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. Author manuscript; available in PMC: 2009 Oct 1.
Published in final edited form as: J Thromb Haemost. 2008 Jul 19;6(10):1789–1795. doi: 10.1111/j.1538-7836.2008.03088.x

The membrane-proximal intermolecular disulfide bonds in glycoprotein Ib influence the receptor binding to von Willebrand factor

Xi Mo 1, Shi-Zhong Luo 1, Adam D Munday 3, Wensheng Sun 2, Michael C Berndt 4, Jose A Lopez 3, Jing-fei Dong 2, Renhao Li 1
PMCID: PMC2671080  NIHMSID: NIHMS99768  PMID: 18647229

Summary

Background

In the platelet glycoprotein (GP) Ib-IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted in the N-terminal domain of the GPIbα subunit. How the other subunits in the complex, GPIbβ and GPIX, regulate the GPIbα-VWF interaction is not clear.

Objectives and Methods

Since GPIbα connects with two GPIbβ subunits via disulfide bonds, we tested whether these inter-subunit covalent links were important to the proper VWF-binding activity of the GPIb-IX complex by characterizing the structure and VWF-binding activity of a mutant GPIb-IX complex that lacked the GPIbα/GPIbβ disulfide bonds.

Results

Mutating both Cys484 and Cys485 of GPIbα to serine prevents GPIbα from forming covalent disulfide bonds with GPIbβ, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIbβ to form a disulfide bond between themselves. Compared to Chinese hamster ovary (CHO) cells stably expressing the wild-type GPIb-IX complex at a comparable level, CHO cells stably expressing the mutant GPIb-IX complex bind to significantly less soluble VWF in the presence of ristocetin and roll on the immobilized VWF under flow with faster velocity.

Conclusions

The disulfide bonds between GPIbα and GPIbβ are necessary for optimal GPIbα binding to VWF. The structural plasticity around the disulfide bonds may also help shed light on the inside-out mechanism underlying GPIbβ modulation of VWF binding.

Keywords: GPIb-IX, membrane-proximal disulfide bonds, VWF binding, inside-out regulation

Introduction

The platelet glycoprotein (GP) Ib-IX complex is an adhesion receptor complex that helps to initiate platelet activation and aggregation under hemostatic and thrombotic conditions[1,2]. A basic feature of the GPIb-IX complex is the restriction of its ligand-binding site to the N-terminal membrane-distal domain in the GPIbα subunit[3–6]. Although the other subunits in the complex GPIbβ and GPIX do not participate in direct binding to VWF, GPIbβ, including its cytoplasmic domain, can modulate the VWF-binding activity of GPIbα[7–12]. Although the underlying molecular mechanism remains unknown, such trans-subunit modulation should entail communication through the interaction interface between GPIbβ and GPIbα.

Other than the two disulfide bonds linking GPIbβ and GPIbα, how they interact with each other is not entirely clear. Without GPIbβ and GPIX, expression of GPIbα in platelets or transfected mammalian cells is compromised, suggesting that GPIbα needs to assemble with GPIbβ and GPIX in the endoplasmic reticulum before being transported to the plasma membrane[13,14]. Recent studies have thus concentrated on identifying the sequences in GPIbβ that are required for efficient GPIbα expression on the cell surface, with the premise that some of these sequences may be directly involved in the interaction with GPIbα. It was recently shown that deletion or replacement of the transmembrane and/or cytoplasmic domains of GPIbβ led to drastic decrease in GPIbα expression in platelets or transfected cells[15–17]. Further analysis has revealed the transmembrane domain as an interaction site between GPIbβ and GPIbα. Peptides corresponding to the GPIbα, GPIbβ and GPIX transmembrane domains interact specifically with one another in detergent micelles (Luo and Li, manuscript submitted). The interaction facilitates formation of the membrane-proximal disulfide bonds between GPIbα and two GPIbβ subunits[18,19]. Mutations in the transmembrane domains that hampered their association resulted in misassembly and/or decreased expression of the GPIb-IX complex[17,19]. Although the extracellular domain of GPIbβ is also required for efficient GPIbα expression, it is largely attributed to its interaction with that of GPIX[20], in which residues 15–32 of GPIbβ were found to participate[21]. It is not clear whether the extracellular domain of GPIbβ directly interacts with that of GPIbα. Nonetheless, the two disulfide bonds linking GPIbα and GPIbβ are covalent bonds and therefore the strongest connections between them. In this study, we have explored whether perturbing the GPIbα/GPIbβ interaction by removing the GPIbα/GPIbβ disulfide bonds would impact on VWF binding of the GPIb-IX complex.

