selective inactivation of the arg-gly-asp-ser (rgds) binding site in

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 5, Issue of February 15, pp. 3409-3415, 1992 Prmted in U.S.A.

Selective Inactivation of the Arg-Gly-Asp-Ser (RGDS) Binding Site in von Willebrand Factor by Site-directed Mutagenesis*

(Received for publication, May 13, 1991)

Dorothy A. Beacham, Robert J. Wise, Susan M. Turci, and Robert I. Handin$ From the Hematology-Oncology Division, Brigham and Women’s Hospital Department of Medicine, Harvard Medical School, Boston, Massachusetts 021 15

In order to assess the requirement for the Arg-Gly- Asp-Ser (RGDS) consensus adhesion sequence in von Willebrand factor (vWF) for v W F binding to platelets and endothelial cells, point mutations were introduced into this sequence by site-directed mutagenesis. A glycine to alanine substitution yielded RADS-vWF, while an aspartate to glutamate substitution resulted in RGES-vWF. Recombinant RADS-vWF and RGES- vWF, purified from transformed Chinese hamster ovary cells, were compared with recombinant wild type vWF (WT-vWF) in functional assays with platelets and human umbilical vein endothelial cells (HU- VECs). High molecular weight RADS-vWF and RGES- vWF multimers inhibited binding of ‘aaI-vWF to a mixture of insolubilized native type I and I11 collagen and competed effectively with ‘261-vWF for binding to formalin-fixed platelets in the presence of ristocetin, indicating functional collagen and platelet glycoprotein Ib binding. However, RADS-vWF and RGES-vWF were unable to displace the binding of ‘251-vWF to thrombin or ADP-activated platelets. The attachment of HUVECs to either RADS-vWF or RGES-vWF coated surfaces was reduced and spreading was almost completely inhibited, compared with WT-vWF. We con- clude that point mutations of the RGDS sequence in vWF selectively impair binding to platelet glycoprotein IIb/IIIa and the HUVEC vitronectin receptor.

The von Willebrand factor (vWF)’ is a large multimeric glycoprotein that facilitates the initial adhesion of platelets to the subendothelial matrix following vascular injury. For stable platelet adhesion under high flow conditions, vWF, present in the subendothelial matrix or the plasma, must bind to the glycoprotein Ib/IX (GPIb/IX) receptor on the platelet

* This work was supported by a grant from the American Heart Association, Massachusetts Affiliate Fellowship (to D. A .B.) and National Institutes of Health Grants T32-HL07623 and POI- HL33014 (to R. I. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence and reprint requests should be ad- dressed Hematology-Oncology Division, Dept. of Medicine, 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; CM, conditioned medium; CHO, Chinese hamster ovary; GPIIb/IIIa, platelet glycoprotein IIb/IIIa; HUVEC, human umbilical vein endothelial cell; MEM, minimal essential medium; DMEM, Dulbecco’s modified Eagle’s medium; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; VNR, vitronectin receptor; BSA, bovine serum albumin; ELISA, enzyme- linked immunosorbent assay; HEPES, 4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid; WT, wild type.

surface. Following platelet attachment and activation, vWF also may bind to the platelet GPIIb/IIIa receptor. GPIIb/IIIa is a member of the integrin superfamily of heterodimeric adhesion receptors, which binds fibronectin, fibrinogen, vWF, and vitronectin (1). Although the binding of fibrinogen to GPIIb/IIIa is the predominant interaction contributing to platelet aggregation and formation of a thrombus, vWF binding to GPIIb/IIIa can support aggregation in the absence of fibrinogen (2) and may regulate the size of platelet thrombi (3) in flow-dependent adhesion models (3,4).

vWF is initially synthesized as pro-vWF which contains a 741-amino acid propeptide that is cleaved in the Golgi appa- ratus during multimer formation (5-7). Binding sites for factor VIII, GPIb/IX, heparin, collagen, and GPIIb/IIIa have been localized to discrete domains in the 2050-amino acid mature vWF protein subunit. As previously shown using Arg- Gly-Asp-Ser (RGDS)-containing peptides, the binding of vWF to activated platelets is dependent on the RGDS consensus cellular adhesion recognition sequence, present at residues 1744-1746 in the mature vWF subunit (8-10). The RGDS-dependent binding of vWF to activated platelets has been attributed to the platelet membrane receptor GPIIb/IIIa ( a r I B P 3 in the integrin family of adhesion receptors) (2). Using an immunological approach, Dejana et al. (11) found that an antibody against the RGDS-containing peptide Gly1737 to Ser1750 could also inhibit adhesion of HUVECs to vWF substrates. In contrast, an antibody raised against the vWF residues adjacent to the RGDS sequence, Gly1740-Ser’742, had no effect on HUVEC adhesion. The authors concluded that HUVEC attachment to a vWF substrate is mediated by RGDS-dependent binding to the HUVEC vitronectin receptor (VNR, W33).

While the peptide and antibody-inhibition studies suggest that the RGDS sequence in vWF is essential for the binding of vWF to platelet GPIIb/IIIa and the endothelial VNR, other regions of the vWF molecule might bind to these receptors if the RGD sequence were abolished. In the case of fibronectin, Obara et al. (12) found a putative “second adhesion site” amino-terminal to the RGDS site which was shown, in a later report, to reside within the type I11 homology domain of the fibronectin molecule (13). Presumably, a “second adhesion site,” distinct from RGDS, could also exist on vWF for GPIIb/ IIIa or the HUVEC analog, the VNR. Such a site might contribute to selective binding of vWF to platelet GPIIb/IIIa or the endothelial VNR.

