Independent Assembly and Secretion of a Dimeric Adhesive Domain of von Willebrand Factor Containing the Glycoprotein Ib-binding Site*

von Willebrand factor (vWF) is a multimeric glycoprotein that supports platelet adhesion on thrombo- genic surfaces as part of the normal hemostatic response to vascular injury. We have employed a domain- specific expression strategy to analyze the biosynthetic processing steps and minimum structural requirements for assembly of the platelet receptor glycoprotein Ib-binding domain of vWF. A chimeric cDNA that codes for the vWF signal peptide and a segment of vWF internal primary sequence, residues 441-730, directs the secretion of a functional vWF fragment from mam- malian cells. The recombinant molecule intrinsically assembles through intermolecular disulfide bond for- mation into a dimeric adhesive domain without contributions from other regions of vWF, including propep- tide, previously indicated as essential for vWF multimer assembly. Prevention of N-linked glycosylation on the recombinant domain does not impair dimer formation or the ability to support platelet aggregation. These results identify a minimum structural element for vWF subunit assembly and provide new insights into the processing steps to produce vWF multimers and adhesive domains. was performed to remove the sequence that separates the third codon of the vWF propeptide and the Arg4" codon. A second round of mutagenesis was performed to add a translation termination codon and an XbaI restriction site following the vWF mature subunit codon Asn7:'". The entire vWF-coding sequence of the resultant M13 recombinant, designated pAD3, was determined to verify the organi-zation of the coding elements and the absence of any spontaneous mutagenic errors. The SalI/XbaI insert of the pAD3 was cloned into the eukaryotic expression plasmid, pcDNAl (Invitrogen), which was previously digested with XhoIIXbaI. The resultant plasmid was designated pAD4/WT. A similar expression plasmid was generated to establish transformants that constitutively secrete the vWF antigen. The SalI/XbaI fragment of pAD3 was cloned into the Sal1 and XbaI sites of pBS/ KS- (Stratagene, La Jolla, CA). The acquired sequence of the pBS/ KS- polylinker sequence allowed the removal of the vWF insert after digestion with XhoI (5' of the vWF initiating Met codon) and Not1 (3' to the artificial vWF stop codon). The XhoIINotI fragment was cloned into the XhoIINotI site of the expression plasmid pcDNAneo, which contains a neomycin resistance gene inserted in the parent plasmid pCDM8 (20). The expression plasmid containing the neo- mycin resistance gene is referred to as pAD5/WT.

among some of the 169 Cys residues of the vWF mature subunit produces multimeric vWF (12). Depending upon the extent of multimer formation, plasma vWF is composed of a population of molecules that have been estimated to range up to 50 individual subunits (1). The extent of vWF multimer formation is directly related to hemostatic efficacy (13,14).
Although vWF exists as a heterogeneous population of molecules, considerable progress has been made in localizing specific regions containing the crucial binding sites for normal hemostasis. When isolated under nonreducing conditions, a homodimeric 116-kDa tryptic fragment of plasma vWF, comprising subunit residues 449-728, inhibits binding of radiolabeled plasma vWF to platelet GP Ib-IX, heparin, and collagen (4,15), and like plasma vWF, can support platelet aggregation through interaction with GP Ib-IX (16). Thus, the 116-kDa tryptic fragment purified as an isolated domain retains crucial functions that are characteristic of native vWF.
To investigate the structural features that govern the adhesive properties of the 116-kDa region of vWF, we constructed a recombinant plasmid to synthesize independently the primary sequence of the 116-kDa tryptic fragment. The expressed molecule undergoes post-translational processing, including intermolecular disulfide bond formation, to produce a homodimeric domain. Moreover, the expressed domain recapitulates native vWF function as demonstrated by its ability to support agonist-induced GP Ib-mediated platelet aggregation and heparin binding. The covalent assembly of a dimeric domain identifies a vWF sequence with the minimum structural elements for assembly into an independent domain and demonstrates an alternative methodology to characterize the processing steps and functional properties of this complex adhesive protein.