Materials and Methods

Reagents and cell lines

Monoclonal antibodies against individual subunits of the GPIb-IX complex, including WM23 (isotype IgG1), SZ2 (IgG1), AK2 (IgG1), AN51 (IgG2a) and FMC25 (IgG1), were either obtained from the hybridoma cell line or purchased from Beckman Coulter, Dako, Chemicon or Santa Cruz Biotechnology. Human VWF and ristocetin were purchased from American Diagnostica Inc. and Sigma Diagnostics, respectively.

The pDX vector encoding the wild-type or C484S/C485S mutant GPIbα cDNA was co-transfected with the pREP-4 vector (Invitrogen) into CHOβIX cells that stably express GPIbβ and GPIX[14,22]. Transfected cells were sorted for GPIbα expression and cultured as described[22].

Structural characterization of the GPIb-IX complex

Protein expression and disulfide formation were detected by flow cytometry and Western blot as previously described[18]. For flow cytometry, the cells were stained with 2 μg/mL primary antibody for 30 min at room temperature and with FITC-conjugated secondary antibody for 25 min. For immunoprecipitation, the cells were lysed in the buffer containing either 1% digitonin or 1% triton X-100, and the soluble portion was treated with desired antibodies and protein G-agarose beads as described[19].

GPIb-VWF interaction

Ristocetin-induced binding of transfected CHO cells with human VWF was detected as described[23,24]. Briefly, cells resuspended in phosphate buffered saline containing 0.5% bovine serum albumin at 106 cells/ml were incubated with human VWF of various concentrations and 1mg/ml ristocetin. The mixture was vortexed for 2 min for VWF binding and the unbound VWF was subsequently removed by centrifugation. The amount of VWF bound to the surface of CHO cells was measured by flow cytometry after anti-VWF antibody staining.

Rolling of GPIb-expressing CHO cells on VWF-coated surface under flow conditions was detected in a parallel-plate flow chamber[24,25]. Cells in suspension were perfused through the chamber and allowed to settle for 2 minutes on the coverslip coated with 30 μg/mL VWF before resumption of the flow. Rolling of cells on immobilized VWF under various wall shear stresses was recorded by 61 frames in 2 minutes and analyzed with MetaMorph software (Universal Images, West Chester, PA). Groups were compared using the nonpaired t test.

Results

Generation of stable cell lines

Since both Cys484 and Cys485 of GPIbα form disulfide bonds with GPIbβ in the GPIb-IX complex[18], two GPIbα cDNAs, wild type (GPIbαCC) and the C484S/C485S mutant (GPIbαSS), were transfected separately into CHO cells stably expressing GPIbβ and GPIX (CHOβIX cells). The resulting two cell lines stably expressing GPIbαCC and GPIbαSS are referred in this paper as CHOαCCβIX and CHOαSSβIX cells, respectively.

Expression of the GPIb-IX complex in both cell lines was first assessed by flow cytometry. The conformation-insensitive monoclonal antibody WM23 binds to an epitope in the macroglycopeptide region of GPIbα[26], and it has been widely used as a standard to measure the level of GPIbα expressed on the cell surface[24,25]. Both CHOαCCβIX and CHOαSSβIX cells bound to comparable amount of WM23, indicating very similar, if not the same, levels of GPIbα expression in both cells (Fig. 1A). GPIX was stably expressed on the surface of CHOβIX cells as expected[14,22]. Transfection of GPIbαCC and GPIbαSS into CHOβIX cells both significantly enhanced expression of GPIX (Fig. 1A). Moreover, the increased expression level of GPIX assessed by monoclonal antibody FMC25 was approximately the same in both cell lines, indicating that the double cysteine mutation in GPIbα did not affect expression of GPIX on the cell surface.