To determine the importance of the RGDS sequence in the intact vWF molecule and to extend previous adhesion studies, site-specific vWF mutant cDNAs were prepared and recombinant vWF expressed in Chinese hamster ovary (CHO) cells. There are several theoretical advantages to this approach. First, a single amino acid substitution within the RGDS sequence should abolish its functional activity without affect-

3409

3410 The uon Willebrand Factor RGDS Binding Site

ing other vWF activities. Second, by inactivating the RGDS site in multimeric vWF with point mutations, functional changes can be traced to single, well defined amino acid substitutions. Third, the vWF containing an RGDS mutation can be expressed in CHO cells which do not synthesize vWF.

In this report we utilized the RGDS mutants of vWF to study binding to GPIb/IX on fixed platelets in the presence of ristocetin and the binding of the RGDS-mutant vWF to GPIIb/IIIa on activated platelets. The adhesion of HUVECs onto an RGDS-mutant vWF substrate was also examined. Our results demonstrate that new insights into the mechanism of HUVEC adhesion to vWF can be gained from these RGDS mutants.

MATERIALS AND METHODS

Mutagenesis and Plasmid Construction-The RGDS sequence re- sponsible for vWF binding to GpIIb/IIIa is located at amino acid residues 1744-1747 in the mature protein, which corresponds to residues 2507-2510 in pro-vWF (5, 6, 14). Mutations were made in vWF cDNA by changes at positions corresponding to residue 1745 (glycine + alanine, G-A) in the RADS-vWF mutant and 1746 (aspartic acid -+ glutamic acid, D-E) in the RGES-vWF mutant. Oligonucleotide-directed mutagenesis was performed as described (15). A 1642-base pair BglII-EcoRI vWF cDNA fragment, containing the RGDS-coding sequence, was subcloned into M13 mp19. Recom- binant phage were grown in a dut- ung- F' E. coli strain to generate single-stranded MI3 template containing uracil residues (15). A 5'- phosphorylated mutagenic oligonucleotide primer was then annealed to the recombinant template. For second strand synthesis, the mutagenic primer was extended with T4 DNA polymerase and the ends were ligated using T4 DNA ligase. The resultant heteroduplex was subsequently transformed into a dut' ung+ E. coli strain. The RADS- vWF mutant was generated with the mutagenic oligonucleotide 5'- CTG GGA GTC CG*C CCG AGG TGA GCC-3', and the RGES- vWF mutant with the mutagenic oligonucleotide 5'-CTG GGA T*TC CCC CCG AGG TGA GCC-3'. Both oligonucleotides are complemen- tary to the sequence 7609-7632 (14) except for the single-base mutation indicated by the asterisk and were synthesized using an Applied Biosystems oligonucleotide synthesizer. The mutant vWF cDNA inserts were sequenced by the dideoxy chain-termination method (16) to confirm the presence of the appropriate nucleotide substitutions. The plasmid DNA of the RGDS mutants was isolated and digested with BstYI and EcoRV to remove it from the M13 mp19 vector. The respective mutant DNA was then ligated into the appropriate BglII- EcoRV partially digested full-length vWF cDNA in pUC13 and subsequently shuttled into the expression vector pMT2 for expression in COS-1 and CHO cells (6).

wild type (WT) vWF cDNA in pMT2 was transfected into COS-1 Transfections-To express vWF transiently, RGDS-mutant and

cells using a DEAE-dextran protocol with a dimethyl sulfoxide shock (17). COS-1 cells were rinsed twice with DMEM (Dulbecco's modified Eagle's medium) prior to the addition of 10 pg of DNA in 4 ml of 10 mM Tris/DEAE-dextran; final concentration 250 pglml, pH 7.3. After 6 h, cells were rinsed with DMEM and shocked for 2 min with 10% dimethyl sulfoxide in DMEM containing 25 mM HEPES, pH 7.3. Cells were allowed to recover in DMEM, 10% fetal calf serum.

To produce stable cell lines, the vWF expression vectors were transfected into dihydrofolate reductase-deficient CHO cells (CHO- DUKX) using lipofection reagent (BRL/Life Technologies, Inc.) in serum-free Opti-Mem I medium essentially as described by the man- ufacturer. Selection for vWF-dihydrofolate reductase producing CHO cells was carried out in a minimal essential medium (MEM) without nucleosides. Production of vWF was quantitated by enzyme-linked immunosorbent assay (ELISA) (American Diagnostica, Inc.) using WT-vWF and plasma vWF as a positive control.

Cell Labeling-COS-1 cells were labeled with [35S]methionine and [35S]cysteine 48-72 h after recovery from dimethyl sulfoxide shock. For pulse-labeling of vWF in either COS-1 or CHO cells, confluent monolayers were plated in 1.5 of ml serum-free MEM deficient in methionine and cysteine (Gibco) containing 0.15% tissue culture grade BSA (Sigma), insulin/transferrin/sodium selenite mixture (GIBCO), aprotinin (Sigma), glutamine, penicillin/streptomycin, and 250 pCi each of [35S]methionine and [35S]cysteine (Amersham Corp.) per 10-cm dish. After 1 h, 1 ml of complete serum-free medium was added. Conditioned medium (CM) was collected after 18 h and

centrifuged to remove cellular debris. Additional aprotinin, 0.01% NaN3, and 5 mM Na2EDTA were added to the CM prior to storage at 4 "C.

