©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of the von Willebrand Factor (vWF) with Collagen
LOCALIZATION OF THE PRIMARY COLLAGEN-BINDING SITE BY ANALYSIS OF RECOMBINANT vWF A DOMAIN POLYPEPTIDES (*)

Miguel A. Cruz , Huabing Yuan , Joseph R. Lee , Robert J. Wise , Robert I. Handin (§)

From the (1) Hematology-Oncology Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The von Willebrand factor (vWF) mediates platelet adhesion to the vascular subendothelium by binding to collagen, other matrix constituents, and the platelet receptor glycoproteins Ib/IX and IIb/IIIa. Although substantial progress has been made in defining vWF structure-function relationships, there are conflicting data regarding the location of its collagen-binding site(s). Possible collagen-binding sites have been localized in the A1 and A3 domains of vWF. To study the proposed binding sites, we have expressed cDNA sequences encoding the A1 and A3 domains of vWF in Escherichia coli and purified the resulting proteins from bacterial inclusion bodies. In addition, a chimeric molecule containing residues 465-598 of the vWF A1 domain polypeptide (vWF-A1) fused in frame to residues 1018-1114 of the vWF A3 domain polypeptide (vWF-A3) was also expressed. Each of the three recombinant proteins purified as a monomer and contained a single disulfide bond. As previously reported (Cruz, M. A., Handin, R. I., and Wise, R. J. (1993) J. Biol. Chem. 268, 21238-21245), recombinant vWF-A1 inhibited ristocetin-induced platelet agglutination, but did not compete with vWF multimers for collagen binding. In contrast, vWF-A3 inhibited the binding of multimeric vWF to immobilized collagen, but did not inhibit ristocetin-induced platelet agglutination. Metabolically labeled vWF-A3 bound to immobilized collagen in a saturable and reversible manner with a Kof 1.8 10 M. The vWF-A1/A3 chimera was bifunctional. It inhibited vWF binding to platelet glycoprotein Ib/IX with an IC of 0.6 10 M and inhibited vWF binding to collagen with an IC of 0.5-1.0 10 M. These results, taken together, provide firm evidence that the major collagen-binding site in vWF resides in the A3 domain.


INTRODUCTION

The von Willebrand factor (vWF)() is a multimeric plasma glycoprotein that plays an important role in primary hemostasis (1, 2) . vWF mediates the adhesion of platelets to exposed subendothelium by forming a bridge between collagen, heparin-like glycosaminoglycans and other components of the subendothelium, and platelet receptor sites on glycoproteins Ib/IX and IIb/IIIa. vWF stabilizes adherent platelets under conditions of high flow and shear stress (3, 4, 5, 6) . Although the sites on vWF that bind to platelet GPIb/IX and GPIIb/IIIa have been well characterized, the collagen-binding site(s) within vWF is not well defined. Results vary with the source of vWF, the type of collagen, and the nature of the binding assay. Previous investigators have reported that vWF binds to collagen types I, III, and VI (7, 8, 9, 10, 11, 12, 13, 14, 15) . Studies with proteolytic fragments of vWF have defined three potential collagen-binding sites in vWF. One is localized in the propeptide, which is cleaved during the assembly of vWF multimers and is unlikely to play a major role in platelet adhesion (16) . The other two sites have been localized to amino acids 542-622 and 948-998 of the mature vWF subunit polypeptide (17) .

Analysis of vWF cDNA and its predicted amino acid sequence shows an interesting pattern of homologous repeats (20, 21, 22) . There is strong evidence that the vWF-A1 repeat, which encodes amino acids 479-717, contains binding sites for GPIb/IX, glycosaminoglycans, sulfatides, and collagen (23, 24, 25, 26, 27) . The vWF-A3 repeat, which encodes amino acids 910-1111 (28) , is also reported to have a collagen-binding site (15, 17, 18, 29, 30) . Thus, two of the triplicated A repeats contain sequences that have been implicated in collagen binding. In addition to their sequence similarity, the A1 and A3 repeats each contain a single intrachain disulfide bond. The A1 disulfide bond links Cys-509 and Cys-695, and the A3 disulfide links Cys-923 and Cys-1109, forming a 185-amino acid ``loop'' structure in each domain (19, 31) . Given the similarities in primary and secondary structure, the A1 and A3 domains could easily have overlapping functions.

