of the role viii* of cleavage in binding and stabilization · association of factor vi11...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol . 266, No. 32, Issue of November 15, PP.

The Role of von Willebrand Factor Multimers and Propeptide Cleavage in Binding and Stabilization of Factor VIII*

(Received for publication, April 24, 1991)

Robert J. Wise$, Andrew J. Dorner, Maryann Krane, Debra D. Pittman, and Randal J. Kaufmanj From the Genetics Institute, Cambridge, Massachusetts 02140

von Willebrand factor (vWF) is a multimeric glycoprotein that promotes platelet aggregation and stabilizes coagulation factor VI11 in the plasma. vWF is also required for the stable accumulation of recombinant factor VI11 secreted from cells in a heterologous expression system. In this report, we show that vWF can promote the in vitro reconstitution of factor VI11 activity from dissociated heavy and light chains of factor VIII, suggesting that vWF may act to promote stable assembly of factor VI11 subunits at the site of secretion. The structural requirements for vWF propeptide cleavage and for vWF multimerization in its binding and stabilization of factor VI11 was examined using specifically altered recombinant vWF. The mutant vWF molecules were also assayed for their function in ristocetin-induced platelet agglutination mediated through the platelet receptor GPIb. Deletion of the vWF propeptide produced a dimeric vWF molecule that failed to mediate platelet agglutination, suggesting that multimerization is required for vWF to attain functional GPIb binding. This mature dimeric form of vWF, however, was fully capable of binding to and supporting stable secretion of factor VIII. A vWF mutant with an altered propeptide cleavage site formed large multimers of uncleaved pro-vWF that functioned in platelet agglutination. However, this noncleavage mutant neither bound to or supported stable accumulation of factor VIII. Analysis of the vWF propeptide, expressed independently, demonstrated that it could not bind factor VI11 or stabilize its secretion. These results show that the dimeric mature vWF subunit is sufficient to bind and stabilize factor VI11 and that the presence of uncleaved vWF propeptide inhibits both factor VI11 binding and stabilization.

The factor VI11 complex is composed of a noncovalent association of factor VI11 procoagulant protein (factor VIII) with von Willebrand factor (vWF).’ The factor VI11 molecule is an important regulator of the blood coagulation cascade.

* This work was supported by the Genetics Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Hematology Division, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115.

J To whom correspondence and reprint requests should be ad- dressed 87 Cambridge Park Dr., Cambridge, MA 02140. Tel.: 617-

The abbreviations used are: vWF, von Willebrand factor; a-MEM, CY modification of Eagle’s minimal essential medium; CHO, Chinese hamster ovary; GPIb, platelet glycoprotein Ib; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; MES, 2-(N-mor- pho1ino)ethanesulfonic acid.

876-1170.

Its deficiency is the cause of the X-linked bleeding disorder hemophilia A. von Willebrand factor is a multifunctional glycoprotein that plays a central role in primary hemostasis. In response to vascular injury, vWF mediates the adhesion of platelets to the subendothelium by its binding to glycoprotein Ib (GPIb) on the platelet surface. A deficiency or abnormality of vWF results in the autosomally linked bleeding disorder known as von Willebrand’s disease. The relationship between hemophilia A and von Willebrand’s disease was confused for many years, because severe von Willebrand’s disease is asso- ciated with factor VI11 deficiency and early preparations of “pure” factor VI11 were shown to correct the platelet aggregation defects of plasma from patients with von Willebrand’s disease. I t is now known that these two proteins circulate as a complex in plasma and that their activities are intimately intertwined (for review, see Ref. 1). vWF plays a crucial role in regulating factor VI11 levels in plasma, as well as factor VI11 activity. vWF protects factor VI11 from inactivation by activated protein C and prevents activation by activated factor X (2-4). Although vWF does not protect factor VI11 from activation by thrombin (5), recent studies suggest that vWF can promote thrombin-mediated cleavage of the factor VI11 light chain (6). In addition, vWF prevents factor VI11 from binding to phospholipids and cell surfaces such as activated platelets (7-9). At the site of factor VI11 synthesis, vWF may regulate the stable assembly of factor VI11 heavy and light chains and may possibly promote factor VI11 release from the cell by competing for phospholipid binding to the factor VI11 light chain (10). Understanding the mechanism by which vWF regulates the biosynthesis and metabolism of factor VI11 has been difficult, in part due to the lack of a natural cell line capable of synthesizing factor VIII.

A model system has been developed in which factor VI11 synthesis and secretion can be studied in a heterologous cell. By introduction of an expression vector containing human factor VI11 cDNA into Chinese hamster ovary (CHO) cells, factor VIII-producing cell lines were established (10). An initial observation was that these factor VIII-producing cells required the presence of serum for the stable accumulation of factor VI11 activity and that the primary component provided by serum was vWF (10, 11). Here we describe the use of this model system to identify structural determinants of vWF that govern its interaction with factor VIII.