Preparation of Concentrated CM-For large scale production of CM, CHO cells expressing either WT-vWF, RADS-vWF, or RGES- vWF were grown on microcarrier beads (Cytodex 3, Pharmacia) in Spinner culture. After the beads reached confluence, they were centrifuged and resuspended in serum-free a-MEM. The CM was collected at 24 h, centrifuged to remove microcarriers, saturated am- monium sulfate was added to a final concentration of 40%, and the precipitate collected by centrifugation at 1000 X g for 30 min. The pellet was resuspended in Tris-buffered saline (TBS; 100 mM Tris, 150 mM NaC1, pH 7.4). The concentration of vWF before and after concentration was determined by ELISA.

Immunoprecipitation-vWF was immunoprecipitated from CM essentially as described (18). Briefly, 100 pl of 10 x immunoprecipitation (IP) buffer (100 mM Tris-HC1, 5% deoxycholate, 5% Triton X- 100, 10 mM NazEDTA, 1.5 M NaCl, pH 7.2) was added to 900 p1 of CM and 10 p1 of rabbit anti-vWF antiserum. After shaking for 4 h at 4 "C, 20 pl protein A-Sepharose beads (Pharmacia) were added and the mixture incubated overnight a t 4 "C. Beads were washed 5 times with cold IP buffer and once with TBS, prior to resuspension in sample buffer for gel electrophoresis.

Preparation of Rabbit Anti-u WF Antibodies-vWF purified from plasma as previously described (19) was separated by SDS-PAGE on 6% gels under reducing conditions and transferred to nitrocellulose. The M, = 220,000 protein band, which corresponded to mature vWF, was emulsified with complete Freund's adjuvant and injected into New Zealand White rabbits. For the second, third and fourth injec- tions, a mixture of native and heat-denatured purified vWF was injected subcutaneously. Antisera was obtained which reacted with vWF at a 1:2000 dilution on an immunoblot.

Immunoaffinity Chromatography of u WF-The IgG fraction of the rabbit anti-vWF antiserum was purified on protein A-Sepharose by elution with 0.1 M glycine, pH 2.5. After dialysis against Dulbecco's phosphate-buffered saline (NaCl, 8 g/liter; KCl, 0.2 g/liter; Na2HP04, 1.15 g/liter; KH2P04, 0.2 g/liter), the IgG fraction was coupled to

bilization kit (Pierce Chemical Co.) and used to purify plasma vWF Aminolink columns using the Immunopure antigen/antibody immo-

from cryoprecipitate and recombinant vWF from concentrated CM. The column was washed in TBS, pH 8.0, and CM containing RADS- vWF, RGES-vWF, or WT-vWF was continuously circulated over the column for 2 h at room temperature or overnight a t 4 "C. The column was washed with 1 X IP buffer to remove nonspecifically bound proteins, then vWF was eluted in 0.1 M glycine, pH 2.5.

Isolation of u WF by Size Exclusion Liquid Chromatography-More recently, recombinant vWF was purified from CM using a Waters 650E protein purification system. CM was dialyzed uersus HzO, lyophilized, and resuspended in column buffer (Trizma base, 20 mM; 20 mM c-aminocaproic acid (0.15 M NaCl, 20 mM Naacitrate, pH 6.5). The sample was loaded onto a Sepharose CL-4B (Pharmacia) column from which vWF eluted in the void volume. Both immunoaffinity- purified and chromatographically purified recombinant vWF were shown to be pure by silver stain analysis of SDS-PAGE gels, and each behaved identically in attachment assays.

Gel Electrophoresis of uWF-SDS-PAGE (20) of reduced samples was performed on 6% polyacrylamide using a 11O:l ratio of acrylamide to bisacrylamide as described (18). Immunoprecipitates were heated to 95 "C for 5 min prior to loading onto gels. SDS-agarose gel electroDhoresis was Derformed to separate vWF multimers as described (18).

Iodination of u WF-Iodo-beads were we-eauilibrated with TBS and added to a' tube containing 1 mCi of NalZ5i (Amersham, specific activity 1.5 pCi/pg). After 5 min, 100 pg of purified vWF was added and the reaction continued for 15 min. The vWF solution was then applied to a Sephadex G-25 column, and fractions that were >80% trichloroacetic acid-precipitable were collected (21).

Zsolation of Platelets-Human blood samples were collected into acid/citrate/dextrose and centrifuged to remove red blood cells. Plate- lets were isolated using an albumin gradient technique to remove contaminatingplasma proteins (22). To prevent endogenous secretion of activated platelets, 0.1 unit/ml apyrase, 500 mM creatine phosphate, and 2100 units/ml creatine phosphokinase were added to Tyrode's buffer (5 ml of solution 1 (2.75 M NaC1,54 mM KC1,60 mM NaHzP04.H20), 5 ml of solution 3 (0.2 M HEPES), 100 mg of dextrose), pH 7.4. Platelets were resuspended in Tyrode's buffer and excess albumin was removed by applying the platelet suspension to a Sepharose CL-2B column (22). The platelet suspension was counted

The von Willebrand Factor RGDS Binding Site 3411 on a particle counter (Coulter, model Z,,,), and adjusted to 2.5 X lo5 platelets/pl in the absence of secretion inhibitors prior to binding. Fixed platelets were prepared for analyzing binding in the presence of ristocetin as described (21).