Figure 1. GPIbαSS containing the C484S/C485S is well expressed and takes on largely native-like conformation in transfected CHO cells.

Figure 1

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(A) Comparable expression levels of the mutant GPIb-IX complex expressed in CHOαSSβIX cells (dashed line) and the wild type complex in CHOαCCβIX cells (solid line). Surface expression of GPIbα and GPIX were measured by flow cytometry with monoclonal antibodies WM23 and FMC25, respectively. Dark grey peak: CHO cells; light grey peak: CHOβIX cells. (B,C) Conformation of GPIbαSS expressed in CHOαSSβIX cells is largely native-like as the wild type in CHOαCCβIX cells. GPIbα expressed in transfected CHO cells were stained with the indicated conformation-sensitive monoclonal antibodies and measured by flow cytometry. Representative histograms of antibody binding was shown in (B). The mean fluorescence intensities (MFI) were measured from 4–6 independent experiments, normalized to the wild type level and presented as the mean ± SD (C). Groups were compared using the non-paired t test; **, p < 0.01. Binding of mutant GPIbαSS to AK2, another conformation-sensitive antibody, was significantly increased compared to the wild type GPIbαCC, suggesting a local conformational change around GPIbα residues 35– 59, the epitope region recognized by AK2.

We also probed the conformation of GPIbα expressed in transfected CHO cells using conformation-sensitive antibodies. Antibodies SZ2, AN51 and AK2 recognize distinctly the conformation around the ligand-binding domain of GPIbα[27,28]. As shown in Fig. 1B, both CHOαCCβIX and CHOαSSβIX cells bound to these monoclonal antibodies at similar levels, which suggested that most, if not all, of the mutant GPIbαSS expressed on the cell surface was well folded and took on native-like conformation as GPIbαCC. On the other hand, the conformation of GPIbαSS may not be indistinguishable from that of GPIbαCC, because a modest but reproducible difference was observed in their binding to AK2 (Fig. 1B).

Structural changes in the GPIb-IX complex caused by the C484S/C485S mutation

Our determination of the mutational effects on the structure and integrity of GPIb-IX complex started with the GPIbα/GPIbβ disulfide bonds. Cell lysates of CHOαCCβIX and CHOαSSβIX cells were resolved in SDS-PAGE and GPIbα visualized by WM23 blotting (Fig. 2). Under reducing conditions, GPIbαSS migrated in the SDS gel at the same speed as wild type GPIbαCC, indicating that neither synthesis nor subsequent glycosylation of GPIbα was altered by the mutation. Under non-reducing conditions, GPIbαCC migrated slower in the SDS gel than it did under reducing conditions because it was disulfide-linked to GPIbβ in CHOαCCβIX cells. Unlike GPIbαCC, no apparent difference was observed for GPIbαSS in SDS gel under non-reducing or reducing conditions. Thus, the C484S/C485S mutation deprived GPIbαSS of its ability to form disulfide bonds with GPIbβ in stably transfected CHO cells.

Figure 2. The C484S/C485S double cysteine mutation in GPIbα abolishes formation of disulfide bonds between GPIbα and GPIbβ in transfected CHO cells.

Figure 2

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Lysates from CHOαCCβIX (CC) and CHOαSSβIX (SS) cells were resolved in a SDS gel under non-reducing (N.R.) or reducing (R.) conditions, and the blotted membrane was probed with WM23. The figure is a representative of 5 independent experiments.