EXPERIMENTAL PROCEDURES
Plasmid Construction-A recombinant plasmid, pAD4/WT, was constructed to direct the synthesis in mammalian cells of vWF mature subunit residues Arg4"-Asn'"o , corresponding closely to the monomeric subunit of the vWF 116-kDa tryptic fragment (residues Val44y-Lys7'"). pAD4/WT contains a chimeric vWF cDNA fragment with the following five structural elements in a 5' to 3' direction: 1) a Kozak consensus translation initiation sequence, CCACC (17); 2) the initiating vWF methionine codon, g , and coding sequence for the remainder of the 22-residue vWF signal peptide; 3) coding sequence for 3 amino acid residues from the amino terminus of the vWF propeptide; 4) coding sequence for vWF amino acid residues Arg"'-Asn':''; and 5) a translation termination codon, E. The coding sequence for vWF signal peptide was included in the cDNA fragment to direct cellular secretion of the synthesized molecules, and the coding sequence for the first 3 residues of vWF propeptide was included to leave the signal peptidase recognition sequence intact.
T o generate pAD4/WT, a vWF full-length cDNA clone (provided by Dr. Dennis Lynch, Dana Farber Cancer Institute, Boston MA) was used as template in a polymerase chain reaction (PCR) (18) to generate a 376-base pair fragment containing a Sal1 restriction site, the Kozak sequence, and coding sequence for precursor vWF residues 1-122. Oligonucleotide vWFKoZak ("GTCGACCCACCATGATTCCT GCCAGA") contains a Sal1 restriction site (italics) 5' to the Kozak sequence (underlined) followed by the vWF Met' (ATG) codon. The second oligonucleotide of the PCR, vWF1Z2 (5'CCTCAGTTTCTA--GATACAGCCCT"), corresponds to nucleotides 2/523-2/546 (according to numbering scheme of Ref. 11) and contains an XbaI restriction site (underlined). The PCR product was cleaved with Sal1 and XbaI, cloned into M13mp18 and designated pAD1.
The coding sequence corresponding to vWF mature subunit residues Arg441-Asn73n was also generated by PCR using oligonucleotides that add BamHI restriction sites to the ends of the amplified frag-TTTGCCTCAGGA"' and "GGGATCCACCATGGAGTTCCTCTTG-ment. The two oligonucleotides for this PCR ("ACGGATCCGGCGT-GG") correspond to nucleotide numbers 23/196-23/214 and 24/999-24/1018, respectively (the additional 5' nucleotides in italics were included to add BamHI restriction sites on the 5' end of each molecule). The amplified fragment was cloned into the BamHI site of PAD1 (BamHI site of PAD1 is within the polylinker of M13mp18) in an orientation that keeps the vWF-coding sequences within the two independently amplified fragments oriented in the same direction. The resultant clone was referred to as pAD2.
Uracil-containing single-stranded template of pAD2 was generated using Escherichia coli CJ236 for site-directed mutagenesis (19). Two subsequent mutagenesis experiments were performed to complete the formation of the chimeric expression insert. First, a loop-out mutagenesis was performed to remove the sequence that separates the third codon of the vWF propeptide and the Arg4" codon. A second round of mutagenesis was performed to add a translation termination codon and an XbaI restriction site following the vWF mature subunit codon Asn7:'". The entire vWF-coding sequence of the resultant M13 recombinant, designated pAD3, was determined to verify the organization of the coding elements and the absence of any spontaneous mutagenic errors. The SalI/XbaI insert of the pAD3 was cloned into the eukaryotic expression plasmid, pcDNAl (Invitrogen), which was previously digested with XhoIIXbaI. The resultant plasmid was designated pAD4/WT.