Binding of vWF to Native Collagen-Binding of vWF to native types I and I11 collagen was performed as described (21). Briefly, 1.87 mg/ml acid-soluble calf skin collagen (Worthington) was adsorbed to microtiter wells in 20 mM sodium citrate, pH 6.1, for 90 min. The unadsorbed collagen was then removed by washing with TBS, pH 7.4, followed by blockage of nonspecific sites by incubation in 0.1% BSA/TBS for 60 min. Wells were then incubated with "'I-vWF in the presence of increasing concentrations of unlabeled plasma vWF or concentrated CM from WT-vWF, RADS-vWF, or RGES-vWF.

Binding to Fixed Platelets in the Presence of Ristocetin-To a total volume of 75 pl, 1 X 10' formalin-fixed platelets, 1 mg/ml BSA, 1 mg/ml ristocetin, 2.6 pg/ml '251-vWF, and increasing concentrations of unlabeled vWF ranging from 0.18 to 80 pg/ml were added in Tyrode's buffer. Nonspecific binding was determined by adding a 20- fold excess of unlabeled plasma or WT-vWF or by omitting ristocetin. After mixing for 30 min a t room temperature, 50 pl of the platelet suspension was layered onto 500 pl of 50% sucrose/Tyrode's buffer and spun at 12,000 X g for 4 min in a microcentrifuge. The tube was quick-frozen in liquid nitrogen and the tip containing the pellet cut off and counted in a y counter. A statistical analysis of the binding data was performed using the EBDA and LIGAND binding analysis programs (Biosoft, Inc.)

Binding to Activated Platekts-Competition of '2'II-~WF binding to stimulated platelets with unlabeled plasma vWF, WT-vWF CM, RADS-vWF CM, or RGES-vWF CM was carried out as described above except that thrombin or ADP was used to stimulate freshly isolated platelets. Thrombin was added to the assay mixture at a final concentration of 0.1 unit/ml and after 10 min, hirudin was added at a 16-fold excess to neutralize thrombin. ADP was added to the assay mixture a t a final concentration of 2.5 X io+ M.

HUVEC Adhesion and Spreading-HUVECs were grown to confluence in T-75 flasks in medium 199 supplemented with 15% fetal calf serum, 100 pg/ml porcine intestinal heparin, and 50 pg/ml bovine endothelial cell growth factor (Biomedical Technologies, Inc.). Cells were labeled for 4 h following the addition of 150 pCi/ml ["S] methionine and 150 pCi ["SJcysteine. HUVECs were harvested ac- cording to the procedure of Dejana et al. (11) by brief exposure to trypsin/EDTA solution, followed by a 10-min recovery in serum-free medium, and resuspended in serum-free M199 for use in the adhesion assay. Microtiter wells were prepared by incubation with affinity- purified vWF in phosphate-buffered saline a t a final concentration of 30 pg/ml. Excess vWF was aspirated, and nonspecific binding sites were blocked with 0.1% BSA in phosphate-buffered saline for 60 min. Fifty thousand labeled cells were aliquoted into microtiter wells in the presence of 10 pg/ml cycloheximide and 0.7 pg/ml monensin. HUVECs were incubated a t 37 "C in a humidified incubator and attachment determined a t 5, 15, 30, and 60 min. Unattached cells were removed by three washes in phosphate-buffered saline. Attached cells were lysed with 100 pl of 10% SDS heated to 65 "C, and radioactivity counted in a liquid scintillation spectrometer. Attach- ment to WT-vWF was compared with the vWF mutants using a Student's t test. To assess HUVEC cell spreading, affinity-purified WT-vWF or RGDS-mutant vWF was adsorbed to microtiter wells as described above, and cells were examined by phase contrast microscopy 1 h after plating.

RESULTS

Multimers from RGDS-Mutant v WF Are Indistinguishable from WT-vWF-RADS-vWF, RGES-vWF, and WT-vWF cDNAs were initially transfected into COS-1 cells to study the processing and multimerization of RGDS-mutant vWF. Stable CHO cell lines were then established in order to isolate sufficient vWF from CHO CM for the attachment and spreading studies. When assayed by ELISA CHO cell lines produced 2 and 4 pg/ml of RGES-vWF and RADS-vWF, respectively. In order to analyze vWF synthesis, transfected COS-1 and CHO cell proteins were radiolabeled with [3sS]methionine and ["S]cysteine and CM was collected. As shown in Fig. lA, the processing of CHO RADS-vWF (lane 1 ), RGES-vWF ( l a n e 2), and WT-vWF ( l a n e 3) was indistinguishable by SDS- PAGE. All three vWF-producing CHO cell lines yielded a