Despite the data obtained with tryptic fragments of vWF (13, 15, 17, 18, 19) , there is still some uncertainty regarding the existence of a collagen-binding site in the A1 domain. One early study, utilizing unpurified bacterial lysate, reported an interaction between recombinant vWF-A1 protein in the bacterial lysate and collagen (32) . Other investigators who have subsequently expressed and studied the function of purified vWF-A1 protein have reported conflicting results (33, 34, 35) . For example, our laboratory recently reported that a highly purified monomeric vWF-A1 protein bound to platelet GPIb/IX and heparin, but did not bind to collagen (36) . In addition, it has been reported that recombinant vWF from which the entire A1 domain has been deleted, which forms the normal spectrum of multimers, no longer binds to platelet GPIb/IX, but still binds to collagen (37) .

To resolve these conflicting results and to learn more about the collagen-vWF interaction, we have cloned and expressed vWF A1 and A3 domain polypeptides and compared their biochemical properties with a chimeric vWF A1 domain polypeptide containing sequences derived from the vWF A1 and A3 domains (vWF-A1/A3). The studies reported here clearly demonstrate that vWF-A3 protein and the vWF-A1/A3 chimera both bind to type I collagen. Results obtained with vWF-A1/A3 help to localize the collagen-binding sequence in the A3 domain to a sequence between amino acids 1018 and 1114. We have concluded that the vWF-A3 binding site can account for all of the interactions between multimeric vWF and type I collagen and that this site probably represents the physiologically relevant collagen-binding site in vWF.


EXPERIMENTAL PROCEDURES

Construction of vWF Expression Vectors

A map for each of the three vWF cDNAs expressed is shown in Fig. 1. For expression of the vWF A3 domain in Escherichia coli, a cDNA fragment encoding amino acids 908-1111 of mature vWF was constructed by mutagenesis of vWF cDNA in M13 with two oligonucleotides. Oligonucleotide 1 spanned codons 1666-1674 (28) and introduced a BamHI restriction site (encoding Gly and Ser) at codons 1670 and 1671. Oligonucleotide 2 spanned codons 1870-1882 and introduced a termination codon and a HindIII restriction site. Following sequence confirmation, the vWF-A3 cDNA fragment was isolated by digestion with BamHI and HindIII and inserted into the expression vector pQE9 (QIAGEN Inc.). Insertion of the vWF-A3 fragment in pQE9 produces an amino-terminal fusion protein containing the vWF sequence fused in frame to 10 amino acids (6 histidines) contributed by the vector. vWF-A1 cDNA was prepared as described previously and used to transform pQE9 as well (36) .


Figure 1: Maps of the recombinant vWF-A1, vWF-A3, and vWF-A1/A3 constructs used for bacterial expression studies. A, the vWF-A1 sequence (amino acids 475-709) is shown in black. B, the vWF-A3 sequence (amino acids 908-1111) is shown in white. C, the vWF-A1/A3 chimera (amino acids 475-598/1018-1114) is shown with respective A1 and A3 domain contributions in black and white. The native vWF-A1 disulfide bond (Cys-509-Cys-695), the native vWF-A3 disulfide bond (Cys-923-Cys-1109), and the proposed disulfide bond in the vWF-A1/A3 chimera (Cys-509-Cys-1109) are also depicted.



Chimeric vWF-A1/A3 cDNA was constructed with a polymerase chain reaction-based mutagenesis strategy. In the first round of amplification, the amino-terminal half of the A1 domain (residues 475-598) and the carboxyl-terminal half of the A3 domain (residues 1018-1114) were amplified. The 3`-primer for amplifying the A1 domain was designed to contain 12 extra bases of A3 domain sequence (5`-TCGCACAGCAAACAAGACCTCGCTGGTGGA-3` (vWF-A1 sequence is underlined)). Thus, the amplified A1 cDNA contained a short A3 sequence at its 3`-end. Similarly, A3 cDNA contained a short A1 sequence at its 5`-end (5`-AGCGAGGTCTTGGCTGTGCGATACTTG-3` (vWF-A1 sequence is underlined)). The two chimeric cDNA fragments were then incubated together so that they were annealed via their overlapping sequences. The annealed material was used as the template for the second round of polymerase chain reaction. The chimeric DNA fragment was amplified using 5`-A1 (5`-CCTCACCTGTGAAGGATCCCAGGAGCCGGGAG-3`) and 3`-A3 (5`-CATTCCAAGCTTGAATTCATCAAGATCTAACAAATCCAGAGC-3`) primers designed to introduce BamHI and HindIII restriction sites, respectively, for cloning as described previously above. The chimeric vWF-A1/A3 cDNA fragment was isolated by digestion with BamHI and HindIII and used to transform pQE9.

