©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Platelet Glycoprotein V with Glycoprotein Ib-IX Regulates Expression of the Glycoproteins and Binding of von Willebrand Factor to Glycoprotein Ib-IX in Transfected Cells (*)

Sylvie C. Meyer (1) (2), Joan E. B. Fox (1) (2) (3)(§)

From the (1)Children's Hospital Oakland Research Institute, Oakland, California 94609 and the (2)Cardiovascular Research Institute, (3)Department of Pathology, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The goal of the present study was to determine whether platelet glycoprotein (GP) V interacts directly with the von Willebrand factor receptor GP Ib-IX and, if so, whether it affects the expression and function of this receptor. A melanoma cell line that does not contain actin-binding protein was transfected with the cDNAs coding for GP V and for each of the three subunits of GP Ib-IX. GP V co-immunoprecipitated and co-localized with GP Ib-IX. Although GP V could be expressed in the absence of GP Ib-IX, the amount incorporated in the membrane was markedly increased when GP Ib-IX was present. Similarly, there was an enhanced expression of GP Ib-IX on the cell surface in the presence of GP V. The binding affinity of botrocetin-induced von Willebrand factor to GP Ib-IX was unaffected by the presence or absence of GP V. However, the binding capacity was increased by the presence of GP V. We conclude that GP V interacts directly with GP Ib-IX, that GP V must associate with GP Ib-IX to be efficiently expressed in the membrane, and that GP V increases the binding capacity of the cells for von Willebrand factor by enhancing the surface expression of the GP Ib-IX complex.


INTRODUCTION

Platelets play a fundamental role in the formation of the hemostatic plug at sites of vascular injury(1, 2) . One of the critical interactions mediating the initial contact of platelets with the damaged vessel wall is the interaction of the platelet glycoprotein (GP)()Ib-IX complex with von Willebrand factor (vWf)(3, 4) .

The GP Ib-IX complex consists of two disulfide-linked subunits, GP Ib (M = 140,000), and GP Ib (M = 25,000) that are noncovalently associated in a 1:1 ratio with GP IX (M = 22,000)(5, 6) . Each of these glycoproteins is a transmembrane protein(7, 8, 9) . An interesting feature of the three components of the GP Ib-IX complex is that they each contain leucine-rich motifs in their extracellular domains(7, 8, 9) . A fourth platelet membrane glycoprotein, GP V (M = 82,000), is an additional member of this family of leucine-rich glycoproteins(10, 11, 12, 13) . All three subunits of the GP Ib-IX complex are missing or are present in significantly decreased amounts in platelets from Bernard-Soulier patients(14, 15, 16) ; the finding that the amount of GP V is also decreased in the membranes of these platelets(15, 16, 17, 18) raised the possibility that GP V may be part of a complex with the other three leucine-rich glycoproteins. Evidence that such a complex may exist comes from a report that GP V can be co-immunoprecipitated with GP Ib-IX from platelets lysed with the mild detergent digitonin (19). However, GP Ib-IX associates with actin-binding protein, thus mediating attachment of this adhesion receptor to the cytoskeleton. Actin-binding protein and GP Ib-IX interact with such high affinity that they remain associated in all of the detergents used to lyse platelets(20, 21, 22, 23, 24, 25) . Thus, actin-binding protein and associated cytoskeletal proteins always co-immunoprecipitate with GP Ib-IX from detergent-lysed platelets, making it difficult to determine whether GP V and GP Ib-IX co-immunoprecipitate because they associate directly with each other or because they are both associated with the cytoskeleton.

The function of GP V is not known. Because it is cleaved by thrombin, it was originally thought to be the functional thrombin receptor(26, 27, 28) . However, it is now known that it is cleaved from the surface of activated platelets by a protease other than thrombin; thrombin appears to cleave it after it has been removed from the platelet membrane by other mechanisms (for review, see Ref. 29). Based on the finding that decreases in GP Ib-IX in patients with Bernard-Soulier syndrome are also accompanied by decreased surface expression of GP V(15, 16, 17, 18) , it appears possible that GP Ib-IX is required for the expression of GP V on the surface. Conversely, it appears possible that if GP V and GP Ib-IX form a complex, GP V might regulate the expression of the GP Ib-IX subunits on the surface. If so, this would not only provide the first known function of GP V, but it would also raise the possibility that some cases of Bernard-Soulier syndrome could potentially arise from mutations in GP V rather than from mutations in one of the subunits in GP Ib-IX.

In the present studies, GP V and GP Ib-IX were expressed in a melanoma cell line that lacks actin-binding protein(30) . This approach eliminated the potential problem of GP Ib-IX and GP V co-immunoprecipitating because they are both associated with the cytoskeleton and allowed us to determine whether GP V interacts with GP Ib-IX. Further, confocal microscopy was used to determine whether the glycoproteins co-localize in the intact cell, an approach that is not feasible in platelets because of the small size of the cells. By transfecting cells with combinations of cDNAs coding for the glycoproteins, we have determined whether the expression of one glycoprotein influences the expression of others. We show that GP V interacts directly with GP Ib-IX in the membrane of the cells and that this interaction is needed for the efficient expression of both GP V and GP Ib-IX in the membrane. Further, our studies suggest that GP V increases the binding capacity of the cells for von Willebrand factor by increasing the surface expression of the GP Ib-IX complex.