MATERIALS AND METHODS

Expression Vectors and Mutugenesis-Construction of the wild- type vWF (vWF-WT) expression vector pMT2-ADA-vWF was previously described (12). In this vector, the vWF-coding sequence is under control of the adenovirus major late promoter with an SV40

under control of an SV40 early promoter. For expression of the vWF enhancer, and a separate adenosine deaminase transcription unit is

mutant, vWF-KKS, the vWF-WT cDNA insert in pMT2-ADA-vWF was replaced by the vWF-KKS mutant cDNA. The vWF-APRO and vWF-KRX mutant cDNAs were inserted into the vector pMT3-

21948

von Willebrand Factor Binding to Factor VIII 21949

SV2ADA (13), which is identical with pMT2-ADA, except that the dihydrofolate reductase-coding region immediately 3' to the cloning site has been removed. The site-directed mutagenesis of vWF cDNA was previously described (14).

Cell Line Selection and Culture-The plasmid vectors containing vWF cDNAs were introduced into dihydrofolate reductase-deficient CHO-DUKX cells by protoplast fusion as described (12). Transform- ants were selected in (a-MEM) containing nucleosides and 10% fetal calf serum, plus 11-AAU (1.1 mM adenosine, 50 mM alanosine, 1 mM uridine) and 0.03 p~ 2'-deoxycoformycin, as described by Kaufman et al. (11, 15). Selected clones were further selected for resistance to increasing levels of 2'-deoxycoformycin. The final cloned cell lines, named vWF-WT, vWF-KKS, vWF-APRO, and vWF-KRX, were selected for resistance to 0.1, 1.0,5.0, and 1.0 p M 2'-deoxycoformycin, respectively. The factor VIII-producing CHO cell line, 10A1, has been described (10). This cell line was maintained in a-MEM without nucleosides containing 10% dialyzed fetal calf serum and 1.0 mM methotrexate.

Factor VIII and u WF Assays-Factor VI11 levels were determined by the Kabi COATEST method (Kabi Diagnostica). A specific activity of 5000 units/mg was used to convert factor VI11 activity to weight. The concentration of vWF in CHO-conditioned medium was assayed by enzyme-linked immunosorbent assay (American Diagnostica) using normal pooled plasma (George King Bio-Medical) as a standard of 5-10 pg/ml. The ristocetin cofactor activity of recombinant vWF was assayed using reagents purchased from Helena Laboratories. The lyophilized, washed, and fixed human platelets were resuspended in one-half the recommended volume. Reactions were performed in siliconized glass cuvettes constantly stirred at 37 "C in an aggregometer (Sienco). Each cuvette contained 200 p1 of resuspended platelets and 200 pl of CHO-conditioned medium. Reactions were started by addition of 50 p1 of 10 mg/ml ristocetin reagent. Agglutination was measured by monitoring the increase in light transmittance.

Purification of u WF and Factor VIII Subunits and Reconstitution of Factor VIII Actiuity-Plasma vWF was purified as described (16), except for the final gel filtration step in which 50 mM MES, pH 6.15, 0.25 M MgCI, was used to assure the complete dissociation of vWF from factor VIII. Recombinant vWF, derived from CHO cell-conditioned medium, was purified by immunoaffinity chromatography, using monoclonal anti-vWF IgG (VW/13.7.9, VW/16.9.2, VW/24.5.8) produced by the Genetics Institute' and coupled to Affi-Gel 10 (Bio- Rad). vWF was eluted from the affinity column with glycine HCI, pH 2.5, 150 mM NaCl, 0.1% Triton X-100, and then neutralized with Tris and dialyzed against 50 mM Tris-HC1, pH 7.6, 150 mM NaCI. Recombinant factor VIII, purified as described (17), was treated with 50 mM EDTA, pH 8.0, for 30 min at room temperature. The pH was then adjusted to pH 6.0, and the mixture was applied to a cation exchanger Mono S fast protein liquid chromatography column (Phar- macia LKB Biotechnology Inc.) equilibrated with 0.2 M NaCI. After washing with 0.2 M NaCI, the light chain was eluted with 0.5 M NaCl. The Mono S column flow-through, which contained the heavy chain, was applied to a Mono Q fast protein liquid chromatography column after adjusting the pH to 7.5. This column was washed with 0.2 M NaCl to remove any residual light chain contamination. The heavy chain was eluted with 0.5 M NaCI. The purity of the isolated protein chains was determined by silver stain analysis of SDS-PAGE gels before and after thrombin digestion. Reaction mixtures for the reconstitution of factor VI11 activity contained 50 mM Tris, pH 6.8, 0.35 M NaCl, 2.5 mM CaC12, 50 mM MnCl', and 1 mM phenylmethanesulfonyl fluoride. The concentrations of factor VI11 heavy and light chain were 28 and 30 pg/ml, respectively. The concentration of vWF varied from 10 to 50 pg/ml. Mixtures were incubated at 37 "C, and portions were removed at the indicated times for determination of factor VI11 activity.