Purification of Recombinant Proteins

E. coli M15(pREP4) cells (QIAGEN Inc.) containing pQE9-vWF-A3, pQE9-vWF-A1, or pQE9-vWF-A1/A3 were cultured overnight at 37 °C in 8 liters of 25 g/liter Tryptone, 15 g/liter yeast extract, 5 g/liter NaCl, pH 7.3, containing 100 µg/ml ampicillin and 25 µg/ml kanamycin. The overnight culture was diluted 1:20 and grown to A = 0.7. The culture was adjusted to 1.5 mM IPTG and incubated for 5 h at 37 °C. The cells were then harvested, resuspended in 125 ml of lysis buffer (50 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, pH 8.0) containing lysozyme at a final concentration of 250 µg/ml, and allowed to stand for 1 h at 4 °C. The bacterial cells were lysed in the presence of 1.25 mg/ml deoxycholic acid and 7 µg/ml DNase I. The lysate was centrifuged at 12,000 g for 15 min, and the resulting pellet was washed with lysis buffer containing 0.5% Triton X-100 and 10 mM EDTA, followed by recentrifugation.

For purification of vWF-A3 protein, the washed pellet was solubilized by the addition of 7.5 M urea in 25 mM Tris-HCl, pH 8.8, and the solubilized proteins were dialyzed against 25 mM Tris-HCl, pH 8.2. The solubilized proteins were passed over a Q-Sepharose column (Pharmacia Biotech Inc.) equilibrated with 25 mM Tris-HCl, pH 8.2. vWF-A3 eluted from the column with sodium chloride. The fractions containing vWF-A3 were pooled, concentrated by ultrafiltration, and dialyzed against Tris-buffered saline (TBS; 25 mM Tris-HCl, 150 mM NaCl, pH 7.4)

Recombinant vWF-A1 or chimeric vWF-A1/A3 protein was expressed in E. coli as described above. They were both purified as described previously (36) . Briefly, after induction with IPTG and collection of inclusion bodies from lysed E. coli cells, the washed inclusion bodies were solubilized in 6 M guanidine hydrochloride, dialyzed into 6 M urea, and purified by fast protein liquid chromatography on an S-Sepharose column (Pharmacia Biotech Inc.). Urea was removed by slow dialysis, and the recombinant proteins were dialyzed against TBS (36) . Each of the purified recombinant proteins was then concentrated by adsorption to and elution from a heparin column (Bio-Rad) and/or ultrafiltration and dialyzed against TBS.

Radiolabeling of vWF-A3 Protein

E. coli cells were grown overnight and diluted in medium described above. When grown to A = 0.7, the cells (500 ml) were pelleted by centrifugation, washed with M9 minimal medium (6 g of NaHPO/liter, 3 g of KHPO/liter, 0.5 g of NaCl/liter, 1 g of NHCl/liter, 1 mM MgCl, 0.1 mM CaCl, and 0.2% glucose), and pelleted again. The pellet was resuspended in 500 ml of M9 minimal medium supplemented with a 0.02% concentration of 18 amino acids except Met and Cys. Bacteria were grown for 45 min; IPTG was added; and the medium was supplemented with 1.5 µCi/ml [S]HSO (DuPont NEN) overnight at 37 °C. Labeled vWF-A3 was purified as described above. Specific activity of S-labeled vWF-A3 was 3.6 10 cpm/µg.

Production and Purification of Multimeric Recombinant vWF

Recombinant vWF was purified from the conditioned medium of Chinese hamster ovary cells that had been stably transformed with full-length vWF cDNA as described previously (21, 39, 40) . Confluent cells were rinsed and incubated for 24-48 h in -minimal essential medium containing 0.05% bovine serum albumin, insulin/transferrin/sodium selenite supplement (Sigma), and 1% (v/v) aprotinin (Sigma, A-6279). The conditioned medium was collected and centrifuged to remove cellular debris, and NaEDTA and phenylmethanesulfonyl fluoride were added to final concentrations of 5 and 2 mM, respectively. To isolate vWF wild-type protein, Chinese hamster ovary cell-conditioned medium was concentrated in dialysis tubing by using Aquacide II (Calbiochem) and passed over a Sepharose CL-4B gel filtration chromatography column (Pharmacia Biotech Inc.; 5 100 cm). Fractions containing vWF protein were pooled, concentrated by Aquacide II, and dialyzed against TBS. For radiolabeling of vWF produced in Chinese hamster ovary cells, Cys- and Met-deficient serum-free medium was supplemented with 10 µCi/ml [S]Met and [S]Cys (39, 40) . Radiolabeled vWF was purified as described above. Specific activity of S-labeled vWF was 2.0 10 cpm/µg.