A similar expression plasmid was generated to establish transformants that constitutively secrete the vWF antigen. The SalI/XbaI fragment of pAD3 was cloned into the Sal1 and XbaI sites of pBS/ KS-(Stratagene, La Jolla, CA). The acquired sequence of the pBS/ KS-polylinker sequence allowed the removal of the vWF insert after digestion with XhoI (5' of the vWF initiating Met codon) and Not1 (3' to the artificial vWF stop codon). The XhoIINotI fragment was cloned into the XhoIINotI site of the expression plasmid pcDNAneo, which contains a neomycin resistance gene inserted in the parent plasmid pCDM8 (20). The expression plasmid containing the neomycin resistance gene is referred to as pAD5/WT. Cell Culture, DNA Transfection, and Selection of Stable Transformants-COS-1 (Green monkey kidney) and CHO-K1 (Chinese hamster ovary) cells were maintained in 5% CO, and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (10%-DMEM), 0.5 mM nonessential amino acids, and 2 mM L-glutamine (Whittaker Bioproducts). DNA was introduced into cells using a calcium phosphate-mediated transfection procedure (21). Cells were subcultured a t 1.5 X 105/60-mm dish 24 h prior to transfection. Ten pg of DNA/dish was applied and the cells were maintained in 10% DMEM after transfection.
For transient expression experiments (Fig. 2B), 40 h post-transfection, cells were rinsed with phosphate-buffered saline (PBS), incubated for 30 min in 4 ml of DMEM lacking methionine (GIBCO/ Bethesda Research Laboratories), and then supplemented with [35S] methionine (100 pCi/ml, Amersham Corp.) for 4 h. Cells were rinsed with PBS and incubated with 10%-DMEM for an additional 16 h or the indicated times for the chase experiment (Fig. 2). Culture media was harvested into 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride and centrifuged (1300 X g) to remove cellular debris.
To establish stable transformants, cells were split on the second day after transfection and grown for 10-14 days in the presence of 0.8 mg/ml of the antibiotic G418 (Sigma). Independent colonies were picked into 12-well plates and grown to confluence. Transformed cells expressing vWF antigen were identified by spotting cell lysates on nitrocellulose or immunoblotting after SDS-PAGE and testing for the presence of vWF antigen using monoclonal antibody LJ-RG46. To prepare cell lysates, cells were washed with PBS, resuspended in disruption buffer (10 mM Tris-HC1, pH 7.8, 150 mM NaC1, 5 mM EDTA, 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40) and lysed with three repetitive freeze-thaw cycles. Cellular debris was removed by centrifugation for 5 min (15,000 X g). Samples were reduced with 100 mM D T T (5 min a t 100 "C). Following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or spotting on nitrocellulose, samples were reacted with the anti-vWF monoclonal antibodies followed by an "'1-labeled rabbit anti-mouse IgG and subjected to autoradiography. vWF antigen-positive clones were expanded for large scale r116 immunopurification.
Immunopurification-An antibody-affinity column was prepared by coupling purified anti-vWF monoclonal antibody NMC-4 to CNBr-activated Sepharose 4B beads (Pharmacia LKB Biotechnology Inc.). Prior to r116 immunoaffinity purification, the column was equilibrated with 50 mM Tris, pH 7.4, 0.5 M LiCl, and 0.05% sodium azide. In a typical experiment, approximately 500 ml of 10% DMEM collected from confluent cells after a 24-h incubation was loaded onto the column followed by extensive washing (approximately 15 column bed volumes) with equilibration buffer. r116 was eluted from the column with 3 M sodium thiocyanate after which the column eluate was concentrated by ultrafiltration (Amicon) and dialyzed extensively against Hepes-buffered saline (20 mM Hepes, pH 7.4, with 0.15 M NaCI). Protein concentrations were determined using a bicinchoninic acid assay (Pierce Chemical Co.) according to the manufacturer's instructions. r116 purity was assessed by Coomassie staining after SDS-PAGE under nonreducing and reducing conditions. Typical yields from 500 ml of 10%-DMEM were approximately 400 pg of r116.
GP Ib, Heparin, and Collagen Inhibition Assays-Plasma vWF was purified from cryoprecipitate as previously described (23,24), labeled with Iz5I (Amersham Corp.) using IODO-GEN (Pierce Chemical Co.) (25), and used a t a final concentration of 2 pg/ml in all the inhibition assays. The molar concentration of dimeric r116 was calculated from the molecular mass of the constituent subunit (50 kDa).