1 2 3 4 1 2 3 4 1 2 3 4 5

FIG. 1. Gel electrophoresis of recombinant vWF. A, immunoprecipitation of RGDS-mutant vWF expressed in CHO cells. RADS-vWF and RGES-vWF cDNA, inserted into the pMT2 expression vector, were transfected into CHO-DUKX cells. CHO cells producing either RADS-vWF, RGES-vWF, or WT-vWF were labeled with ["SJmethionine and ["Slcysteine and CM collected as described under "Materials and Methods". vWF was immunoprecipitated with rabbit anti-vWF from CM of cells transfected with RADS-vWF (lane I); RGES-vWF (lane 2); WT-vWF (lane 3); and untransfected CHO- DUKX cells, or mock (lane 4 ) . B, SDS-agarose gel electrophoresis of vWF from COS-1 CM. vWF was isolated from radiolabeled COS-1 CM, immunoprecipitated, and run under nondenaturing conditions in 1% agarose electrophoresis gels to determine the vWF multimer pattern. The multimer distribution of RADS-vWF is shown (lane I); RGES-vWF is depicted (lane 2); WT-vWF is illustrated (lane 3); and mock-transfected cDNA from the pMT2 vector alone is included (lane 4) . C, SDS-agarose gel electrophoresis of vWF from CHO CM. Immunoprecipitates of CHO-derived vWF were run on agarose gels to determine their multimeric pattern. The multimer distribution of RADS-vWF is shown (lane 1 ), RGES-vWF is depicted (lane 2); WT- vWF from the PM5F CHO cell line (6) (lane 3 ) and untransfected, mock CHO-DUKX CM (lane 4 ) . Slight differences in spacing of multimer bands are due to the WT-vWF sample being run on a separate gel from RADS-vWF and RGES-vWF.

mixture of uncleaved pro-vWF, MI = 270,000, and cleaved mature vWF, MI = 220,000. Interestingly, following SDS- PAGE of the RGDS mutants, the 270,000 M, band typically appeared fainter than the 220,000 MI band, suggesting that the two RGDS mutants may be processed more efficiently than WT-vWF.

To determine the multimeric pattern of RADS-vWF, RGES-vWF, and WT-vWF transfected into COS-1 cells, vWF was immunoprecipitated and subjected to SDS-agarose gel electrophoresis. As shown in Fig. lB, both RADS-vWF and RGES-vWF, when transfected into COS-1 cells, multimerized to the same extent as WT-vWF, indicating that the point mutations did not affect the ability of vWF to multi- merize. As panel C indicates, RADS-vWF, RGES-vWF, and WT-vWF derived from CHO cells (6) multimerized to a similar extent, as did endogenous vWF from HUVECs.

RGDS-Mutant v WF Is Compared with WT-v WF in Platelet and Collagen Binding Assays-To determine whether recombinant WT-vWF, RADS-vWF or RGES-vWF could inhibit ristocetin-dependent binding of '251-vWF to platelets, concentrated CM was prepared as described, and concentrations ranging from 0.1 to 80 pg/ml were used to inhibit the binding of 2.6 pg/ml '2sI-~WF to fixed platelets. As shown in Fig. 2 4 , CM from the WT-vWF, RADS-vWF, or RGES-vWF cell lines inhibited ristocetin-dependent binding of l2'1-vWF to fixed platelets. At the highest concentrations tested, binding was reduced to the level obtained in the absence of ristocetin (5- 10% of total). In contrast, CM from untransfected CHO- DUKX cells did not inhibit '251-~WF binding (data not shown). A statistical analysis of the Scatchard plot yielded a KD= 20 f 4 x lo-' M for WT-vWF; 1 -1- 1 x lo-' M for RADS-

3412 The von Willebrand Factor RGDS Binding Site

FIG. 2. RGDS-independent binding of RGDS-mutant vWF. A, inhibition of vWF binding to fixed platelets by RADS-vWF and RGES-vWF in CHO CM. The ristocetin-dependent binding of 2.6 pg/ml '251-vWF to fixed platelets was determined in the presence of increasing amounts of vWF from concentrated CHO CM from RGES- vWF (open triangles), WT-vWF (open squares), and RADS-vWF (open circles). Both RGES-vWF and RADS-vWF were able to inhibit binding of '251-vWF to an extent similar to that of WT-vWF, indicating that ristocetin-dependent binding to platelets is not affected by the RGDS mutants. B, inhibition of vWF binding to native type I collagen by RADS-vWF, RGES-vWF, and WT-vWF CHO CM. The binding of 1 pg/ml 12'I-vWF to collagen immobilized onto polystyrene microtiter wells was tested in the presence of increasing amounts (corresponding to 1-80 pg/ml) of WT-vWF CM (open squares), RADS-vWF (open circles), and RGES-vWF 1 (closed triangles), and RGES-vWF 2 (open triangles). RADS-vWF as well as the two CHO RGES-vWF lines were able to compete with plasma vWF for binding to collagen, similar to WT-vWF.

vWF (23); and 3 +: 3 X lo-' M for RGES-vWF with a single class of binding sites. These results are comparable with studies using plasma vWF which have reported a KO = 0.5 X lo-' M (24, 25). In a previous study from this laboratory, we obtained a value of 4.2 X lo-' M assuming an average molecular weight of 1.2 x lo6 for multimeric vWF (21).