MATERIALS AND METHODS

Cell Culture and Transfections

The melanoma cells used in these studies were derived from a human malignant melanoma lacking actin-binding protein(30) . Actin-binding protein-deficient cells were used in the immunoprecipitation experiments described below. For all other experiments, cells that had been stably transfected with the cDNA coding for actin-binding protein were used(30) . Both the actin-binding protein-deficient cells and the actin-binding protein-containing cells were kindly provided by Dr. C. Cunningham (Brigham Women's Hospital, Boston, MA). The actin-binding protein-deficient cells were grown in Dulbecco's Modified Eagle Medium (Life Technologies, Inc.) with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.) at 37 °C in a 5% CO atmosphere. The actin-binding protein-containing cells were grown in the same medium supplemented with 0.5 mg/ml G418. The constructs containing the cDNAs coding for GP Ib, GP Ib, and GP IX subcloned into the eukaryotic expression vector pDX were a gift from Dr. J. López (Gladstone Institutes, San Francisco, CA)(31) . The cDNA encoding GP IX (9) was previously obtained from Dr. G. Roth (Seattle Veterans Administration Hospital). The cDNA encoding GP V was provided by Dr. F. Lanza (Centre Régional de Transfusion Sanguine, Strasbourg, France) (12) and was subcloned into the pDX vector.

Stable transfections were performed by the calcium phosphate method described by Graham and van der Eb(32) . The cells were co-transfected with 10 µg of each linearized construct containing the cDNAs encoding GP Ib, GP Ib, and GP IX or GP V and 0.5 µg of an appropriate selection marker. Individual colonies were isolated using a cloning cylinder. About 20 clones were grown and evaluated by flow cytometry for surface expression of the GP Ib-IX complex or GP V. The clones with the higher expression levels of the proteins of interest were used in the experiments described below.

In one experiment, as indicated in the text, GP V-expressing cells were transiently transfected with 3 µg of each expression plasmid containing the cDNAs coding for GP Ib, GP Ib, and GP IX and 40 µl of liposome suspension (LipofectAMINE, Life Technologies, Inc.)(33) .

Fluorescence and Confocal Microscopy

The cells were plated on Lab-Tek tissue culture chamber slides (Nunc, Inc., Naperville, IL), fixed in the absence of detergent, and stained as described previously (31). The cells were labeled with a monoclonal antibody against GP Ib (monoclonal antibody Ib-23 (34) courtesy of Dr. B. Steiner, Hoffmann-La Roche, Basel, Switzerland) and/or serum against GP V (courtesy of Dr. D. Phillips, COR Therapeutics Inc., San Francisco, CA). The polyclonal GP V antibody was detected with Texas red-labeled goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA), and the monoclonal GP Ib antibody was detected with biotinylated goat anti-mouse IgG and fluorescein isothiocyanate (FITC)-conjugated streptavidin (Amersham International plc, Buckinghamshire, United Kingdom). When the labeling was performed only with the serum against GP V, biotinylated goat anti-rabbit IgG (Amersham), and FITC-conjugated streptavidin were used. Fluorescence microscopy was performed on an inverted microscope (DIAPHOT-TMD, Nikon, Japan). Confocal microscopy was performed using a Sarastro 1000 confocal microscope (Molecular Dynamics, Sunnyvale, CA).

Flow Cytometry and Cell Sorting

Cells were harvested by treatment with 1 mM EDTA (Sigma), washed in PBS, and incubated with the relevant polyclonal or monoclonal antibody for 30 min at 4 °C. The cells were washed and then incubated for 30 min at 4 °C with FITC-conjugated goat anti-mouse or anti-rabbit IgG (5 µg/ml in PBS containing 1% bovine serum albumin) (Vector Laboratories). Following washes, the samples were resuspended in PBS and analyzed by a FACScan flow cytometer (Becton Dickinson, San Jose, CA). In some cases, cell sorting was performed using a modified FACS 440 cell sorter (Becton Dickinson). The 5% of cells with the highest levels of fluorescence intensity were collected.

Western Blot Analysis

Cells (2 10) were solubilized in 40 µl of Laemmli sample buffer(35) , and solubilized proteins were electrophoresed through a 7.5% SDS-polyacrylamide gel. The proteins were transferred onto nitrocellulose paper(36) , which was incubated with a serum against GP V or with a monoclonal antibody against GP Ib, monoclonal antibody Ib-4 (34) (courtesy of Dr. B. Steiner). The nitrocellulose paper was sequentially incubated with either a biotinylated goat anti-rabbit IgG or an anti-mouse IgG and streptavidin-horseradish peroxidase conjugate (Amersham). The signal was detected by enhanced chemiluminescence using Amersham's ECL system.

Co-immunoprecipitation of GP Ib-IX with GP V

Polyclonal antibodies against GP V or rabbit IgG (Sigma) were covalently coupled to polyacrylamide immunobeads (Bio-Rad Laboratories, Richmond, CA) (average diameter, 10 µm) according to the manufacturer's instructions. The cells were harvested with EDTA, washed twice with PBS and lysed in an ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.4, 1% digitonin (Sigma), 5 mM CaCl (Sigma), 1 mg/ml DNase I (Boehringer Mannheim), 1 mg/ml leupeptin (Vega Biotechnologies, Tucson, AZ), 1 mM phenylmethylsulfonyl fluoride (Sigma), 50 mM benzamidine (Sigma), 1 mM sodium orthovanadate (Fisher Scientific, Fair Lawn, NJ), 20 µg/ml soybean trypsin inhibitor (Sigma) for 1 h at 4 °C under agitation (2.5 10 cells/ml of lysis buffer). The samples were centrifuged at 14,000 g for 20 min at 4 °C. The supernatant (1 ml) was agitated for 1 h at 4 °C with 50 µl of rabbit IgG-coupled immunobeads. The beads were sedimented by centrifugation at 14,000 g for 4 min at 4 °C. The precleared supernatant was agitated with 50 µl of anti-GP V-coated beads or rabbit IgG-coated beads for 16 h at 4 °C. The immunobeads were washed four times in a buffer similar to the lysis buffer but containing only 0.1% digitonin and lacking DNase I. Immunoprecipitated proteins were removed from the beads by boiling the samples in an SDS-containing buffer (35) and were analyzed on Western blots.