The inhibitory effect of immunopurified 1-116 on 12sII-~WF binding to GP Ib was determined with formalin-fixed platelets (26) in the presence of ristocetin (final concentration 1 mg/ml, Sigma) as described (16). r116 was added to the platelet mixture at the indicated concentrations prior to the addition of 12sI-labeled vWF. After a 30min incubation, 50 pl of the mixture was centrifuged through a 20% sucrose gradient to separate bound radiolahled ligand from free ligand, and the bound radioactivity was determined in a y-scintillation spectrometer (23).
The heparin inhibition assay has also been described in detail elsewhere (6,15). Briefly, heparin-Sepharose CL-GB gel (Pharmacia LKB Biotechnology Inc.), "'1-labeled vWF, and r116 were added together in a final volume of 125 pl, incubated for 30 min (room temperature), and bound radiolabled ligand was separated from free radiolabeled ligand by centrifugation as described above.
Collagen assays were performed with a commercial preparation of pepsin-solubilized bovine dermal type I collagen (Vitrogen 100, Collagen Corp.) prepared as fibrillar collagen (4,6). After a 20-min (room temperature) incubation, the mixture of r116, 'zsI-~WF, and fibrillar collagen (final volume 75 pl) were pelleted by centrifugation (30 min, 12,000 X g), and the radioactivity within the insoluble collagen pellet was quantitated by y-scintillation counting. The anti-vWF antibody, MR5, has been previously characterized as an inhibitor of vWFcollagen binding (3).
Platelet Aggregation-Platelet aggregation was examined in siliconized glass cuvettes placed in a Lumi-aggregometer (Chrono-Log Corp.) a t 37 "C with constant stirring (1,200 rpm). Aggregation was measured by monitoring the increase in light transmittance through the stirred platelet suspension. Human platelets were prepared as described (27) and used at a final concentration of 2 X 108/ml in the presence of ristocetin. For antibody inhibition studies, purified IgG Domain-specific Expression of von Willebrand Fuctor of the anti-GP Ihn monoclonal antibody LJ-Ibl (28) or the anti-GP IIh/IIIa antibody LJ-CPR (29) was added a t a final concentration of 100 pg/ml and incubated with platelets for 1 min a t 37 "C before the addition of ristocetin. Tunicamycin Treatment of Stabb Transformants-Stable transformants secreting vWF antigen were incubated overnight in 10%-DMEM containing tunicamycin (0.8 pglml) (Sigma). Cells were washed twice with PBS and incubated in DMEM containing tunicamycin (0.4 pg/ml) for another 24 h. Culture medium was harvested and concentrated up to 300-fold by centrifugation (Centricon 30, Amicon) prior to assay. Control stable transformants were treat.ed identically except that the culture medium lacked tunicamycin. For platelet aggregation assays from tunicamycin-treated and untreated cells (Fig. .5B), the quantity of NMC-4 antigen was determined from densitometric scans of a Western blot autoradiograph (Fig. 5A ). As determined from the densitometric scans, equivalent amounts of NMC-4 antigen were used in the platelet aggregation assay.

RESULTS
Expression of the 116-kDa Domain of vWF-To direct the independent expression and secretion of the 116-kDa domain of vWF, we constructed the expression plasmid pAD4/WT (Fig. 1). The chimeric vWF cDNA fragment within pAD4/ W T contains coding sequence for vWF signal peptide and the first 3 residues of vWF propeptide fused to the coding se-  (6). The monomeric subunit of the 116-kDa fragment is a heterogeneous 52/48-kDa fragment extending from of the vWF mature subunit and is illustrated below the corresponding region of the mature subunit (15). Portions of the vWF cDNA were fused to generate the recombinant plasmid, pAD4/ WT, for synthesizing the primary sequence of the 116-kDa fragment. Briefly, the coding sequence for the vWF initiating methionine (ATG), signal peptide, and three N-terminal residues of propeptide were joined to coding sequence closely corresponding to the 52/48-kDa monomeric subunit fragment of the 116-kDa tryptic fragment. A translation termination codon (TCA) was added 3' to the Asn""' codon. The expression plasmid contains a cytomegalovirus promotor ( C M V promoter) and a polyadenylation signal (polyfA) signal) to drive the expression of the vWF fragment.