We next investigated whether the loss of disulfide bonds between GPIbαSS and GPIbβ disrupted assembly of the GPIb-IX complex. When CHOαSSβIX cells were lysed in the buffer containing 1% digitonin, both GPIbαSS and GPIbβ were co-precipitated by anti-GPIX antibody FMC25, indicating that even with the double cysteine mutation the integrity of the GPIb-IX complex was preserved (Fig. 3A). By contrast, only GPIbβ, but not GPIbαSS, was co-precipitated in the cell lysis buffer containing 1% triton X-100 (Fig. 3B). The differential abilities of digitonin and triton X-100 in maintaining the integrity of the mutant GPIb-IX complex were consistent with an earlier study documenting the contribution of polar residues in transmembrane domains to the GPIbα/GPIbβ interaction[19], because digitonin, but not triton X-100, can preserve the polar-polar interactions between transmembrane domains[29,30]. The association between GPIbβ and GPIX in the mutant complex survived treatment of both detergents, because the interaction among the extracellular domains of GPIbβ and GPIX[21] is unlikely to be affected by nonionic detergents. The largely maintained integrity of the mutant complex, especially the non-covalent interactions among the transmembrane domains of the three subunits, led to efficient surface expression of the complex. Overall, these observations led us to conclude that, in CHOαSSβIX cells, although the covalent links between GPIbα and GPIbβ were abolished by the C484S/C485S double mutation, the non-covalent interaction were largely in place to hold the GPIb-IX complex together.

Figure 3. Structural integrity of the mutant GPIb-IX complex.

Figure 3

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Cell lysates from CHOαCCβIX (CC) and CHOαSSβIX (SS) cells were immunoprecipitated with FMC25 in the buffer containing either 1% digitonin (A) or 1% triton X-100 (B), and resolved in a SDS gel under reducing conditions. After transfer, the membrane was probed separately for GPIbα, or with polyclonal antibodies for GPIbβ or GPIX. Each figure is a representative of 3–4 independent experiments.

Although GPIbαSS in the mutant GPIb-IX complex was unable to form any inter-subunit disulfide bonds because of the mutation, GPIbβ, devoid of any mutations, was still capable of forming a disulfide bond with another molecule. To determine the redox state of GPIbβ in the mutant complex, CHOαSSβIX cell lysate was immunoprecipitated with WM23 in the presence of 1% digitonin, resolved by SDS-PAGE under non-reducing conditions and eventually immunoblotted for GPIbβ with a polyclonal antibody. As controls, the same lysate was either directly loaded for electrophoresis or treated separately with anti-GPIX antibody and mouse IgG. As shown in Fig. 4A, all GPIbβ that associated with GPIbα was present in a band with a molecular mass of approximate 45 kDa. This band was also the major species associated with GPIX. Additional bands were also present for the GPIX-immunoprecipitated sample, which likely contain non-native GPIbβ that accumulated in the cytoplasm. Since the apparent molecular mass of GPIbβ monomer in SDS gel is about 25 kDa, much smaller than the observed 45 kDa, GPIbβ must form a disulfide bond with another protein in this 45-kDa complex. To determine the identity of the other protein, the same immunoprecipitated sample was resolved in a non-reducing/reducing 2-dimensional SDS gel and all the proteins visualized by silver staining (Fig. 4B–C). Under reducing conditions, the 45-kDa band was converted to only one band with an approximate 25-kDa molecular mass, which could be recognized by anti-GPIbβ antibody (Fig. 4D). Thus, we concluded that the 45-kDa protein band is made of two GPIbβ molecules connected via a disulfide bond. In other words, without their native disulfide-linked partner in GPIbα, two GPIbβ subunits in the mutant GPIb-IX complex formed a disulfide bond between themselves. Due to the strict spatial constraint of a disulfide bond, the change of inter-subunit disulfide bonds induced by the double cysteine mutation should induce additional structural changes around the affected cysteine residues and in the nearby macroglycopeptide and transmembrane regions.

Figure 4. Formation of a disulfide-linked GPIbβ dimer in the GPIb-IX complex containing the C484S/C485S mutation.