Binding of Botrocetin-induced von Willebrand Factor to the Transfected Cells

Purified vWf (generously provided by Dr. J. Moake, Baylor College of Medicine, Houston, TX) (37) was labeled by the lactoperoxidase method(38) . The cells (10 cells in 400 µl of PBS containing 1% bovine serum albumin) were incubated in the presence of 5 µg/ml botrocetin (Pentapharm, Basel, Switzerland), and various concentrations of I-labeled vWf (specific activity approximately 6 10 cpm/µg). Samples were rocked gently at room temperature for 1 h. 225-µl aliquots were removed and layered onto 400 µl of ice-cold 30% sucrose in PBS containing 0.1% bovine serum albumin. The samples were centrifuged at 15,600 g for 4 min at 4 °C. The sucrose solution was aspirated carefully, and the amount of I-labeled vWf associated with the sedimented cells was determined in a counter (Iso-Data, Rolling Meadows, IL). Nonspecific binding at each vWf concentration was determined by measuring I-labeled vWf binding to melanoma cells that had not been transfected with GP Ib-IX. These counts were subtracted from those obtained with the corresponding transfected cells. A molecular weight of 1.1 10 was assumed for vWf. The data were analyzed by Scatchard analysis.


RESULTS

To determine whether GP V associates with GP Ib-IX, cells were stably transfected with the cDNA for GP V and the three cDNAs encoding the subunits of GP Ib-IX (GP Ib, GP Ib, and GP IX). Cells were lysed, and GP V immunoprecipitated from the lysates. Because GP Ib-IX associates with actin-binding protein, thus mediating attachment of this adhesion receptor to the cytoskeleton, actin-binding protein and associated cytoskeletal proteins always co-immunoprecipitate with GP Ib-IX from detergent-lysed cells(20, 21, 22, 23, 24, 25) . To circumvent this problem, the present experiments were performed using a lysis buffer that included DNase I to depolymerize actin filaments(20, 21) . However, when actin filaments are depolymerized and GP Ib-IX is released into the detergent-soluble fraction, the glycoprotein complex remains associated in a high affinity interaction with actin-binding protein(21) . To eliminate the possibility that the glycoproteins co-immunoprecipitate simply because they are both associated with actin-binding protein, the present experiments were performed with a previously described cell line that lacks actin-binding protein(30) . Western blot analysis showed that GP Ib co-immunoprecipitated with GP V from these cells (Fig. 1B, lane2). The finding that GP Ib-IX co-immunoprecipitated with GP V even when actin-binding protein was not present indicates that there is a direct interaction of GP V with one or more of the glycoproteins in the GP Ib-IX complex. Some proteolysis of the GP Ib chain apparently occurred during lysis of the cells yielding a fragment of M = 90,000-100,000 (Fig. 1B). This fragment was not immunoprecipitated by a monoclonal antibody that blocked von Willebrand factor binding to GP Ib (monoclonal antibody Ib-23) (data not shown), suggesting that it represented GP Ib that has been cleaved at a protease-sensitive site in the extracellular amino-terminal end of the molecule(39) . This fragment lacks the 45-kDa domain that contains the leucine-rich repeats and the ligand-binding site(7, 40, 41, 42, 43, 44) ; like intact GP Ib it co-immunoprecipitated with GP V (Fig. 1B, lane2).


Figure 1: Co-immunoprecipitation of GP Ib-IX with GP V in actin-binding protein-deficient melanoma cells. Melanoma cells that were expressing GP Ib-IX and GP V but were not expressing actin-binding protein were lysed in a digitonin-containing lysis buffer in the presence of Ca and DNase I to induce actin filament depolymerization (see ``Materials and Methods''). After centrifugation the digitonin-soluble fractions (lanes1) were incubated with either anti-GP V-coupled beads (lanes2) or rabbit IgG-coupled beads (lanes3). Immunoprecipitated proteins were electrophoresed through 7.5% SDS-polyacrylamide gels under reducing conditions and then transferred to nitrocellulose. The blots were probed with a polyclonal serum against GP V (A) or a monoclonal antibody against GP Ib (B). The mobility of prestained marker proteins (kDa) is indicated on the rightside of each panel.



To determine whether GP V associates with the GP Ib-IX complex in intact cells, we analyzed the location of the proteins by confocal microscopy (Fig. 2). Each micrograph represents a thin optical section through the cells. There was a precise co-localization of GP V (Fig. 2A) with GP Ib (Fig. 2B).


Figure 2: Confocal microscopy images showing co-localization of GP Ib-IX and GP V in the membrane of transfected cells. Melanoma cells stably transfected with the cDNAs encoding GP Ib, GP Ib, GP IX, and GP V were incubated with serum against GP V and a monoclonal antibody against GP Ib. The polyclonal GP V antibody was detected with Texas red-labeled goat anti-rabbit IgG (A), and the monoclonal GP Ib antibody was detected with biotinylated goat anti-mouse IgG and FITC-conjugated streptavidin (B).