Rudiokzbeling, Immunoprecipitations, and Gel Electrophoresis- CHO cell-secreted proteins were radiolabeled by a 2-h pulse in serum- free medium deficient in Met and Cys but supplemented with [%] Met and [35S]Cys (Amersham Corp.) followed by a 12-h chase in complete a-MEM containing nucleosides, 0.15% bovine serum albu- min, insulin-transferin-sodium selenite supplement (Sigma), and 1% (v/v) aprotinin solution (Sigma, A-6279). Radiolabeled conditioned medium was collected and cellular debris removed by centrifugation. Immunoprecipitation and gel electrophoresis of vWF was as described (14). Following electrophoresis, gels were treated with EnHance (Du Pont-New England Nuclear) and dryed for fluorography. For analysis of factor VI11 accumulation in the presence of CHO-derived vWF,

' W. B. Foster and E. Alderman, unpublished work.

conditioned medium samples were dialyzed against 20 volumes of serum-free a-MEM medium and then filtered through a 0.45-pm cellulose acetate microfilter (Schleicher & Schuell). For factor VI11 binding assay, factor VI11 purified as described above was added to radiolabeled CHO cell-conditioned medium containing 1 mM phenylmethanesulfonyl fluoride and incubated for 1 h at 37 "C. After incubation, samples were immunoprecipitated as described (18) without the addition of detergent. Anti-vWF polyclonal antibody was purchased from DAKO and directly coupled to Affi-Gel 10. The anti- factor VI11 monoclonal antibody (F8)' was coupled to Sepharose CL- 4B beads (Pharmacia). For precipitation with the anti-vWAgII monoclonal antibody, rabbit anti-mouse IgG (Zymed Laboratories) and protein A-Sepharose (Pharmacia) was included. After centrifugation of the immunoprecipitates, the supernatants were removed for factor VI11 activity assay, and the pellets were washed in ice-cold 10 mM Tris-HCI, pH 7.5, 150 mM NaCI, 2.5 mM CaCl', 1% Triton X-100 for SDS-PAGE.

RESULTS

von Willebrand Factor Promotes Association of Factor VIII Heavy and Light Chains in Vitro-One mechanism by which vWF may support the secretion and stabilization of factor VI11 is by promoting factor VIII chain assembly. To test this, recombinant factor VIII, purified free of vWF, was dissociated into its subunit heavy and light chains by treatment with EDTA. Each subunit was isolated by ion-exchange chromatography and its purity evaluated by silver-stained SDS- PAGE gels. The isolated heavy and light chain preparations were freed of vWF and exhibited the expected molecular mass and thrombin digestion cleavage pattern (data not shown). Plasma-derived vWF was purified to homogeneity and contained no detectable factor VI11 activity. Reconstitution of factor VI11 activity was assessed by incubating a mixture of isolated heavy and light chains in the presence or absence of the purified vWF. Incubation of heavy and light chains without vWF for 60 min resulted in a 5-fold increase in factor VI11 activity, whereas light chain incubated alone showed no increase in activity (Fig. 1). This indicated that some reconstitution of factor VI11 activity occurred in the mixture, suggesting the spontaneous assembly of heavy and light chain factor VIII. The rate and extent of reconstitution of factor VI11 activity was increased by the addition of purified vWF. At a vWF concentration of 50 pg/ml, factor VI11 activity was increased 20-fold over that observed in the absence of vWF. Peak increases in activity were usually obtained at the 60- min time point. These results indicate that vWF promotes the reconstitution of factor VI11 activity from dissociated heavy and light chain subunits and suggests that vWF acts to accelerate the rate and/or improve the efficiency and stability of subunit association. These observations provided the basis for experiments aimed at determining structural features of vWF required for its mediation of factor VI11 assembly and stabilization.

Derivation of Chinese Hamster Ovary Cell Lines That Ex- press vWF Mutants-Site-directed mutagenesis was used to specifically alter the primary structure of prepro-vWF. The mutants used in this study were previously shown to affect propeptide processing and, subsequently, vWF multimer assembly when expressed in COS-1 monkey kidney cells (14). These mutants are diagramed in Fig. 2 . The mutant, desig- nated vWF-APRO, contains a deletion of the entire propeptide-coding region so that the signal peptide, which is correctly processed, directs the secretion of mature vWF. A second mutant, vWF-KKS, contains an amino acid substituion (R + K) at the propeptide cleavage site that blocks propeptide cleavage in COS-1 cells. The vWF-KRX mutant contains a deletion of the mature coding region so that the vWF propeptide is expressed alone.

These mutant vWF cDNAs were inserted into mammalian

uon Willebrand Factor Binding to Factor VIII

pg/ml vWF

50

20

10

HL

L I

60

Time (mid FIG. 1. Reconstitution of factor VI11 activity in the presence

of vWF. Purified factor VI11 heavy chain (H) at 28 pg/ml and factor VI11 light chain ( L ) at 30 pg/ml were incubated at 37 "C alone (0) or in the presence of 10 pg/ml (O), 20 pg/ml (A), or 50 pg/ml (A) of purified plasma-derived vWF. An incubation with factor VIII light chain alone (0) was also included as control. Portions of the reaction mixes were removed at 0, 15, 30, and 60 min for factor VI11 activity determination (expressed as units/ml), as described under "Materials and Methods."

expression vectors and introduced into Chinese hamster ovary (CHO) cells as described for wild-type vWF (12). Upon selection, amplification, and cloning of the vWF-producing CHO cell lines, the vWF secreted into serum-free conditioned media was quantitated, examined by gel electrophoresis, and assayed for platelet-binding activity. The secreted vWF was quantitated by enzyme-linked immunosorbent assay and by protein staining of immunoprecipitated material on SDS-PAGE gels. Typical levels obtained in 24-h conditioned medium were 5- 10 pg/ml for vWF-WT, 25-50 pg/ml for vWF-APRO, 10-20 pg/ml for vWF-KKS, and 5-10 pg/ml for vWF-KRX.