V~I.I.I!I to Lys: ' x quence for vWF subunit residues Arg441-Asn7:'". The signal peptide and 3 residues of the propeptide were included to facilitate secretion and minimize structural constraints that might block signal peptidase cleavage and secretion as a result of the chimeric attachment of residues 441-730. pAD4/WT also contains a termination codon immediately following the Asn""' codon. COS-1 cells were transfected with pAD4/WT, radiolabeled with [%]methionine, and the culture medium was immunoprecipitated with the anti-vWF monoclonal antibody, NMC-4. The epitope recognized by NMC-4 is dependent upon a disulfide-bond-dependent conformation present within the vWF 116-kDa tryptic fragment and native vWF (16,22). SDS-PAGE analysis of the immunoprecipitation products under nonreducing conditions revealed a predominant species of approximately 116 kDa secreted by cells transfected with pAD4/WT, whereas control transfections usingpcDNAl were negative for vWF antigen (Fig. 2 A ) . Electrophoresis under reducing conditions revealed a 52-kDa but not the 116-kDa species, as expected from the dissociation of dimeric 116-kDa molecules into their monomeric components ( Fig. 2A). The radiolabeled 116-kDa antigen could be detected as early as 15 min after the removal of [""Slmethionine from transfected cells (Fig. 2B). Thus, the vWF signal peptide directs the secretion of the internal vWF domain, designated r116, and the molecule is immunoprecipitated with an anti-vWF monoclonal antibody that recognizes native vWF conformation. The presence of a t least one intermolecular disulfide bond is demonstrated by a predicted increase in electrophoretic mobility after reduction of disulfide bonds.
Functional Properties of rll6-To characterize r116 more completely, we inserted the vWF chimeric insert of pAD4/ WT into a similar vector, pcDNAneo, which allows the selection of stable transformants resistant to the aminoglycoside antibiotic, G418. The resultant r116 expression plasmid, pAD5/WT, was transfected into CHO-K1 cells and stable transformants were selected that constitutively secrete r116. r116 was immunopurified from the culture medium of stable transformants using an -NMC-4 immunoaffinity column. As illustrated in Fig. 3, immunopurified r116 can successfully compete with ""I-vWF for binding to formalin-fixed platelets in the presence of ristocetin with an ICso value of approximately 1 PM. Likewise, r116 can inhibit "'I-vWF binding to heparin with an ICso value of approximately 7.5 p~ (Fig. 3).

LJ-CP8
I the a-subunit of the GP Ib-IX complex and blocks vWF binding to GP Ib-IX (28), abolished the aggregation response, whereas incubation with the anti-GP IIb/IIIa antibody LJ-CP8 (29) did not, consistent with a vWF-GP Iba-specific interaction (Fig. 4B).
Prevention of N-Linked Glycosylation on r116 Does Not Impair Dimer Formation or Platelet Aggregation-The presence of N-linked carbohydrate side chains within r116 was investigated by treating pAD5/WT stable transformants with tunicamycin. Western blot analysis demonstrates that the expressed r116 from untreated cells can be resolved into species with slightly different electrophoretic mobilities, whereas r116 synthesized in the presence of tunicamycin is a single major species (Fig. 5A). The results demonstrate that the presence of N-linked carbohydrate side chains is not a prerequisite for the formation of intermolecular disulfide bonds to produce r116. The heterogeneity of species observed in the absence of tunicamycin demonstrates that N-linked glycosylation on r l l 6 is a heterogeneous process, presumably due to incomplete processing of N-linked side chains or complete lack of N-linked side chains on some of the monomeric immunopurified r116 does not compete with '''I-vWF for binding to type I collagen. In the same collagen inhibition assays, an inhibition of 80% was observed with addition of the anti-vWF monoclonal antibody MR5 (100 pg/ml), a known inhibitor of vWF-collagen binding (3). A similar 80% inhibition of ""I-vWF binding could be demonstrated by MR5 in the presence of 12 p~ r116 further illustrating the apparent lack of interaction between r116 and fibrillar collagen or between r116 and MR5 (data not shown). Thus, of the three reported functional properties of the tryptic 116-kDa fragment, binding to platelet GP Ib, heparin, and collagen, r116 recapitulates two of these functions but has no apparent affinity for type I collagen. Immunopurified r116 was further evaluated for its ability to support platelet aggregation in the presence of ristocetin (Fig. 4). The aggregation response increases with increasing amounts of r116 and is