Since in patients with Bernard-Soulier syndrome, decreases in GP Ib-IX are also accompanied by decreased surface expression of GP V(15, 16, 17, 18) , we wanted to know whether GP V expression requires the presence of the GP Ib-IX complex. Melanoma cells were stably transfected with the cDNA encoding GP V alone. In these and all subsequent experiments, melanoma cells that were expressing actin-binding protein were used. Analysis of solubilized cells on Western blots showed that even in the absence of GP Ib-IX, GP V was synthesized and had a molecular weight(82, 0) similar to that of the protein in platelets (Fig. 3, left panel,lane2). As shown by FACS analysis (Fig. 3, right panel,dashedline) and immunofluorescence of nonpermeabilized cells (Fig. 4B), some GP V was incorporated into the membrane. To determine whether the surface expression of GP V was more efficient if the three subunits of the GP Ib-IX complex were present, the synthesis and surface expression of GP V was also examined in a clone of cells that had been stably transfected with the cDNAs for GP Ib, GP Ib, and GP IX in addition to GP V. Even though this clone synthesized considerably less GP V than the clone that had been transfected with the cDNA for GP V alone (Fig. 3, left panel, compare lane3 with lane2, and ), more GP V was expressed on the surface of these cells (Fig. 3, right panel, compare solidline with dashedline). These results suggest that in the absence of GP Ib-IX intact GP V exists within the cell but that it is more efficiently incorporated into the cell membrane when the GP Ib-IX complex is present. To directly test this idea, a clone of cells expressing GP V alone was transiently transfected with the three cDNAs encoding GP Ib, GP Ib, and GP IX. The cells were harvested 48 h after transfection. The expression of GP Ib-IX was verified by flow cytometry (data not shown). Fig. 5shows that after addition of GP Ib-IX, GP V reached the surface at levels significantly higher (solidline) than those that existed prior to GP Ib-IX addition (dashedline). When GP V was expressed alone, a large amount of protein with the same molecular weight as intact GP V was also found in the cell culture medium (). The amount of GP V in the medium, relative to the amount in the cells, was markedly decreased when GP Ib-IX was also present ().


Figure 3: Western blots and FACS analysis showing that full-length GP V is expressed in transfected cells but that little is expressed in the membrane unless GP Ib-IX is also present. Left panel, melanoma cells (2 10 cells/sample) were solubilized in an SDS-containing buffer. Solubilized proteins were electrophoresed through a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose. The blot was probed with a serum against GP V. Lane1, nontransfected cells; lane2, a clone of cells stably transfected with the cDNA encoding GP V; lane3, a different clone of cells that had been stably transfected with the cDNAs for GP V, GP Ib, GP Ib, and GP IX. The mobility of prestained marker proteins (kDa) is indicated on the right. Right panel, the same cells as those analyzed in the left panel (5 10 cells/sample) were labeled with a serum against GP V followed by FITC-conjugated anti-rabbit IgG and analyzed by flow cytometry. The dottedline represents nontransfected cells, the dashedline represents cells stably transfected with the cDNA encoding GP V only, and the solidline represents cells stably transfected with the four cDNAs encoding GP V, GP Ib, GP Ib, and GP IX. The cells that contained the cDNAs for all four glycoproteins expressed more GP V on the surface than did the cells containing only the cDNA for GP V even though the latter cells contained considerably higher amounts of GP V than did the cells that also expressed GP Ib, GP Ib and GP IX.




Figure 4: Immunofluorescence microscopy of cells expressing GP V. Melanoma cells that were not transfected (A) or that were stably transfected with the cDNA encoding GP V (B) were fixed (in the absence of detergent) and then incubated with serum against GP V, followed by biotinylated goat anti-rabbit IgG, and FITC-conjugated streptavidin. Bar, 15 µm.




Figure 5: Transient expression of GP Ib-IX increases the surface expression of GP V in melanoma cells. Melanoma cells were stably transfected with the cDNA encoding GP V (dashedline). In addition, some cells expressing GP V were transiently transfected with the cDNAs for GP Ib, GP Ib, and GP IX (solidline). The cells were incubated with serum against GP V and FITC-conjugated anti-rabbit IgG, and they were analyzed by flow cytometry. Dottedline, nontransfected cells.



To determine whether the presence of GP V regulates the expression of the GP Ib-IX subunits, the amount of GP Ib-IX present on the surface of the GP Ib-IX-expressing cells was measured by flow cytometry (Fig. 6) and compared with the amount present on the surface of the GP Ib-IX plus GP V-expressing cells. In this experiment, the cells were stained with an antibody against GP Ib, but similar results were obtained by using an antibody against GP IX (clone BL-H6) (45) or a complex-specific antibody, SZ1 (46) (data not shown). As shown in Fig. 6, GP Ib-IX was expressed on the surface in the absence of GP V (dashedline), but GP Ib-IX expression was enhanced in cells that also expressed GP V (solidline). Different clones were analyzed, and the shift in fluorescence was always proportional to the amount of GP V expressed.


Figure 6: Increased surface expression of GP Ib-IX after addition of GP V in the melanoma cells. Melanoma cells (5 10 cells/sample) that were not transfected (dottedline) or were stably transfected with the cDNAs encoding either GP Ib, GP Ib, and GP IX (dashedline) or GP Ib-IX plus GP V (solidline) were incubated with a monoclonal antibody against GP Ib. Following addition of FITC-conjugated goat anti-mouse IgG, the cells were analyzed by flow cytometry.