Figure 4

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(A) GPIbβ in the mutant GPIb-IX complex is linked to another protein through a disulfide bond. Cell lysate from CHOαSSβIX cells was first co-immunoprecipitated with antibodies against GPIbα (Ibα lane), GPIX (IX lane) or mouse IgG (IgG lane) in the lysis buffer containing 1% digitonin, and then resolved in a SDS gel under non-reducing conditions. For comparison, 20% of the cell lysate used in immunoprecipitation was loaded directly in the same SDS gel ( lane). After transfer, the membrane was probed with a polyclonal anti-GPIbβ antibody. Note that only a band with a molecular mass of approximate 45kDa was present in the “Ibα” lane, and the apparent mass of GPIbβ monomer is approximately 25kDa. (B–D) GPIbβ in the mutant GPIb-IX complex is linked to another GPIbβ via a disulfide bond. CHOαSSβIX cell membrane including the embedded proteins was separated from the cytosolic fraction and co-immunoprecipitated by anti-GPIX antibody SZ1. The precipitated fraction was first resolved in a 4–12% Bis-Tris precast SDS gel (Invitrogen) under non-reducing conditions. The gel strip was either (B) directly stained by silver staining or extracted, incubated at 65 °C for 20 min in the SDS sample buffer containing 20% DTT and placed on top of a 12% Tris-glycine SDS gel for electrophoresis under reducing conditions. The proteins in the gel were either (C) visualized by silver staining or (D) transferred to a PVDF membrane and probed with anti-GPIbβ polyclonal antibody. The diagonal molecular weight markers in the 2-D gel were apparent in (C) and marked in (D). The dashed line highlights the positions of the 45-kDa protein complex in the 2-D gel. Silver staining showed that GPIbβ was the only protein in this 45-kDa complex. The protein complex with an apparent mass of approximate 150kDa in (B) is the antibody used for immunoprecipitation and used as the positive control for the 2-D gel. Each figure is a representative of at least 2 independent experiments.

VWF binding to CHOαSSβIX cells is decreased under static and flow conditions

With the changes in complex structure caused by the C484S/C485S double mutation elucidated, we next explored whether the mutation altered the principal function of the GPIb-IX complex binding of VWF. Since stable CHO cell lines have been successfully used to characterize the VWF-binding activity of mutant GPIb-IX complex[7,9,10,23,24,31], here we compared the VWF-binding activities of CHOαCCβIX and CHOαSSβIX cells using two assays. In the first assay, purified human VWF was added to CHO cells resuspended in 1 mg/mL ristocetin, and the VWF bound to the cell was stained with anti-VWF antibody and quantitated by flow cytometry. To enable direct comparison of VWF-binding ability of the GPIb-IX complex expressed in different CHO cell lines, measured VWF binding were normalized against surface expression of GPIbα in the same CHO cells as indicated by WM23 binding. As shown in Fig. 5, mutant CHOαSSβIX cells bound VWF in a dose-dependent manner as CHOαCCβIX cells, but the binding was significantly decreased compared to the latter.

Figure 5. Ristocetin-induced binding of soluble VWF to transfected CHO cells.

Figure 5

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The binding was detected by flow cytometry, and plotted as MFI versus VWF concentration. MFI values of CHOαCCβIX and CHOαSSβIX cells were normalized against the GPIbα expression level as indicated by WM23 binding. All data are presented as the mean ± SD from 4 independent experiments. Groups were compared using the nonpaired t test; *, p < 0.05; **, p < 0.01.

In the second assay, VWF was immobilized on the glass coverslip and the suspension of CHOαCCβIX and CHOαSSβIX cells were allowed to roll on immobilized VWF at various flow rates. The flow rates were chosen to mimic shear stresses in typical venous and arterial vessels[25]. Individual cells rolling in the parallel-plate flow chamber were tracked by microscopy and recorded for analysis. The cell rolling velocity observed in our experiments correlated well with binding of VWF to the GPIb-IX complex expressed in these cells[23]. CHOβIX cells did not express GPIbα on their surface, so they could not tether to VWF-coated surface (not shown). Both CHOαSSβIX and CHOαCCβIX cells could roll on immobilized VWF, with the former at a significantly faster rolling rate than the latter under all shear flow conditions tested (Fig. 6). Overall, these results showed that the binding of the mutant GPIb-IX complex expressed in CHOαSSβIX cells to VWF was significantly decreased compared to the wild type.

Figure 6. Rolling of CHOαCCβIX (black bar) and CHOαSSβIX (white bar) cells on the VWF-coated surface under flow conditions.