In order to assess processing and assembly of the vWF mutants expressed in CHO cells, the secreted proteins were metabolically radiolabeled with 35S-aminoacids, and the vWF, immunoprecipitated from conditioned medium, was examined by gel electrophoresis under reduced and native conditions. SDS-PAGE of disulfide-reduced material identifies the prod- ucts by size and determines the extent of proteolytic processing (Fig. 3A). vWF-WT produced by CHO cells is mostly cleaved to the 225-kDa mature subunit; however, up to approximately 25% remains uncleaved (pro-vWF, lane 2) . In contrast, the vWF-APRO is secreted exclusively as mature vWF (lane 3 ) , and the vWF-KKS mutant is primarily (greater than 90%) uncleaved pro-vWF (lane 4 ) . Using an antibody

APRO = , KKS = 5$<, ,Ij.L p?;pLa-,in.w+$gj ,I

KRX = w FIG. 2. Regions of prepro-vWF that were altered by site-

directed mutagenesis. A, diagram of the primary structure of prepro-vWF. The 2813-residue primary translation product consists of a 22-amino acid signal peptide (black), a 741-amino acid propeptide (gray), and the 2050-residue mature vWF subunit (white). The propeptide cleavage site is marked by an arrow. B, arrangement of the repeated domain structure, determined by internal homologies (Refs. 12 and 48). The molecule contains four cysteine-rich D domains plus a truncated D' domain at the mature amino terminus. The GPIb- binding site is contained in domains D3 and the first of the cysteine- poor A domains (33). The putative factor VIII-binding site (40) is contained in the D' region. The cysteine residues believed to form the intermolecular disulfide bonds that link vWF dimers into multimers are located in the D3 domain and the first A domain (32). C, the vWF mutants. The wild-type (WT) sequence is shown in con- densed form at the top. The amino acids (one-letter code) located at the sites of the signal peptide and propeptide cleavage are indicated. The vWF mutations (described in the text) are diagramed below.

directed against vWAgII (19), the vWF-KRX mutant is detected at approximately 100 kDa (lane 6 ) . SDS-agarose gel electrophoresis of nonreduced samples demonstrates the degree of vWF multimer assembly (Fig. 3 B ) . As expected from previously described transient expression experiments in COS-1 monkey cells (14), native gel electrophoresis demonstrates that vWF-WT and the vWF-KKS mutant are highly multimerized (lanes 2 and 4 ) , whereas vWF-APRO forms only mature dimers (Fig. 3B, lane 3 ) . The functional platelet agglutination activity of vWF-WT and each vWF mutant was determined by ristocetin cofactor assay. This assay measures agglutination of washed formalin-fixed platelets in the presence of vWF and the antibiotic ristocetin, and is a function of vWF binding to platelet GPIb (20). Fig. 4 shows the aggregometer tracings from this assay performed with CHO- conditioned medium containing vWF at the concentrations listed above. Only vWF-WT and vWF-KKS produce platelet agglutination above background, whereas vWF-KRX and vWF-APRO failed to promote agglutination. The result with the vWF-APRO mutant may have been expected because of studies showing that ristocetin cofactor activity correlates with the degree of vWF multimerization (21,22). The positive ristocetin-induced agglutination observed with vWF-WT and vWF-KKS demonstrates that these recombinant vWF molecules expressed by CHO cells are multimerized to a degree that is required for this activity and that the presence of the vWF propeptide does not interfere with this activity.

Accumulation of Factor VIII Supported by Wild-type and Mutant uWF i n the Absence of Serum-The CHO cell line lOAl expresses human factor VI11 and requires the presence of vWF in the culture medium to support its stable accumulation (10, 11). To test the ability of recombinant vWF to support the accumulation of factor VI11 from the CHO-1OA1 cell line, these cells were rinsed and placed in serum-free medium to which purified CHO-derived vWF was added. Factor VI11 accumulation at 24 and 48 h was determined, and the result is shown in Fig. 5A. Only a low level of activity (-40 milliunits/ml) accumulated in serum-free medium in the

A vWF Agl l - CHO Wl APRO KKS CHO KRX

von Willebrand Factor Binding to Factor VIII

” - + pro-vWF

-0 + mature vWF

200 -

propepllde

93 -

1 2 3 4 5 6

B vWF Agl l CHO WT APRO KKS CHO KRX

Dimer*

A 1 2 3 4 5 6

FIG. 3. Gel electrophoresis of CHO cell-derived recombinant vWF. Radiolabeled proteins were immunoprecipitated from the conditioned medium of vWF-producing CHO cells. The antibodies used for immunoprecipitation (indicated at the top of the figure) were either a polyclonal anti-vWF (vWF) or a monoclonal anti-vWAgII (AgZn. The conditioned media used for the immunoprecipitations were derived from the following cell lines: lanes I , CHO cell control; lanes 2, vWF-WT; lanes 3, vWF-PRO; lanes 4 , vWF-KKS; lanes 5, CHO cell control; lanes 6, vWF-KRX. A, SDS-PAGE gel after sample reduction with 8-mercaptoethanol. The migration of “C-molecular mass markers (Amersham Corp.) is indicated on the left. B, SDS- agarose gel without reduction (vWF multimer gel).

