective loss of function, as do mutations at Asp-514 and Lys-549-Arg-552. Segments that are required specifically for botrocetin binding are labeled (Arg-636 and Arg-663-Lys-667). Panel B shows a model that is discussed in the text for interactions within domain A1. Positive regulation is denoted by [b|]m+ and negative regulation by . Inhibitor segments prevent the spontaneous binding of VWF to GPIb, and this repression is relieved by ristocetin. Inhibitor segments may act directly on GPIb binding segments or indirectly, possibly through Activator segments that also are required for ristocetin-induced binding to GPIb. Botrocetin bypasses the putative regulatory segments.



Modulation of VWF binding by ristocetin requires a third region that may contain the discontinuous acidic segments Asp-520-Lys-534 and Glu-626. The requirement for these segments is bypassed by botrocetin. Therefore, this region might act as an endogenous ``botrocetin-like'' activator that stimulates binding to GPIb but that is regulated by the inhibitor regions (Fig. 8). This model will be tested and refined by further mutagenesis and by structural studies of VWF domain A1.


FOOTNOTES

*
This work was supported in part by the Ryoichi Naito Foundation for Medical Research (to T. M.). 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: Howard Hughes Medical Institute, Washington University School of Medicine, 660 South Euclid Ave., Box 8022, St. Louis, MO 63110. Tel.: 314-362-9029; Fax: 314-454-3012.

The abbreviations used are: VWF, von Willebrand factor; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbant assay; GPIb, glycoprotein Ib; PBS, phosphate-buffered saline; rVWF, recombinant von Willebrand factor; TBS, Tris-buffered saline; VWD, von Willebrand disease.

Complete dose response binding data for all constructs are available from the authors upon request.


ACKNOWLEDGEMENTS

We thank Lisa Westfield for preparing oligonucleotides, Dr. David Ginsburg (University of Michigan) for providing 293T cells, Dr. Claudine Mazurier (CRTS, Lille, France) for providing monoclonal antibody 33E12, Dr. Barry Coller (Mt. Sinai Medical Center, NY) for providing monoclonal antibody 6D1, Dr. Midori Shima (Nara Medical College, Japan) for generously providing monoclonal antibody NMC-4, and Dr. Yoshihiro Fujimura (Nara Medical College, Japan) for his gift of highly purified botrocetin. We also thank Dr. Zhengyu Dong for helpful discussions.


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