Concentrated CM containing WT-vWF, RADS-vWF, and RGES-vWF from two separate CHO cell lines, RGES-vWF 1 and RGES-vWF 2, were tested for their ability to inhibit the binding of 1 pg/ml '261-vWF to coIIagen. Fig. 2 8 indicates that the inhibition of lZ5I-vWF binding to collagen by WT- vWF CM is concentration-dependent. Each of the RGDS mutants also inhibited binding of '"I-vWF to collagen as well as WT-vWF CM. These findings suggest that the binding domains on vWF for collagen, located in the A1 and A3 domains of mature vWF (5), are not affected by the RGDS point mutations.

To determine whether binding to platelet GPIIb/IIIa was affected by the RGDS mutations, freshly isolated platelets were used in binding assays following activation with either thrombin or ADP. As indicated in Fig. 3A, following activation with thrombin, the binding of 2.6 pg/ml lZ6I-vWF was inhibited by increasing concentrations of WT-vWF and was reduced to the level of nonspecific binding at concentrations

A l.2 "a 0.6

B 1.4

1.2

1.0

g o.8

0.6

0.4

vWF added @g/ml)

FIG. 3. Binding of WT-vWF and mutant vWF to activated platelets. A, thrombin-dependent binding of vWF to activated platelets in the presence of RADS-vWF, RGES-vWF, or WT-vWF CM. The binding of 2.6 pg/ml '2'I-vWF to thrombin-activated platelets was tested in the presence of increasing concentrations of vWF from WT-vWF (open squares), RADS-vWF (open circles), or RGES-vWF

RADS-vWF CM nor RGES-vWF CM significantly inhibited "'I- (open triangles) CM containing from 0-60 pg/ml of vWF. Neither

vWF at all concentrations tested, whereas WT-vWF abolished binding of 'T-vWF to thrombin-activated platelets a t concentrations >50 pg/ml. B, ADP-dependent binding of vWF to activated platelets in the presence of RADS-vWF, RGES-vWF, or WT-vWF CM. '"I-vWF and ADP-activated platelets were incubated with increasing concentrations of v W F derived from WT-vWF CM (open squares), RADS- vWF (open circles), RGES-vWF (open triangles), or untransfected CHO cells, CHO-DUKX (closed squares). Neither RGES-vWF, RADS-vWF, nor CHO-DUKX CM inhibited binding of I2'I-vWF to ADP-activated platelets, while WT-vWF CM abolished binding of Iz5I-vWF at concentrations >30 pg/ml.

>50 pg/ml. This binding curve corresponded to a KO = 4.5 +- 2 X M, by Scatchard analysis, similar to KD values obtained for the binding of fibrinogen to GPIIbjIIIa (1). In contrast, neither RADS-vWF CM nor RGES-vWF CM could significantly inhibit vWF binding to thrombin-activated platelets (Fig. 3A), suggesting that their ability to compete for binding to GPIIb/IIIa is significantly reduced.

Since thrombin causes the release of vWF from platelet a- granules, which could possibly inhibit lZ5I-vWF binding, platelets were activated with a second agonist, ADP. As shown in Fig. 3B, WT-vWF CM inhibited the binding of lZ61-vWF from ADP-activated platelets in a concentration-dependent man- ner, virtually eliminating specific binding at >30 pg/ml WT- vWF CM. This inhibition corresponded to a KD = 1.6 X lo-' M by Scatchard analysis, suggesting that vWF binds thrombin-activated and ADP-activated platelets with the same affinity. Both RGES-vWF and RADS-vWF could not inhibit '251-vWF binding as demonstrated by a B,/Br 2 1.0, indicating that RGDS-mutant vWF did not bind to activated platelets via the GPIIb/IIIa receptor.

Adhesion and Spreading of HUVECs onto RGDS-Mutant v WF and WT-v WF-To assess HUVEC attachment to vWF

The von Willebrand Factor RGDS Binding Site 3413

substrates, microtiter plates were coated with 10-30 pg/ml purified plasma and recombinant vWF. To quantitate the number of HUVECs attached, cells were labeled with [35S] methionine and [35S]cysteine and incubated in the vWF- coated wells. HUVEC attachment and spreading upon a surface coated with plasma vWF or WT-vWF was concentration- dependent and maximal at 30 pg/ml, similar to results reported by Dejana et al. (11). Attachment to BSA was consist- ently 5-15% of the attachment to vWF. Fig. 4 indicates that HUVEC attachment to recombinant WT-vWF was maximal at 60 min, similar to attachment to plasma vWF (data not shown). In contrast, there is an initial lag in attachment and an overall reduction in attachment a t all times analyzed to RADS-vWF and RGES-vWF. By 60 min, HUVEC attachment to RADS-vWF and RGES-vWF was 30% less than attachment to WT-vWF. The reduction in attachment to the vWF mutants was statistically significant ( p < 0.001).

Since both the rate and the extent of HUVEC attachment to RADS-vWF and RGES-vWF was reduced, but not abolished, there may be a second, possibly RGDS-independent mechanism for HUVEC adhesion to vWF. The platelet binding studies suggested that the carboxyl-terminal RGDS site of vWF no longer bound to platelet GPIIb/IIIa and therefore was unlikely to bind to the HUVEC analog, the VNR (11). However, the residual attachment observed with RADS-vWF and RGES-vWF could conceivably have occurred via the RGD sequence in the vWF propeptide since there was some uncleaved pro-RADS-vWF and pro-RGES-vWF subunits secreted from the CHO cells, which was incorporated into vWF multimers (Fig. 1). To determine whether the RGD sequence in the propeptide contributed to HUVEC attachment to the vWF RGDS mutants, we compared attachment of HUVECs

8oOoo -

0 2 0 4 0 6 0

Minute.