Collagen Binding Assay

A final concentration of 1.8 mg/ml acid-soluble bovine type I collagen (Collaborative Biomedical, Boston) was added to microtiter wells in 20 mM sodium citrate buffer, pH 6.0, for 90 min. An acid-soluble calfskin type I collagen (Sigma) was also used in some experiments. After washing three times with TBS to remove nonadsorbed collagen, wells were blocked with 1% bovine serum albumin in TBS for 30 min. Increasing concentrations of S-labeled vWF proteins were added to the wells and incubated for 60 min at room temperature. For competition assays, a constant concentration (1-4 µg/ml) of S-labeled vWF was added to the wells with increasing concentrations of the unlabeled ligands. Wells were washed with TBS, and bound radioactivity was removed for scintillation counting by overnight incubation in 1% SDS/TBS. Nonspecific binding was determined in the presence of a 40-fold excess of nonradioactive vWF-A3 or a 50-fold excess of nonradioactive vWF. In each case, nonspecific binding was always <5% of total binding. The amount of collagen bound to wells was measured in duplicate as described previously (12) . Bound collagen was removed with 1% SDS and submitted to protein quantitation. At the concentration used in the assays, 6 ± 1.0 µg of collagen bound to each well.

Platelet Agglutination Assay

Ristocetin-induced platelet agglutination was carried out in siliconized glass cuvettes at 37 °C with constant stirring at 1200 rpm in a four-channel aggregometer (Bio/Data Corp.). A suspension of 2 10/ml formaldehyde-fixed platelets containing 8 µg/ml purified vWF (41) and increasing concentrations of the recombinant proteins was prepared. After 5 min of incubation at 37 °C, agglutination was initiated by the addition of ristocetin (Sigma) to a final concentration of 1 mg/ml.

Protein Quantitation

Protein concentrations were determined by the bicinchoninic acid method (Pierce). Purity was assessed by Coomassie Blue staining of SDS-polyacrylamide gels (42) . Radiolabeled protein was visualized by autoradiography utilizing ENHANCE (DuPont NEN). Gel filtration analysis was carried out in a Sephacryl 300-HR column (Sigma; 0.8 30 cm) using a Waters 650E-APPS apparatus (36) .


RESULTS

Production, Purification, and Collagen-binding Activity of vWF-A3

After induction with IPTG, transformed bacteria expressing vWF-A3 cDNA, pQE9-vWF-A3, were lysed, and their inclusion bodies were collected. As shown in Fig. 2 A, when analyzed by SDS-PAGE under reducing conditions, the washed inclusion bodies contained a prominent protein band of 27,000 Da. The inclusion bodies could be solubilized in 7.5 M urea, and all of the protein remained in solution after dialysis against 20 mM Tris, pH 8.2. The soluble proteins were then fractionated by fast protein liquid chromatography using a Q-Sepharose ion-exchange column and a linear NaCl gradient. As shown in Fig. 3, vWF-A3 eluted in a sharp peak at 160 mM NaCl. This peak represented 50% of the total vWF-A3 protein expressed. The remaining 50%, which formed high molecular mass aggregates, eluted in the flow-through volume of the column. The final yield of purified monomeric protein was 8 mg/liter of bacterial culture. To assess the purity of vWF-A3, radiolabeled protein was produced by incubating E. coli containing pQE9-vWF-A3 in medium containing SO and IPTG. Radiolabeled vWF-A3 was then purified by the procedure described above. A single radiolabeled band was seen after purification by SDS-PAGE and autoradiography (Fig. 2 B, lane4).