To determine whether the presence of GP V affects the ability of GP Ib-IX to bind von Willebrand factor, botrocetin-induced binding of I-labeled von Willebrand factor to either the GP Ib-IX-expressing cells (Fig. 7A) or to the GP Ib-IX plus GP V-expressing cells (Fig. 7B) was measured. Nonspecific binding was determined by measuring binding of I-labeled von Willebrand factor to non-GP Ib-IX-expressing cells. The binding was botrocetin-dependent and could be inhibited by a monoclonal antibody directed against the von Willebrand factor binding site on GP Ib (data not shown). Binding of von Willebrand factor to the cells was saturable. The data, presented as a Scatchard plot, fit a model of a single class of binding sites and was representative of four independent experiments. The dissociation constants were similar for the GP Ib-IX-expressing cells and for the cells that also expressed GP V (0.16 ± 0.01 and 0.18 ± 0.02 nM, respectively). Thus, GP V did not affect the binding affinity of von Willebrand factor to the receptor. However, as shown above, GP V enhanced the expression of GP Ib-IX and by doing so increased the binding capacity of the cells for von Willebrand factor. Approximately three times more GP Ib-IX complex was present on the cells that also expressed GP V. The binding capacity of the cells expressing GP V was higher than that of the cells expressing only GP Ib-IX (63 ± 20 pM and 26 ± 5 pM, respectively).


Figure 7: Botrocetin-induced binding of I-von Willebrand factor to GP Ib-IX-expressing melanoma cells in the presence or absence of GP V. 10 cells were incubated in the presence of botrocetin and various concentrations of I-vWf. The specific binding of I-labeled vWf to the cells was measured as described under ``Materials and Methods.'' Each data point represents the mean of a duplicate determination. The experiment shown is representative of four independent experiments. A, binding to cells expressing only GP Ib-IX; B, binding to cells expressing GP Ib-IX plus GP V.




DISCUSSION

Previously, the observation that the platelets from patients with Bernard-Soulier syndrome lacked GP V in addition to GP Ib-IX suggested that GP V and GP Ib-IX may exist in a complex. The finding that the four glycoproteins co-immunoprecipitated from detergent-lysed platelets provided some support for this possibility. However, GP Ib-IX interacts with high affinity with actin-binding protein, so a variety of cytoskeletal proteins are always present in a GP Ib-IX immunoprecipitate. In the present study, we provide several lines of evidence that GP V interacts directly with GP Ib-IX. First, GP V co-immunoprecipitated with GP Ib-IX from lysates of cells that did not contain actin-binding protein. Second, platelets are too small to provide convincing evidences for co-localization of proteins by immunofluorescence microscopy. However, by expressing the glycoproteins in melanoma cells we were able to visualize their distribution by confocal microscopy; this approach revealed a precise co-localization of GP V and GP Ib-IX in the membrane, showing that the interaction between the glycoproteins occurs in the intact cell and is not an artifact induced in cell lysates. Finally, in cells expressing both GP Ib-IX and GP V, the cells expressing most GP Ib-IX on the surface expressed most GP V and vice versa.

These results suggest that GP V is an additional component of the GP Ib-IX complex in intact cells. In the present study, GP V and GP Ib-IX remained associated when the cells were lysed with the mild detergent, digitonin, but dissociated in a stronger detergent, Triton X-100. Modderman et al.(19) also found that GP V and GP Ib-IX co-immunoprecipitated from lysates prepared with digitonin but did not co-immunoprecipitate when lysates were prepared with Nonidet P-40 or octyl glucoside. In contrast, the three subunits of the GP Ib-IX complex remain tightly associated when platelets are lysed in strong detergents such as Triton X-100(6, 23) . Thus, GP Ib, GP Ib, and GP IX associate with high affinity, but GP V appears to associate with the resulting complex with a lower affinity.

Binding studies using monoclonal antibodies have indicated that each platelet contains approximately 24,000 molecules of GP Ib-IX but only 11,000 molecules of GP V(19, 46, 47) . It has been suggested that two GP Ib-IX molecules may associate with one GP V molecule(19) . An alternative possibility might be that some of the GP Ib-IX complexes are not associated with GP V and that GP V confers a special function on a subpopulation of GP Ib-IX. It was originally thought that GP V was the functional thrombin receptor on platelets(26, 27, 28) , but it is now clear that this is not the case (for review, see Ref. 29). Elucidation of other potential functions of GP V awaits further study.

Interestingly, GP V, like GP Ib, GP Ib, and GP IX is a member of the leucine-rich family of proteins(7, 8, 9, 10, 11, 12, 13) . Thus, all four members of this family that are known to be present in platelets exist in a complex with each other. In GP Ib, the leucine-rich repeats are all in the 45-kDa amino-terminal end that contains the binding site for von Willebrand factor(7, 40, 41, 42, 43, 44) . In the present study, we observed that GP V still co-immunoprecipitated with GP Ib-IX in which this 45-kDa domain had been removed proteolytically. Similarly, GP V still co-immunoprecipitated with GP Ib-IX from platelet lysates even after the 45-kDa amino terminus of GP Ib had been removed by treatment of the platelets with human leukocyte elastase(40, 19) . Thus, the leucine-rich repeats of GP Ib do not appear to be needed for the interaction of GP Ib-IX with GP V. Interestingly, many of the known members of the leucine-rich family of glycoproteins are either adhesive receptors or signaling molecules (for review, see Ref. 49). The GP Ib-IX-V complex serves both of these functions; it binds the adhesive ligand von Willebrand factor(3, 4) , and following von Willebrand factor binding it transmits signals across the membrane such that intracellular signaling molecules are activated(50, 51, 52, 53) . The availability of the expression system developed in the present work may allow the importance of these leucine-rich repeats and the significance of the association of the four leucine-rich subunits to be investigated.