absence of vWF. A 5-10-fold increase in factor VI11 activity was obtained when vWF was added a t 1 pg/ml to the serum- free medium. The yield of factor VI11 activity approaches that obtained from CHO-1OA1 cells grown in the presence of 10% serum. Thus, recombinant vWF can support factor VI11 accumulation in serum-free medium. This finding provided the basis for the assessment of each of the vWF mutants.

Experiments were designed to test the ability of the vWF mutants to support factor VI11 accumulation. Serum-free conditioned medium from each CHO cell line was collected and assayed for vWF concentration. The conditioned medium from vWF-APRO was diluted 1:5 in serum-free medium because of its higher level of expression. Following dilution of vWF-APRO, all conditioned medium samples contained be-

J

21951

KRX

r“- CHO

’I I 1

0 2 3 Tlme (min)

FIG. 4. Ristocetin-induced platelet agglutination activity of CHO-derived recombinant vWF. Serum-free conditioned medium from each vWF-producing CHO cell line (described under “Results”) was incubated with washed-fixed platelets in an aggregometer cuvette (see “Materials and Methods”). Ristocetin was added to 1 mg/ml a t the zero time point. Agglutination was monitored by recording changes in light transmittance. Shown are typical aggregometer tracings from repeated assays. Concentrations of recombinant vWF in the reactions were (approximately) 5 pg/ml vWF-WT, 10 pg/ml vWF- KKS, 20 pg/ml vWF-APRO, and 5 pg/ml vWF-KRX.

tween 5 and 20 pg/ml of vWF product. The conditioned medium samples were resupplemented with nutrients and placed on rinsed CHO-1OA1 cells. Factor VI11 activity levels were determined a t 24 h and compared with that obtained in control CHO-cell conditioned medium lacking vWF. Factor VI11 accumulates to 30 and 50 milliunits/ml when CHO-1OA1 cells are placed in serum-free medium or control CHO-conditioned medium, respectively (Fig. 5 B ) . In contrast, addition of conditioned medium from vWF-WT or vWF-APRO cells support increased factor VI11 activity accumulation to 300 and 200 milliunits/ml, respectively. Surprisingly, however, medium from cells expressing the fully multimerized propeptide-containing vWF-KKS mutant failed to support factor VI11 accumulation to levels obtained with WT-vWF or vWF- APRO. Additionally, the vWF-KRX mutant also failed to support factor VI11 activity.

Factor VIII Accumulation Correlates with u WF Binding- In order to determine whether factor VI11 accumulation correlated with vWF binding, an experiment was designed to provide a qualitative assessment of vWF/factor VI11 interaction. Pure, unlabeled human recombinant factor VI11 was added to 3”S-radiolabeled medium containing vWF-WT or mutant vWF. After incubation, the mixture was immunoprecipitated with either a polyclonal antibody specific for vWF or a monoclonal antibody (F8) that reacts with the A2 domain of factor VI11 and does not interfere with vWF interaction.* Co-immunoprecipitation of vWF and factor VI11 was inter- preted to indicate association of factor VI11 and vWF. After immunoprecipitation, factor VI11 activity remaining in the supernatant was determined, and the washed immunoprecip- itate pellet was subjected to gel electrophoresis to identify and quantitate precipitation of the vWF (Fig. 6). The results for vWF-WT, vWF-APRO, and vWF-KKS incubated with 0, 10, or 30 units/ml of pure factor VI11 and immunoprecipitated as described above, along with a nonspecific antibody control,

21952 uon Willebrand Factor Binding to Factor VIIl

mU/ml

* 10% Serum

300- - 0

Recombinant - a VWF - - 200- -

- Serum-Free

X

0 12 24 36 48 I I

Time (hrs)

rnU/ml , i

S F ' CHO . WT APRO KKS KRX ~ 0 . 0 1 ~ 0 . 0 1 10-15 5-10 15-20 -5-10

vWF (pg/ml) FIG. 5. Factor VI11 levels in conditioned medium of the

factor VIII-producing CHO cell line. The ability of recombinant vWF to support factor VI11 activity accumulation from the factor VIII-producing CHO cell line 10A1. A , factor VI11 activity accumulation over 48 h in medium containing 10% serum (O), serum-free

vWF-WT was added to 1 pg/ml (0). B, activity levels at 24 h from medium without vWF (X), and serum-free medium to which purified

CHO-1OA1 cells incubated in serum-free conditioned medium from the vWF-producing CHO cell lines. S-F and CHO, controls of serum- free medium and CHO cell-conditioned medium, respectively; WT, APRO, KKS, and KRX, conditioned medium from the vWF-producing cell lines. The concentration of vWF in each conditioned medium sample is indicated at the bottom.

are shown in Fig. 6A. With vWF-WT, the factor VI11 antibody precipitated the labeled vWF, indicating association of the two proteins. Similar results were obtained with the vWF- APRO mutant; however, vWF-KKS was not co-precipitated with the factor VI11 antibody. Lack of factor VIII binding was also observed with the vWF-KRX mutant (Fig. 6 B ) . Factor VI11 levels remaining in the supernatant (data not shown) correlated with the results in Fig. 6. Precipitation of vWF molecules that showed factor VI11 binding (i.e. vWF-WT and vWF-APRO) effectively removed factor VI11 activity from the incubation mixture, whereas precipitation of vWF-KKS and vWF-KRX failed to remove factor VI11 activity.