Figure 2: SDS-PAGE analysis of recombinant vWF-A3 protein. Bacterial inclusion bodies and purified bacterial vWF-A3 protein were analyzed by SDS-PAGE (12.5%). A, the gel, analyzed under reducing conditions, shows molecular mass markers ( lane1) and washed bacterial inclusion bodies ( lane2) from E. coli transformed with pQE-vWF-A3 and induced with IPTG. B, lanes 1, 2, and 4 were analyzed under reducing conditions. Lane1 shows molecular mass markers. Lane 2 shows vWF-A3 eluted from a Q-Sepharose column. Lane 3 shows vWF-A3 analyzed under nonreducing conditions. Lane 4 shows an autoradiograph of vWF-A3 metabolically labeled with SO.




Figure 3: Ion-exchange chromatography of vWF-A3. Following solubilization of inclusion bodies, vWF-A3 was purified using a Q-Sepharose column equilibrated with 20 mM Tris-HCl, pH 8.2. The bound protein was eluted with a gradient of NaCl. The eluted protein was concentrated by ultrafiltration and dialyzed against TBS. Abs, absorbance.



The calculated molecular mass for the sequence between Ser-908 and Gly-1111 is 21,538 Da. The 10 additional amino acids from the vector sequence add another 1254 Da, bringing the estimated molecular mass to 22,792 Da. This is in good agreement with the estimated molecular mass of the purified material of 27,000 Da (Fig. 2 B, lane2). In addition, purified vWF-A3 eluted from a Sephacryl 300 column with the K of a globular monomeric 24-kDa protein (data not shown). When purified vWF-A3 was analyzed by SDS-PAGE under nonreducing conditions (Fig. 2 B, lane3), it migrated slightly faster than the reduced form, suggesting a compact globular structure that is extended following reduction of the single disulfide bond between Cys-923 and Cys-1109.

As shown in Fig. 4, S-labeled vWF-A3 bound to immobilized soluble type I collagen derived from bovine Achilles tendon or calfskin collagen in a saturable and reversible manner. At the highest concentration of added S-labeled vWF-A3, nonspecific binding accounted for <5% of total bound radioactivity. The relevant binding parameters for vWF-A3 were derived by Scatchard analysis of binding isotherms (Fig. 4, inset). The Scatchard plot demonstrated a single class of binding sites with a Kof 1.8 ± 0.3 µM. At saturation, there were 300 fmol of vWF-A3 bound per µg of immobilized collagen.


Figure 4: Binding of S-labeled recombinant vWF-A3 protein to collagen-coated microtiter wells. A final concentration of 1.8 mg/ml acid-soluble bovine type I collagen was added to microtiter wells in 20 mM sodium citrate buffer, pH 6.0, for 90 min at 37 °C. After washing with TBS to remove nonadsorbed collagen, wells were blocked by the addition of 1% bovine serum albumin for 60 min at room temperature. Increasing concentrations of S-labeled recombinant vWF-A3 were added to the wells and incubated for 30 min at 37 °C. Wells were washed with TBS, and bound radioactivity was removed with 1% SDS/TBS and counted. Nonspecific binding, measured in parallel wells with the addition of a 40-fold excess of unlabeled vWF-A3 protein, was subtracted from each point. The values shown represent specific binding from five separate experiments. B, bound; F, free.



Characterization of Chimeric vWF-A1/A3 Protein

The purified vWF-A1/A3 chimera, when analyzed by SDS-PAGE under reducing conditions, had a band of 32,000 Da. This is in good agreement with the predicted molecular mass of 27,000 Da. The anticipated shift in mobility between reduced and unreduced protein was also demonstrated, suggesting formation of a disulfide bond between Cys-509 and Cys-1109 (data not shown). As shown in Fig. 5, both vWF-A1 and vWF-A1/A3 proteins inhibited ristocetin-induced platelet agglutination in a dose-dependent manner. The IC for both proteins was between 200 and 600 nM. This finding provides additional evidence for proper folding of the chimeric protein and helps to localize the GPIb/IX-binding domain to the first 98 residues of the vWF A1 domain. In contrast, at concentrations up to 2 µM, vWF-A3 did not inhibit ristocetin-induced platelet agglutination.


Figure 5: Inhibition of ristocetin-induced platelet agglutination by vWF-A1 and vWF-A1/A3. Increasing concentrations of vWF-A1, vWF-A1/A3, and vWF-A3 proteins were incubated with 2 10/ml Formalin-fixed platelets in the presence of 8 µg/ml purified vWF for 5 min at 37 °C. Agglutination was initiated by the addition of 1 mg/ml ristocetin. Platelets were stirred continuously at 1200 rpm at 37 °C, and the change in light transmission was recorded. 100% is defined relative to the agglutination of platelets without any recombinant proteins (control). The IC for both vWF-A1 and vWF-A1/A3 was similar (200-600 nM). vWF-A3 did not inhibit ristocetin-induced platelet agglutination at any concentration tested.