dependent upon the addition of ristocetin, a property shared with native vWF. Preincubation of platelets with the monoclonal antibody LJ-Ibl, which binds "Experimental Procedures," and the secreted products were analyzed directly by Western blot analysis. Following SDS-PAGE and immunoblotting, nonreduced samples ( N R ) were reacted with NMC-4, reduced ( R ) samples with W-RG46, and the immunoreactive proteins detected by incubation with an ""I-labeled rabbit anti-mouse IpG and subsequent autoradiography. The results demonstrate that NMC-4 recognizes a population of molecules with slightly different mobilities from untreated (-) cells as compared to tunicamycin treated (+) cells. Under reducing conditions W-RG46 recognizes three major species in untreated cells, whereas after tunicamycin treatment a single major species is observed under reducing conditions. Panel B, platelet aggregation assays were performed from tunicamycin treated (+) and untreated (-) cells using equal amounts of NMC-4 antigen as determined from densitometric scans of the autoradiograph illustrated above ( A ) . r116 synthesized in the presence or absence of tunicamycin can support platelet aggregation. No platelet aggregation was observed in the absence of ristocetin for either sample or with concentrated culture media from nontransfected CHO-K1 cells.

Domain-specific Expression of von Willebrand Factor
subunits of r116. Glycosylation within the analogous 116-kDa tryptic fragment of plasma vWF is also heterogeneous and explains the doublet produced (52/48 kDa) after reduction of the tryptic fragment (5). The functional contribution of N-linked glycosylation on r116 was evaulated in platelet aggregation assays. The relative quantities of r116 from tunicamycin-treated and untreated cells were determined from densitometric scans of the Western blot illustrated in Fig. 5A, and equal amounts of NMC-4 antigen were assayed for their ability to support GP Ibmediated platelet aggregation. The results demonstrate that r116 from tunicamycin-treated cells retains the ability to support GP Ib-mediated platelet aggregation (Fig. 5B), and like r116 produced in the absence of tunicamycin, requires ristocetin to support platelet aggregation. Control culture media from untransformed CHO-K1 cells did not induce platelet aggregation.

DISCUSSION
The role of vWF in hemostasis is to serve as an anchoring substrate for platelet-subendothelium and platelet-platelet interactions. In spite of the structural complexity of multimeric plasma vWF, a single 116-kDa tryptic fragment has been characterized containing vWF-binding sites for the platelet GP Ib-IX receptor complex, collagen, and heparin (3,4,6,15). In the present study, we have obtained evidence that the 116-kDa fragment of vWF can be assembled and secreted as an independent molecule with the ability to: 1) fold into a disulfide-dependent conformation analogous to that of the corresponding domain of native vWF; 2) intrinsically assemble into a dimeric molecule without contributions from adjacent regions of vWF or N-linked carbohydrate side chains; and 3) retain function as shown by its ability to support agonist-induced GP Ib-mediated platelet aggregation and interact with heparin.
The observed self-assembly of the monomeric subunits of r116 provides information on the regulation of intracellular processing to produce multimeric vWF. Previous structural analyses of tryptic vWF fragments had identified the intermolecular disulfide bonds responsible for vWF multimers to be within two regions of vWF, an N-terminal region (residues 283-695) and a C-terminal region (residues 1908-2050) containing 30 and 18 Cys residues, respectively (12). Wagner et al. (30) proposed that the initiating step of multimer formation is dimerization of precursor vWF molecules via C-terminal disulfide bonds. Studies with recombinant molecules suggested that the formation of multimers, after dimer formation, is dependent on the 741-residue vWF propeptide, presumably by promoting the formation of intermolecular disulfide bonds within the N-terminal region (31,32). More recently, a truncated recombinant molecule containing the vWF propeptide and residues 1-470 of the mature subunit has been shown to form dimeric molecules through N-terminal intermolecular disulfide bonds (33). This result demonstrates that dimer formation as a result of C-terminal intermolecular disulfide bonds is not a prerequisite for N-terminal intermolecular disulfide bonds. The expression of r116 demonstrates that vWF residues N-terminal to Gly440, including vWF propeptide, are not required for the synthesis of a functional dimeric domain.