Previously, it has been shown that although GP Ib could be expressed on the surface of transfected cells in the absence of the other subunits(34) , the expression was markedly increased when all three subunits were present(31, 48) . Together with the finding that in patients with Bernard-Soulier syndrome, mutations in GP Ib, GP Ib, or GP IX apparently result in decreased expression of all three subunits of the GP Ib-IX complex, these observations led to the idea that all three subunits of this complex must associate in order to obtain efficient surface expression of any of the subunits. The fact that Bernard-Soulier patients have little GP V on their surface (15, 16, 17, 18) suggests that GP Ib-IX may also play a role in the expression of GP V. The finding that GP V associates with GP Ib-IX suggests that all four subunits of this complex may be needed for efficient expression of the entire complex. To directly test this possibility, we transfected melanoma cells with GP V in the presence or absence of GP Ib-IX. As with GP Ib, expression of GP V as a single polypeptide resulted in the synthesis of a glycoprotein that could be incorporated into the membrane. However, the presence of the three subunits of the GP Ib-IX complex markedly increased the amount of GP V that was incorporated into the membrane. Previous studies have shown that when GP Ib is expressed alone only about 5% of the recombinant protein exists as intact protein(34) . Most of the protein is degraded and exists in the cells or is secreted into the medium as fragments. Like GP Ib, when GP V was expressed alone, only small amounts of the protein were incorporated into the membrane. However, in contrast to GP Ib, the remaining GP V was present in the cell lysates and in the cell medium with the same molecular weight as intact GP V. Thus, although it is not possible to determine whether GP V was present as the intact protein or whether it had been cleaved at the protease-sensitive site adjacent to the membrane insertion site(29) , GP V appears to differ from GP Ib in that it is not degraded to detectable smaller fragments when expressed alone. These findings on the importance of GP Ib-IX in allowing GP V to be expressed in the membrane might explain the fact that surface labeling of platelets showed that GP V was missing or present in a decreased amount in Bernard-Soulier patients with mutations in components of the GP Ib-IX complex(15, 16, 17, 18) . Western blot analysis of these platelets would determine if GP V is synthesized but not incorporated in the platelet membrane, as is the case for transfected cells.

The finding that GP Ib-IX is needed for the efficient surface expression of GP V supports the idea that all four subunits of the GP Ib-IX-V complex are needed for the efficient expression of the others in the membrane. Further support for this came from experiments in which we transfected melanoma cells with the cDNAs encoding GP Ib, GP Ib, GP IX, and then GP V. The addition of GP V increased the cell surface expression of GP Ib-IX. Thus, while it has previously been thought that the three subunits of GP Ib-IX are sufficient for the efficient expression of this complex in the membrane(31) , the present results show that the presence of the fourth component, GP V, increases expression of the complex further.

The finding that GP V increases the surface expression of GP Ib-IX represents the first known function of GP V. This function is important in that it raises the possibility that some cases of Bernard-Soulier syndrome could arise from mutations in GP V rather than in one of the subunits of the GP Ib-IX complex. Further, it raises the possibility that GP V could be important in regulating the ability of cells to bind von Willebrand factor. To address this question, we measured botrocetin-induced von Willebrand factor binding to the GP Ib-IX- and GP Ib-IX plus GP V-expressing cells. The binding affinity of von Willebrand factor to GP Ib-IX was not affected by the presence of GP V. However, the binding capacity of the cells was markedly increased by the presence of GP V. Thus, we suggest that GP V serves to increase the binding of von Willebrand factor by regulating the expression of the GP Ib-IX complex on the surface of the cells. In the present study, the binding of von Willebrand factor was induced by botrocetin; in vivo, the binding of von Willebrand factor to platelets is induced by immobilization of von Willebrand factor and is very dependent on shear forces (for review, see Refs. 54 and 55). While we could not detect an effect of GP V on the binding affinity of von Willebrand factor in the present study, the interaction of GP Ib-IX with von Willebrand factor is especially important at high wall shear conditions (56). It appears possible that GP V might affect the accessibility of von Willebrand factor for the binding site on GP Ib differently in a shear dependent system. Future studies will be needed to address this possibility and to elucidate additional functions of GP V.

  
Table: Amount of GP V secreted in the medium and bound to the cells in the presence or absence of GP Ib-IX

Stably transfected cells expressing GP V or GP V and GP Ib-IX were plated in six-well tissue culture dishes (10 cells/2 ml culture medium/well). The cell supernatant was collected after 72 h, centrifuged at 1,000 g for 10 min to eliminate dead cells, and denaturated in Laemmli sample buffer. The cells were harvested with EDTA, counted, and solubilized in Laemmli sample buffer. The amount of GP V associated with the cells or present in the medium was determined on Western blots using a serum against GP V and quantitated by densitometry. Values shown are the mean (± S.D.) from the number of determinations shown.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL30657. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel: 510-428-3510; Fax: 510-428-3608.

The abbreviations used are: GP, glycoprotein; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; PBS, phosphate-buffered saline; vWf, von Willebrand factor.