Inhibition of S-Labeled Recombinant vWF Binding to Type I Collagen

The ability of the three recombinant proteins to block the binding of multimeric vWF to type I collagen was then examined. Inhibition of S-labeled vWF multimer binding to immobilized type I collagen by unlabeled vWF, vWF-A1, vWF-A3, and vWF-A1/A3 proteins is shown in Fig. 6. Multimeric vWF, vWF-A3, and the vWF-A1/A3 chimera compete with S-labeled vWF for binding to collagen, with varying affinities. The IC for multimeric vWF was 8 ± 2 nM using the molecular mass of its 275-kDa subunit to calculate molarity. The IC for vWF-A3 was 1.0 ± 0.5 µM, which is comparable to the previously determined Kof 1.8 µM established by direct binding assays. The IC for vWF-A1/A3 was 0.5 ± 0.3 µM, which is very similar to the IC for vWF-A3. In contrast, we were unable to inhibit vWF binding to collagen with up to 9 µM vWF-A1.


Figure 6: Inhibition of S-labeled vWF binding to type I collagen. The binding of S-labeled vWF (1-4 µg/ml) to immobilized acid-soluble type I collagen was measured in the presence of purified unlabeled recombinant vWF, vWF-A3, vWF-A1/A3, and vWF-A1 at various concentrations as indicated or the same volume of TBS in the control mixture. 100% is defined as the fraction of added S-labeled vWF bound with no competing ligand. The values shown represent four separate experiments.




DISCUSSION

The interaction between vWF and collagen has been studied most extensively with proteolytic fragments derived from purified plasma vWF. These reports have identified peptides that inhibit collagen binding and that contain either A1 or A3 domain sequences (13, 15, 17, 18, 19, 23, 29) . Despite these data, when highly purified recombinant vWF A1 domain polypeptides were studied in several laboratories, collagen binding could not be demonstrated (33, 34, 35, 36) . This raised the interesting possibility that the major collagen-binding site might be located in the A3 domain of vWF. To study this, we expressed a vWF cDNA encoding Ser-908-Gly-1111 and compared its properties with those of recombinant vWF-A1 and a vWF-A1/A3 chimera. All three proteins were readily purified from E. coli inclusion bodies in quantities sufficient for biochemical studies. Based on our comparative studies, we have concluded that vWF-A3 1) binds to immobilized type I collagen, 2) blocks the binding of multimeric vWF to type I collagen, and 3) does not inhibit ristocetin-dependent platelet agglutination by multimeric vWF. In contrast, vWF-A1 does not compete for collagen-binding sites. Finally, a chimeric molecule, vWF-A1/A3, which contains the amino-terminal half of vWF-A1 fused in frame to the carboxyl-terminal half of vWF-A3, also inhibits vWF binding to collagen. Although not the major focus of this study, the chimeric molecule is bifunctional and also inhibits vWF binding to the platelet GPIb/IX receptor site.

These results extend five previous reports that predicted a collagen-binding site in the vWF A3 domain. Roth et al.(17) first reported that a tryptic fragment of vWF containing residues 948-998 inhibited the binding of vWF to immobilized type III collagen. Three subsequent studies of tryptic fragments spanning residues 730-1114 and/or 911-1365 have demonstrated binding to collagen types I, III, and VI (15, 18, 29) . Jorieux et al.(30) then expressed a recombinant protein containing residues 914-1364 and demonstrated binding to fibrillar collagen types I and III using as ligand a radiolabeled partially purified bacterial extract. Our study extends and clarifies these previous studies by using a highly purified monomeric recombinant protein rather than a proteolytic fragment of plasma vWF or a crude bacterial extract. In addition, our recombinant vWF-A3 protein is considerably smaller than that of Jorieux et al. (206 versus 450 residues). This is of some importance as the recombinant vWF-A3 protein used by Jorieux et al. also contains a portion of the D4 domain, making localization of the collagen-binding site exclusively within the A3 domain less certain.