FIG. 4. Attachment of HUVECs to WT-vWF, RADS-vWF, and RGES-vWF. HUVECs were labeled with [36S]methionine and [36S]cysteine and preincubated with 10 pg/ml cycloheximide and 0.7 pg/ml monensin in serum-free M199 medium to inhibit endogenous protein synthesis and secretion, respectively. Microtiter wells were coated with either recombinant WT-vWF (closed squares), recombinant RADS-vWF (open circles), recombinant RGES-vWF (closed circles), or BSA (closed triangles). As shown, there is an initial lag in attachment of HUVECs to the vWF RGDS mutants compared with recombinant WT-vWF. At 60 min, attachment of HUVECs to plasma vWF or WT-vWF is similar and exceeds attachment to the RGDS mutants by about 30% of WT-vWF levels. Attachment of HUVECs on BSA was 5-15% of the attachment on plasma WT-vWF. Data points represent the average of three separate experiments for which standard deviation was calculated from quadruplicate samples for each point of each individual experiment.

with plasma vWF, which contains no uncleaved pro-vWF subunits, a previously described mutant vWF-KKS, whose propeptide remains completely uncleaved (18), and WT-vWF, which has a mixture of cleaved and uncleaved vWF. As shown in Fig. 5, HUVECs attached equally well to all three substrates. Thus, the propeptide RGD is unlikely to play a major role in HUVEC attachment to vWF. As an additional test, CM containing the propeptide alone, derived from the vWF- KRX cell line (18), was tested for inhibition of HUVEC attachment to plasma or WT-vWF. At a concentration of 5- 10 pg/ml, vWF-KRX CM did not inhibit attachment of HUVECs (data not shown).

Spreading of HUVECs after attachment to affinity-purified RADS-vWF, RGES-vWF, or WT-vWF was observed at 1 h after plating and compared with spreading onto BSA. Fig. 6 indicates that, while cells attach to RADS-vWF (Fig. 6 A ) and RGES-vWF (Fig. 6B), cell spreading is minimal and the cells appear similar to the small number of cells which attach to BSA (Fig. 6C) . In contrast, when plated onto WT-vWF, cells have extended filopodia and flattened onto the WT-vWF (Fig. 6D) substrate after 1 h.

DISCUSSION

We have demonstrated that the introduction of single amino acid substitutions into the carboxyl-terminal RGDS sequence of vWF abolishes binding to platelet GPIIb/IIIa without affecting either multimerization or vWF binding to GPIb/IX and collagen. The failure of RADS-vWF and RGES- vWF to compete with lZ5I-vWF for platelet binding following activation with either thrombin or ADP suggests that binding to platelet GPIIb/IIJa is completely abolished by these two mutations. This is consistent with previous studies demon- strating inhibition of vWF binding to activated platelets by a series of peptides containing RGDS and related sequences (2).

We were interested in whether the mutants would support HUVEC attachment to vWF via a related integrin, the VNR.

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FIG. 5. Attachment of HUVECs to plasma vWF, WT-vWF, and uncleaved pro-vWF (KKS-vWF). HUVECs were labeled and prepared as described in Fig. 4, and in this experiment wells were coated with either plasma vWF (open squares), recombinant WT- vWF (closed squares), recombinant KKS-vWF (open triangles), or BSA (closed triangles). The attachment of HUVECs to KKS-vWF was virtually identical to plasma WT-vWF and proceeded to the same extent as both plasma WT-vWF and recombinant WT-vWF by 60 min.

3414 The von Willebrand Factor RGDS Binding Site RADS-vWF RGES-vWF

BSA WT-vWE

FIG. 6. Spreading of HUVECs to WT-vWF, RADS-vWF, RGES-vWF, and BSA. HUVECs were plated onto substrata coated with 30 pg/ml WT-vWF or RGDS-mutant vWF. Spreading was observed by phase contrast microscopy at 1 h after plating onto affinity-purified RADS-vWF (A ) , RGES-vWF ( B ) , BSA (C), or WT- vWF (D). Magnification: 220X. HUVECs demonstrated cell flatten- ing and extension of filopodia onto WT-vWF after 1 h, whereas HUVECs plated onto RADS-vWF ( A ) or RGES-vWF ( B ) resembled the blunted appearance of HUVECs on BSA (C).

Previous studies (11) had indicated that binding of a monoclonal antibody to the vWF RGDS site or the addition of high concentrations of peptides containing the RGDS sequence inhibited the attachment of HUVECs to insolubilized vWF. Since the vWF domain recognized by HUVECs is believed to be identical to that recognized by GPIIb/IIIa, RGDS-dependent adhesion to vWF via the homologous VNR should also be inhibited. Unexpectedly, attachment of HUVECs to a RADS- vWF or RGES-vWF substrate was not completely abolished. At 60 min there was only a 30-35% reduction in HUVEC attachment to RADS-vWF and RGES-vWF when compared with recombinant WT-vWF or plasma-derived vWF. To en- sure that HUVEC vWF containing an intact RGDS sequence was not being synthesized and secreted by the HUVECs, cycloheximide and monensin were routinely included in the preincubation and assay medium.