ACKNOWLEDGEMENTS

We are grateful to Drs. C. Cunningham and J. Hartwig for providing the melanoma cells; Dr. J. López for the constructs coding for GP Ib-IX; Dr. F. Lanza for the GP V cDNA; Drs. B. Steiner, D. Phillips, P. Modderman, and K. Fujimura for antibodies; and Dr. J. Moake for purified vWf. We thank Dr. S. Ruzin for assistance with confocal microscopy and W. Hyun for assistance with cell sorting. We are grateful to S. Zuerbig for technical assistance and graphic services, G. Santos for his help with the confocal microscopy, and Drs. P. Gascard and J. Cunningham for critical review of the manuscript.


REFERENCES
  1. Weiss, H. J., Tschopp, T. B., Baumgartner, H. R., Sussman, I. I., Johnson, M. M., and Egan, J. J.(1974) Am. J. Med.57, 920-925 [Medline] [Order article via Infotrieve]
  2. Weiss, H. J., Turitto, V. T., and Baumgartner, H. R.(1978) J. Lab. Clin. Med.92, 750-764 [Medline] [Order article via Infotrieve]
  3. George, J. N., Nurden, A. T., and Phillips, D. R.(1984) N. Engl. J. Med.311, 1084-1098 [Abstract]
  4. Berndt, M. C., and Caen, J. P.(1984) in Progress in Hemostasis and Thrombosis (Spaet, T. H., ed) Vol. 7, pp. 111-150, Grune & Stratton, Orlando, FL [Medline] [Order article via Infotrieve]
  5. Phillips, D. R., and Agin, P. P.(1977) J. Biol. Chem.252, 2121-2126 [Medline] [Order article via Infotrieve]
  6. Berndt, M. C., Gregory, C., Kabral, A., Zola, H., Fournier, D., and Castaldi, P. A.(1985) Eur. J. Biochem.151, 637-649 [Abstract]
  7. López, J. A., Chung, D. W., Fujikawa, K., Hagen, F. S., Papayannopoulou, T., and Roth, G. J.(1987) Proc. Natl. Acad. Sci. U. S. A.84, 5615-5619 [Abstract]
  8. López, J. A., Chung, D. W., Fujikawa, K., Hagen, F. S., Davie, E. W., and Roth, G. J.(1988) Proc. Natl. Acad. Sci. U. S. A.85, 2135-2139 [Abstract]
  9. Hickey, M. J., Williams, S. A., and Roth, G. J.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 6773-6777 [Abstract]
  10. Shimomura, T., Fujimura, K., Maehama, S., Takemoto, M., Oda, K., Fujimoto, T., Oyama, R., Suzuki, M., Ichihara-Tanaka, K., Titani, K., and Kuramoto, A.(1990) Blood75, 2349-2356 [Abstract]
  11. Roth, G. J., Church, T. A., McMullen, B. A., and Williams, S. A.(1990) Biochem. Biophys. Res. Commun.170, 153-161 [Medline] [Order article via Infotrieve]
  12. Lanza, F., Morales, M., de La Salle, C., Cazenave, J. P., Clemetson, K. J., Shimomura, T., and Phillips, D. R.(1993) J. Biol. Chem.268, 20801-20807 [Abstract/Free Full Text]
  13. Hickey, M. J., Hagen, F. S., Yagi, M., and Roth, G. J.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 8327-8331 [Abstract/Free Full Text]
  14. Nurden, A. T., Dupuis, D., Kunicki, T. J., and Caen, J. P.(1981) J. Clin. Invest.67, 1431-1440 [Medline] [Order article via Infotrieve]
  15. Clemetson, K. J., McGregor, J. L., James, E., Dechavanne, M., and Luscher, E. F.(1982) J. Clin. Invest.70, 304-311 [Medline] [Order article via Infotrieve]
  16. Berndt, M. C., Gregory, C., Chong, B. H., Zola, H., and Castaldi, P. A. (1983) Blood62, 800-807 [Abstract]
  17. Clemetson, J. M., Kyrle, P. A., Brenner, B., and Clemetson, K. J. (1994) Blood84, 1124-1131 [Abstract/Free Full Text]
  18. Simsek, S., Admiraal, L. G., Modderman, P. W., van der Schoot, C. E., and von dem Borne, A. E. G. K.(1994) Thromb. Haemostasis72, 444-449 [Medline] [Order article via Infotrieve]
  19. Modderman, P. W., Admiraal, L. G., Sonnenberg, A., and von dem Borne, A. E. G. K.(1992) J. Biol. Chem.267, 364-369 [Abstract/Free Full Text]
  20. Fox, J. E. B.(1985) J. Clin. Invest.76, 1673-1683 [Medline] [Order article via Infotrieve]
  21. Fox, J. E. B.(1985) J. Biol. Chem.260, 11970-11977 [Abstract/Free Full Text]
  22. Okita, J. R., Pidard, D., Newman, P. J., Montgomery, R. R., and Kunicki, T. J.(1985) J. Cell Biol.100, 317-321 [Abstract]
  23. Wicki, A. N., and Clemetson, K. J.(1987) Eur. J. Biochem.163, 43-50 [Abstract]
  24. Fox, J. E. B., Boyles, J. K., Berndt, M. C., Steffen, P. K., and Anderson, L. K.(1988) J. Cell Biol.106, 1525-1538 [Abstract]
  25. Ezzell, R. M., Kenney, D. M., Egan, S., Stossel, T. P., and Hartwig, J. H.(1988) J. Biol. Chem.263, 13303-13309 [Abstract/Free Full Text]