While the recombinant vWF A3 domain polypeptide used in this study bound saturably and reversibly to immobilized type I collagen, its affinity ( K= 1.8 µM) is substantially lower than the estimated Kfor multimeric vWF (12, 13, 17, 18) . This suggests that other structural features within multimeric vWF may enhance binding and that the sequences present in vWF-A3, while necessary, are not sufficient for optimal binding. It is also possible that the presence of multiple collagen-binding sites in each vWF multimer enhances the overall affinity of multimeric vWF for collagen.

The failure of monomeric recombinant vWF-A1 to compete with vWF for binding to type I collagen remains a puzzle. The most likely explanation, and the one that is supported by our data, is that the vWF A1 domain does not, in fact, contain a collagen-binding site. This is supported by the observation that a recombinant form of multimeric vWF from which the A1 domain sequence has been deleted still binds to collagen normally (37) . There is also a recent preliminary report confirming that vWF from which the A1 domain has been deleted, which no longer binds to GPIb/IX, still binds to collagen (43) . This study also showed that deletion of the A3 domain preserves the GPIb/IX interaction, but abolishes collagen binding. It is widely appreciated that deletion mutagenesis experiments must be interpreted cautiously since deletion of large portions of the vWF subunit could induce major conformational changes in the remaining portion of the molecule; however, the selectivity of the two deletions suggests that this is not the cause for the observed losses of function. Finally, our data show that recombinant vWF-A3 can completely inhibit vWF multimer binding to collagen (Fig. 6). If both the A1 and A3 domains of vWF were binding to collagen, blockade of one domain should either have no effect on binding or cause only partial inhibition. The data we have obtained show that preventing binding via the vWF A3 domain completely abolishes vWF binding to type I collagen.

Further proof for the role of the vWF A3 domain in collagen binding comes from the characterization of a vWF-A1/A3 chimera containing the amino-terminal half of the A1 domain (amino acids 475-598) and the carboxyl-terminal half of the A3 domain (amino acids 1018-1114). As expected, the vWF-A1/A3 chimera inhibited ristocetin-induced platelet agglutination with an IC that was identical to that of vWF-A1 (0.2-0.6 µM). This observation confirms the importance of residues 474-488 (25) and 514-542 (38) for binding to GPIb/IX. The binding of the chimera to collagen, however, conflicts with previous studies of Roth et al.(17) , who reported that collagen-binding sequences in vWF lie between amino acids 542 and 622 (vWF-A1) and between amino acids 948 and 998 (vWF-A3). Although the chimera contains a portion of the reported vWF-A1 collagen-binding sequence (amino acids 542-598), its relevance to collagen binding is unclear since the native vWF A1 domain does not bind to type I collagen. In contrast, the collagen-binding sequence in vWF-A3 originally proposed by Roth et al. (residues 948-998) is not present in the chimera. However, the observation that the fusion of residues 1018-1114 from vWF-A3 to a vWF-A1 sequence that previously did not interact with collagen reconstitutes collagen-binding activity is fairly strong evidence that this sequence contains the collagenbinding site.

One limitation of our study is that the analysis was limited to a single collagen subtype. There is, in fact, evidence that vWF interacts with multiple collagen subtypes and may also bind to noncollagenous components of the extracellular matrix. For example, Rand et al.(14) identified type VI collagen as one potential binding protein for vWF in the vascular subendothelium. Denis et al.(15) recently analyzed the binding of proteolytic fragments of vWF to the extracellular matrix and to type VI collagen. Their study documents that vWF binds to the endothelial matrix via sequences in the A1 domain and that both the A1 and A3 domains contain a binding site for type VI collagen (15) .

Thus, while there is little doubt that the vWF A3 domain is essential for the binding of vWF to type I collagen, it is possible that other regions of vWF, including sequences within the A1 domain, may mediate vWF interactions with other collagen subtypes or other matrix components. The techniques described here for the expression, purification, and biochemical analysis of recombinant A domain polypeptides can be used in future studies to clarify which vWF domains interact with other collagen subtypes. They may also provide interesting models for designing agents that can selectively inhibit flow-dependent platelet adhesion, the initial event in normal hemostasis as well as in arterial thrombosis.


FOOTNOTES

*
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: Hematology-Oncology Div., Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-5840; Fax: 617-732-5706.

The abbreviations used are: vWF, von Willebrand factor; GP, glycoprotein; vWF-A1, vWF A1 domain polypeptide; vWF-A3, vWF A3 domain polypeptide; vWF-A1/A3, recombinant vWF polypeptide containing sequences from the A1 and A3 domains; IPTG, isopropyl--D-thiogalactopyranoside; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis.


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