The efficiency of co-precipitation of vWF and factor VI11 can be estimated by comparison (Fig. 6 A ) of the vWF band intensity obtained by immunoprecipitation with the factor VI11 antibody to the vWF band intensity obtained by precip-

itation with the vWF antibody, because each immunoprecipitation was performed in antibody excess and was shown to be quantitative. In the absence of added factor VIII, no precipitation of any of the vWF forms was detected with the factor VI11 antibody (lane 1 ). With the addition of 10 and 30 units/ml of factor VIII, the majority of the mature vWF-WT and, also, vWF-APRO is precipitated by the factor VI11 antibody, whereas vWF-KKS mutant is not detectably precipitated (Fig. 6A, lanes 2 and 3 ) . It appears significant that, with vWF-WT, a larger proportion of mature species (lower band) is co-precipitated with factor VI11 compared with the slower migrating species pro-vWF (compare Wild Type, lanes 2 and 3 with lunes 5 and 6). This supports the conclusion that the pro-vWF species is defective in its ability to associate with factor VIII. Precipitation with a nonspecific control antibody (lanes 7-9) showed no significant background precipitation, except for the nonreproducible background band seen on this gel for Wild Type, lane 9. The same experiment using medium from the vWF-KRX mutant cell line (Fig. 6 B ) showed no detectable association of the propeptide of vWF with factor VI11 at 30 units/ml (compare lunes 3 ) or 10 units/ml (data not shown). In summary, the finding that only vWF-WT and vWF-APRO form an association with factor VI11 correlates to the ability of these two forms of vWF to stabilize factor VI11 upon secretion from the cell.

DISCUSSION

The importance of von Willebrand factor in the stablization of factor VI11 has long been suggested by the markedly decreased levels of factor VI11 in patients with severe von Willebrand's disease and by the increase in factor VI11 levels in these patients following infusion of cryoprecipitate. Sub- sequent in vivo and i n vitro observations have demonstrated that vWF stabilizes and transports factor VI11 in the plasma (23-25). We propose that, in addition to stabilizing and trans- porting circulating factor VIII, vWF plays a role in factor VI11 biosynthesis by promoting the assembly of the factor VI11 complex upon secretion from the cell. This suggestion is based on our previous characterization of factor VI11 biosynthesis in Chinese hamster ovary (CHO) cells (10). In support of this proposal, we have demonstrated, in aggrement with a previous study (26) , that vWF can reconstitute factor VI11 activity from isolated subunits in vitro. In order to assess the structural features of vWF that are important to its interactions with factor VIII, we established a model system in which specifically altered recombinant vWF was present in the medium of CHO cells that produce factor VIII.

The structure and function of the recombinant vWF mutants used in this study was assessed by gel electrophoresis and platelet agglutination assay. First, the processing and assembly of the vWF expressed in CHO cells was examined by gel electrophoresis. The vWF-WT and vWF-mutants were synthesized, assembled, and secreted from CHO cells in a manner previously described for expression in COS-1 cells (14, 27). The results confirm the requirement of the propeptide for vWF multimerization. Next, because both the factor VIII- and the platelet GPIb-binding sites map to the amino terminus of mature vWF (28, 29), we examined our CHO- derived vWF for function in a ristocetin cofactor platelet agglutination assay that depends on vWF binding to GPIb (20). The appropriate function of vWF-WT in ristocetin- induced platelet agglutination suggests that recombinant vWF may have potential therapeutic use. Of the mutant vWF molecules containing the GPIb-binding site, only vWF- APRO, which is composed of mature dimers, failed to exhibit ristocetin cofactor activity. Although the relationship between


FIG. 6. Assessment of factor VIII- vWF association by co-immunoprecipitation with specific antibodies. Radiolabeled conditioned medium from vWF-producing CHO cell lines was incubated with purified factor VI11 and immunoprecipitated with the following antibodies (indicated at the top of the figure): VZZZ (or F8), monoclonal anti- factor VIII; u WF, polyclonal anti-vWF; AgZZ, monoclonal anti-vWAgII (anti- propeptide); Control, an unrelated mu- rine monoclonal antibody coupled to Sepharose. A, reduced SDS-PAGE results using conditioned medium from vWF-WT ( WT), vWF-KKS (KKS) , and vWF-APRO ( P R O ) . Migration of the 200-kDa molecular mass marker is indicated at the left of each panel. B, results using vWF-KRX-conditioned medium and 30 units/ml of factor VI11 (KRX, lanes 3), along with a CHO cell-conditioned medium control (CHO, lanes I ) and vWF-WT-conditioned medium ( WT, lanes 2) for comparison. The arrow in the center shows the 100-kDa vWF- KRX protein (see Fig. 3A).