As defined by Titani et al. (34), plasma vWF contains 1 Nlinked and 8 O-linked carbohydrate side chains associated with each monomeric subunit of the 116-kDa tryptic fragment. The large disulfide-loop within each monomeric subunit is flanked by two clusters of 0-linked carbohydrate side chains, four within Thr484-Ser500 and four within Thr705-Thr7'*. Some heterogeneity of vWF glycosylation has been reported (35) and explains the doublet produced after reduction of the 116-kDa tryptic fragment (5). A structural role for N-linked glycosylation within plasma vWF has been suggested based on tunicamycin treatment of cultured human endothelial cells and the observed lack of native vWF dimer formation and subsequent multimer formation (36). Our results from the tunicamycin treatment of transformed CHO-K1 cells demonstrate that N-linked glycosylation of r116 is also heterogeneous but the structural and/or functional consequences of N-linked glycosylation within r116 are not apparent. r116 lacking N-linked side chains is still secreted as a functional dimeric domain.
The use of r116 in GP Ib inhibition assays is similar to previous assays performed with vWF tryptic fragments. Using a 116-kDa tryptic fragment, Mohri et al. (16) reported an IC50 value of 0.05 PM as compared to 5 PM reported by Fujimura et al. (22) using a similar fragment. A 100-fold discrepancy in values between two independently prepared tryptic fragments could reflect the extent of proteolysis within the purified fragments. By comparison, r116 had an value of approximately 1 PM and represents an intermediate value as compared to the tryptic fragments. The requirement of agonist to support r116 binding to GP Ib is analagous to the situation with plasma vWF and demonstrates that r116 mimics a characteristic functional property of plasma vWF. We have recently demonstrated that a r116 domain containing an amino acid substitution associated with type IIB von Willebrand's disease can bind GP Ib in the absence of agonist (37) analogous to reduced and S-carboxymethylated molecules lacking the disulfide bond-dependent conformation (15,37).
Based on the previous characterization of vWF tryptic fragments (3, 4, 6, 38), an unexpected result is the inability of r116 to inhibit binding of '251-vWF to type I collagen. Several possible explanations for this result can be proposed and will be pursued in our continuing characterization of r116. An obvious explanation would be structural differences, such as glycosylation or tertiary structure, between r116 and the tryptic 116-kDa fragment. It is possible that during the immunopurification, as compared to the procedures associated with the isolation of a tryptic fragment, r116 has been structurally altered resulting in a dysfunctional collagen-binding site. It should also be remembered that vWF contains another collagen-binding domain within residues 911-1114 (4) (corresponding closely to the vWF A3 domain), and this sequence contains the epitope recognized by an anti-vWF monoclonal that inhibits vWF-collagen binding (3). If the collagen-binding site within the A3 domain is a high affinity binding site, it is possible that trace contaminants of this sequence or even trace contaminants of multimeric vWF within the tryptic 116-kDa preparations may have erroneously identified a collagen-binding site using the purified tryptic fragment. The results are further complicated when the purity of the collagen tested is evaluated. We demonstrated that r116 binds heparin, and it is reasonable to assume that this domain might contain binding sites for additional heparinlike molecules or constituents of connective tissue that may contaminant crude preparations of collagen. Such a phenomenon would help explain why some reports have suggested that native conformation is not required for this domain to interact with collagen (3,24,38).
Site-directed mutagenesis of the r116 domain will provide an opportunity to precisely define the structural elements that are responsible for normal vWF function. In the expression systems that we have characterized, approximately 50-fold more r116 is synthesized by pAD5/WT, than constructs expressing prepro-vWF. The increased expression levels insure that sufficient quantities of variant recombinant molecules can be produced for detailed structural and functional studies.