There are at least two explanations for the residual adhesion of HUVECs to RGDS-mutant vWF. First, another endothelial membrane protein might mediate adhesion to other regions of the vWF subunit. For example, there is evidence that HUVECs synthesize and express GPIb/IX (26), an im- portant platelet adhesion receptor, which could bind to the A1 domain of vWF. Second, the HUVEC VNR may bind to vWF at a previously cryptic “second adhesion site” on vWF. Fibronectin contains a second adhesion site, amino-terminal to its RGDS sequence, which did not become apparent until RGDS-deletion mutants were produced and expressed (12, 13). It is of interest that the addition of 1 mM GRGDS peptide did not inhibit adhesion of HUVECs to the vWF RGDS mutants, whereas we have shown that attachment of HU- VECs to recombinant WT-vWF and plasma vWF is completely inhibited by the GRGDS peptide.’ This suggests that the RGDS-independent component of HUVEC adhesion to vWF, like fibronectin, is only observed when the RGDS site

‘D. A. Beacham, R. J. Wise, S. M. Turci, and R. I. Handin, unpublished observations.

is eliminated by mutation. Although our data indicate that a second binding site is not recognized by GPIIb/IIIa on ADP- or thrombin-stimulated platelets, we cannot eliminate the possibility that such a second site is selectively recognized by the VNR on HUVECs.

One of the most interesting and unexpected observations was the dissociation between initial cell attachment and subsequent spreading on the mutant forms of vWF. There is abundant evidence in other systems that attachment and spreading, although temporally related, are separate phenom- ena. Cells in culture are thought to go through three stages in the process of spreading and establishing an apical-basolateral membrane (27): 1) initial attachment of plasma membrane receptors to extracellular matrix components; 2) clustering and/or conformational changes in the receptors prior to their interaction with cytoskeletal components; 3) a final stage when fully spread cells form more complex cell-substrate adhesion complexes. In keeping with this model, the initial attachment and subsequent spreading of hepatocytes are distinct and separable responses (28). Furthermore, the spreading but not the initial attachment of B16-Fl0 melanoma cells on a laminin substrate is mediated by a distinct laminin receptor, the enzyme galactosyltransferase (29).

There are also distinct differences in RGD-dependent spreading and attachment when peptides are compared with intact adhesive macromolecules. For example, when bound to a surface, the peptide GRGDS supports attachment but only minimal spreading of HeLa cells onto type I collagen (30). Similarly, normal rat kidney fibroblasts attached equally well to GRGDS peptide or fibronectin (31) but showed fewer focal adhesions (a characteristic of fully spread cells) on the GRGDS peptide substrate when compared with spreading of the same cells on intact fibronectin (31). These results are consistent with the idea that attachment and spreading are mediated by distinct mechanisms and suggest that attachment may be subdivided into an RGDS-dependent and RGDS- independent component.

Although our results may appear to contradict those of Dejana (l l) , the reagents used to perturb adhesion and the experimental designs were somewhat different. Their study employed high concentrations of synthetic peptides and monoclonal antibodies directed against the carboxyl-terminal RGDS site on vWF, whereas our study employed recombinant vWF containing point mutations in the RGDS sequence. Epitope-specific monoclonal antibodies are very useful reagents but can theoretically block adjacent interaction sites by steric hindrance. Similarly, the relatively high concentrations of peptides used in the previous study might bind to low affinity or nonspecific sites on the HUVEC surface and thereby block attachment. Our adhesion assay was similar to Dejana et al. (11) with respect to the number of cells plated and concentration of vWF used to coat the plastic surface but used 3sS-labeled cells instead of a spectrophotometric assay to quantitate attachment. In addition, attachment was mon- itored at 2 and 4 h in the previous study, a time when both attachment and spreading should have proceeded to comple- tion. We selected earlier time points to observe attachment independent of spreading. Any of these differences could account for the discrepant findings of complete inhibition of attachment previously observed (11) opposed to the lag and reduction in attachment observed in our study.

In conclusion, using site-directed mutagenesis, the GPIIb/ IIIa binding domain of vWF was selectively inactivated without altering the biosynthesis, multimerization, or binding of vWF to collagen and GPIb/IX. Although the major thrust of the current study was an analysis of the attachment and

The von Willebrand Factor RGDS Binding Site 3415

spreading of HUVEC on mutant vWF, vWF is also a critical ligand for platelet adhesion to the blood vessel under conditions of high shear stress (4). Since vWF can bind to both the GPIb/IX and GPIIb/IIIa receptors, the interaction of vWF with either receptor could contribute to platelet adhesion under high shear force (3). The RADS-vWF and RGES-vWF mutants can also be used to further enhance our understand- ing of vWFs role in platelet adhesion under varying levels of shear stress. Since the mutant forms of vWF have a selective loss of RGDS-dependent interactions, they are the ideal reagent to probe all the potential RGD-dependent functions of vWF multimers.

Acknowledgments-We thank Miguel Cruz for assistance in vWF purification and Randal Kaufman for providing the pMT2 expression vectors.

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