A Wild Type KKS A pro Vlll vWF Control Vlll vWF Control Vlll vWF Control

0 10 30 0 10 30 0 10 30 0 10 30 0 10 30 0 10 30 0 10 30 0 10 30 0 10 30 Ulml Vlll

"- "m "- "- 200 20( 200-

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 Y 1 2 ; 1 4 3 8 3 / t J Y

B vWF I Agll F 8

CHO WT KRX CHO WT KRX

vWF multimer formation and hemostatic function is poorly understood, the lack of ristocetin cofactor activity of our vWF- APRO mutant is consistent with the observation that platelet- binding activity correlates with degree of multimerization (21, 22). Thus, our results would suggest that a dimeric form of vWF cannot support ristocetin-induced platelet agglutination. Other studies, however, have shown that a dimeric 116-kDa tryptic fragment, derived from multimeric plasma vWF, re- tains ristocetin cofactor activity a t concentrations equivalent to that used for vWF-APRO (30, 33). Because this dimeric fragment, which contains two GPIb-binding sites, is active in platelet agglutination, we might have expected a protein like vWF-APRO to show similar agglutination activity. We propose that this apparent discrepancy may be explained by hypothesizing that the process of multimerization is required to create functional GPIb-binding sites (see Fig. 7). Since the propeptide of vWF promotes the formation of vWF multimers (14, 27), it must at some level influence the formation or rearrangment of disulfide bonds. In fact, it has been proposed that the vWF propeptide may possess a protein disulfide isomerase activity (31). Since the vWF-APRO mutant is synthesized in the absence of propeptide, the disulfide pairing of the Cys residues near the GPIb-binding domain of vWF in the vWF-APRO dimer may reflect the pairing present in the intracellular precursor prior to disulfide bond rearrangement required for multimerization. These differences likely occur near the GPIb-binding domain of vWF because Cys residues in this region are known to form intermolecular disulfide

- 200 -

4 - .I ... -. -93-

"-

1 2 3 1 2 3

bonds (32,33). We postulate that the multimerization process, which is directed by the propeptide, is essential to establish the conformation (i.e. tertiary and/or quaternary structures) appropriate to display ristocetin-induced platelet agglutination.

To test the utility of our CHO cell model system for analysis of factor VIII/vWF interaction, we first established that wild- type recombinant vWF can promote the stable accumulation of factor VI11 activity in serum-free cell culture. As shown in Fig. 5A, the presence of vWF in the CHO cell medium at 1 pg/ml will support the accumulation of 250 milliunits/ml of factor VIII. We have observed in other experiments3 that CHO cell-derived factor VI11 generally forms an approximately 1:50 (w/w) ratio with vWF added to the culture medium. In addition, a roughly 1:50 ratio is acquired in our i n uitro reconstitution experiments (Fig. l), where a much higher factor VI11 activity level is achieved. We believe that these observations reflects the role of vWF in regulating factor VI11 levels i n uiuo because, although the factor VIII-binding sites on vWF may be saturated i n uitro (34-36), in the plasma the ratio of factor VI11 to vWF is maintained at approximately 1:50. How this ratio is regulated in uiuo is not understood, but these observations suggest that the 1:50 ratio observed i n uivo may be established by the way vWF interacts with factor VI11 at the site of synthesis.

The vWF-APRO mutant was capable of stabilizing factor VI11 upon secretion and was capable of binding factor VI11

L. Wasley and R. Kaufman, unpublished data. ~~

21954

WT

APRO

von Willebrand Factor Binding to Factor VIII c c

FIG. 7. Model for factor VI11 and GPIb platelet-binding sites in wild-type and mutant vWF. Diagram of the protomeric vWF dimer and its postulated arrangement in a multimeric complex. Dimers are formed from vWF subunits (gray) linked by disulfide bonds near the carboxyl terminus. Disulfide bonds near the amino- terminal end of mature vWF link dimers of vWF-WT ( WT, top) and vWF-KKS (KKS, bottom) into multimers. The same sulfhydryl groups in the vWF-APRO molecule (APRO, middle) are shown either free (left side) or linked in intramolecular bonding (right side). The sulfhydryl groups depicted are located in the D3 domain and the first A domain of vWF (Refs. 12, 32, 33, and 48). A functional GPIb-binding site is present on WT and KKS, as indicated by the circular indentation and the bound GPIb molecule (gray circle). A functional factor VIII-binding site is present on WT and APRO, as indicated by the indented white bar near the amino terminus. The factor VIII-binding site on KKS is not accessible because of the presence of the uncleaved vWF propeptide (black). This model was derived from results reported here, as well as in previous studies using plasma-derived vWF (reviewed in Ref. 49) and does not distinguish between functional GPIb-binding and ristocetin interactions resulting in platelet agglutination.