  26. Phillips, D. R., and Agin, P. P.(1977) Biochem. Biophys. Res. Commun.75, 940-947 [Medline] [Order article via Infotrieve]
  27. Mosher, D. F., Vaheri, A., Choate, J. J., and Gahmberg, C. G.(1979) Blood53, 437-445 [Abstract]
  28. Berndt, M. C., and Phillips, D. R.(1981) J. Biol. Chem.256, 59-65 [Abstract/Free Full Text]
  29. Fox, J. E. B.(1994) Blood Coagulation and Fibrinolysis5, 291-304 [Medline] [Order article via Infotrieve]
  30. Cunningham, C. C., Gorlin, J. B., Kwiatkowski, D. J., Hartwig, J. H., Janmey, P. A., Byers, H. R., and Stossel, T. P.(1992) Science255, 325-327 [Medline] [Order article via Infotrieve]
  31. López, J. A., Leung, B., Reynolds, C. C., Li, C. Q., and Fox, J. E. B.(1992)J. Biol. Chem.267, 12851-12859 [Abstract/Free Full Text]
  32. Graham, F. L., and van der Eb, A. J.(1973) Virology52, 456-467 [Medline] [Order article via Infotrieve]
  33. Hawley-Nelson, P., Ciccarone, V., Gebeyehu, G., Jessee, J., and Felgner, P. L.(1993) Focus (Gaithersburg, MD) 15, 73-79
  34. Meyer, S., Kresbach, G., Haring, P., Schumpp-Vonach, B., Clemetson, K. J., Hadváry, P., and Steiner, B.(1993) J. Biol. Chem.268, 20555-20562 [Abstract/Free Full Text]
  35. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  36. Towbin, H., Staehelin, T., and Gordon, J.(1979) Proc. Natl. Acad. Sci. U. S. A.76, 4350-4354 [Abstract]
  37. Moake, J. L., Turner, N. A., Stathopoulos, N. A., Nolasco, L. H., and Hellums, J. D.(1986) J. Clin. Invest.78, 1456-1461 [Medline] [Order article via Infotrieve]
  38. Marchalonis, J. J.(1969) Biochem. J.113, 299-305 [Medline] [Order article via Infotrieve]
  39. Fox, J. E. B., Aggerbeck, L. P., and Berndt, M. C.(1988) J. Biol. Chem.263, 4882-4890 [Abstract/Free Full Text]
  40. Wicki, A. N., and Clemetson, K. J.(1985) Eur. J. Biochem.153, 1-11 [Abstract]
  41. Handa, M., Titani, K., Holland, L. Z., Roberts, J. R., and Ruggeri, Z. M.(1986) J. Biol. Chem.261, 12579-12585 [Abstract/Free Full Text]
  42. Vicente, V., Kostel, P. J., and Ruggeri, Z. M.(1988) J. Biol. Chem.263, 18473-18479 [Abstract/Free Full Text]
  43. Vicente, V., Houghten, R. A., and Ruggeri, Z. M.(1990) J. Biol. Chem.265, 274-280 [Abstract/Free Full Text]
  44. Katagiri, Y., Hayashi, Y., Yamamoto, K., Tanoue, K., Kosaki, G., and Yamazaki, H.(1990) Thromb. Haemostasis63, 122-126 [Medline] [Order article via Infotrieve]
  45. Von dem Borne, A. E. G. K., Modderman, P. W., Admiraal, L. G., and Nieuwenhuis, H. K.(1989) in Leucocyte Typing IV (Knapp, W., Dorken, B., Gilks, W. R., Rieber, E. P., Schmidt, R. E., Stein, H., and von dem Borne, A. E. G. K., eds) pp. 951-966, Oxford University Press, Oxford
  46. Du, X., Beutler, L., Ruan, C., Castaldi, P. A., and Berndt, M. C. (1987) Blood69, 1524-1527 [Abstract]
  47. Coller, B. S., Peerschke, E. I., Scudder, L. E., and Sullivan, C. A. (1983) Blood61, 99-110 [Abstract]
  48. López, J. A., Weisman, S., Sanan, D. A., Sih, T., Chambers, M., and Li, C. Q.(1994) J. Biol. Chem.269, 23716-23721 [Abstract/Free Full Text]
  49. Kobe, B., and Deisenhofer, J.(1994) Trends Biochem. Sci.19, 415-420 [CrossRef][Medline] [Order article via Infotrieve]
  50. Kroll, M. H., Harris, T. S., Moake, J. L., Handin, R. I., and Schafer, A. I.(1991) J. Clin. Invest.88, 1568-1573 [Medline] [Order article via Infotrieve]
  51. Chow, T. W., Hellums, J. D., Moake, J. L., and Kroll, M. H.(1992) Blood80, 113-120 [Abstract]
  52. Ikeda, Y., Handa, M., Kamata, T., Kawano, K., Kawai, Y., Watanabe, K., Kawahami, K., Sakai, K., Fukuyama, M., Itagaki, I., Yoshioka, A., and Ruggeri, Z. M.(1993) Thromb. Haemostasis69, 496-502 [Medline] [Order article via Infotrieve]
  53. Jackson, S. P., Schoenwaelder, S. M., Yuan, Y., Rabinowitz, I., Salem, H. H., and Mitchell, C. A.(1994) J. Biol. Chem.269, 27093-27099 [Abstract/Free Full Text]
  54. Roth, G. J.(1991) Blood77, 5-19 [Medline] [Order article via Infotrieve]
  55. Booth, W. J., Andrews, R. K., Castaldi, P. A., and Berndt, M. C.(1990) Platelets1, 169-176
  56. Turitto, V. T., Muggli, R., and Baumgartner, H. R.(1977) Ann. N. Y. Acad. Sci.283, 284-292

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.