measured by co-immunoprecipitation. This observation is in direct contrast to results recently reported by Leyte et al. (37). These investigators detected no factor VI11 binding to a vWF mutant similar to vWF-APRO. Several significant differences in the experimental approaches may explain the inability of their dimeric APRO molecule to bind to factor VIII. First, the APRO mutation analyzed by Leyte et al. (37) contained an extra amino acid (Ala) at the signal peptide cleavage site compared with the vWF-APRO described here. This could interfere with appropriate signal peptide cleavage and/or fold- ing of the mature vWF. The amino terminus of the mutant analyzed by Leyte et al. was not determined. However, if signal peptide cleavage were to occur, the predicted amino terminus would have an extra amino acid that may alter the structure of the amino-terminal region of the vWF subunit. This may be relevant because a naturally occurring mutation within the first 30 amino acids of mature vWF has been shown to interfere with factor VI11 binding (38). Amino-acid sequence analysis has demonstrated that the mature aknino terminus of the vWF-APRO described in this report is identical with that of plasma vWF (14). Another potentially significant difference is that we have expressed the vWF mutants in Chinese hamster ovary cells, whereas Leyte et al. have used AtT-20 cells.

The vWF-APRO mutant demonstrates that mature vWF dimers are sufficient for functional factor VI11 interaction and that multimers or the multimerization process is not required (see Fig. 7). Structural mapping studies (29,39) have demonstrated a major factor VIII-binding domain located in the amino-terminal 272 amino acids of the mature vWF subunit. More recently, the factor VIII-binding region has

been further resolved to amino acids 78-96 (40). This domain is amino-terminal to the disulfide bonds that link dimers into multimers (32). Disulfide bonding within this region is, however, crucial for factor VI11 binding (29). These disulfide bonds, which are all intramolecular, therefore may not be involved in the hypothesized disulfide rearrangements directed by the propeptide during multimerization (14,31). Our results demonstrate that a vWF dimer is sufficient to bind to and support the stable secretion of factor VIII. Foster et al. (29) have shown that a monomeric fragment of vWF can bind to factor VI11 in uitro. Whether this fragment could promote factor VI11 secretion from cells has not been determined. Our results do not rule out the requirement for both of the factor VI11 binding sites in vWF-APRO for critical factor VI11 interactions at the site of secretion.

The vWF propeptide (VWAgII), which is present in approximately 1% of circulating vWF, has been suggested to contain a factor VIII-binding site (19). Our results demonstrate that the vWF propeptide expressed alone does not detectably interact with factor VI11 and cannot support factor VI11 accumulation upon secretion. In addition, the vWF propeptide cleaved from vWF-WT was not co-immunoprecipitated with factor VI11 (see Fig. 6A, Wild Type). Thus, the vWF propeptide, whether expressed alone or generated through the multimerization/secretion process, appears not to interact with factor VIII. Our findings predict, therefore, that circulating vWAgII plays no role in assembly or stabilization of the factor VI11 complex.

The vWF-KKS mutant, although fully multimerized and active in the platelet agglutination assay, also failed to support factor VI11 accumulation. The functional ristocetin cofactor


activity of this mutant supports the notion that this mutant undergoes the correct disulfide bonding required for GPIb recognition and platelet agglutination. The simplest expla- nation for the lack of factor VI11 association with the vWF- KKS mutant is to propose that the uncleaved propeptide prevents access to the factor VIII-binding site (Fig. 7). Alter- natively, removal of the propeptide may result in a confor- mational change to expose the factor VIII-binding site. Based on the hypothesis that uncleaved vWF propeptide interferes with factor VI11 binding, we predict that the factor VIII- stabilizing activity of our recombinant wild-type vWF may be less than maximal because of the persistence of pro-vWF due to the inefficient cleavage of the vWF propeptide in CHO cells (see Fig. 3A). Montgomery et al. (41) reported individuals with hereditary persistence of pro-vWF. Our hypothesis would predict that these individuals may have lower than normal factor VI11 levels. However, since the in vivo ratio of factor VI11 to vWF is approximately 1:50, it may be that the vWF multimers in these heterozygous individuals would be composed of a mixture of mature and pro-vWF and therefore may contain enough available factor VIII-binding sites to support normal factor VI11 levels. Mutations within the factor VIII- binding domain of vWF have been proposed to explain patients with an autosomal inheritence of the hemophilia A phenotype (42-47). The vWF from these patients exhibits decreased factor VI11 binding (44-46) and, recently, the molecular defects in two of these patients have been identified as point mutations either within or amino-terminal to the defined factor VIII-binding region of vWF (38, 47). Our findings predict that homozygous vWF mutations that block propeptide cleavage may also produce a similar phenotype.

Acknowledgments-We thank Ed Alderman and Barry Foster for providing F8 antibody and vWF monoclonal IgG, David Fass for providing purified plasma vWF, Phil Fay for providing the anti- vWAgII monoclonal antibody, Jack Wang for purification of factor VI11 subunits, David Ginsburg for communicating unpublished infor- mation, Ike Blue and Louise Wasley for technical help, Miguel Cruz and Robert Handin for helpful discussions, and Joanne Janeiro for assistance in preparing this manuscript. We also gratefully thank Stuart Orkin, who provided laboratory facilities and support at the inception of this work.

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of the role viii* of cleavage in binding and stabilization · association of factor vi11...

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