Figure 6

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The cells were perfused into the parallel-plate flow chamber and settled for 2 minutes on the VWF-coated coverslip before being subjected to various flow shear stresses. CHOβIX cells did not tether on the VWF-coated coverslip (not shown). The rolling velocity was defined as the distance a cell travels during a defined period. The data are presented as the mean ± SEM from measurements of 92–174 cells under each condition. Groups were compared using the nonpaired t test; **, p < 0.01.

Discussion

In this study, we have investigated the impact of inter-subunit disulfide bonds on the VWF-binding ability of the GPIb-IX complex using a well-established transfected cell model[9,23]. Although removing these membrane-proximal disulfide bonds through the C484S/C485S mutation in GPIbα clearly weakened association between GPIbα and GPIbβ, the complex integrity was largely maintained. Binding of VWF to the mutant complex expressed in CHO cells was significantly decreased under both static and flow conditions. Therefore, the disulfide bonds between GPIbα and GPIbβ are required for optimal VWF binding of the GPIb-IX complex.

The decreased binding of VWF to the mutant GPIb-IX complex expressed in CHO cells was not due to a decrease in the number of GPIbα available for VWF binding, because the expression level of GPIbαSS in CHOαSSβIX cells was comparable to that of the wild type. Moreover, the C484S/C485S mutation did not cause misfolding of the ligand-binding domain in GPIbαSS as GPIbαSS could readily bind to multiple conformation-sensitive monoclonal antibodies at a similar level as the wild type complex. It was noteworthy that the binding of GPIbαSS to AK2, also a conformation-sensitive monoclonal antibody, was markedly higher than the wild type, suggesting that the mutation may induce a local alteration in the conformation at or around GPIbα residues 35–59, the epitope recognized by AK2[28]. The double cysteine mutation in GPIbαSS also led to formation of a new disulfide bond between two GPIbβ subunits in the mutant GPIb-IX complex, indicating a likely conformational rearrangement around the disulfide bonds near the membrane. Thus, although the molecular mechanism underlying the decrease in binding of VWF to the mutant GPIb-IX complex requires further clarification, it appears to entail conformational changes in regions around membrane-proximal disulfide bonds between GPIbα and GPIbβ as well as the membrane-distal ligand-binding domain in GPIbα.

Several earlier studies have documented the involvement of cytoplasmic domains of GPIbα and GPIbβ in regulating the GPIbα/VWF interaction, thus uncovering an inside-out regulatory mechanism for the GPIb-IX complex[7,9–11]. Moreover, perturbing the conformation of the extracellular regions near the cell membrane, through either sequence alteration in GPIbα[31] or antibody binding to GPIbβ[12], can influence the VWF-binding activity of the complex. Here, we have provided evidence suggesting that perturbing the conformation at disulfide bonds linking GPIbα and GPIbβ may also modulate VWF binding. Since these disulfide bonds are located adjacent to transmembrane domains of the GPIb-IX complex and between the cytoplasmic and extracellular regions implicated in ligand binding regulation, we speculate that inside-out regulation of VWF binding may be governed by a common allosteric pathway that traverses all the regions discussed above.

If the putative allosteric regulation in the GPIb-IX complex takes on the form of a puzzle, the cross-talk between GPIbα and GPIbβ must be an essential part of its solution. Such cross-talk is difficult to probe because it is important for efficient expression of GPIbα in the plasma membrane[17,19]. Interfering with the GPIbα/GPIbβ interaction by mutagenesis often significantly decrease GPIbα expression, rendering any interpretation of mutational effects on receptor function defective. But this may not always be the case. In this study, abolishing disulfide bonds between GPIbα and GPIbβ by the double cysteine mutation in GPIbα clearly perturbed the GPIbα/GPIbβ interaction, exemplified by the new disulfide bond between two GPIbβ in the complex. However, GPIbα expression was not compromised by the mutation as overall integrity of the complex was maintained. Details of changes in the structure and possibly the surface distribution of the mutant complex, and how these changes alter VWF-binding activity, await further investigation. Nonetheless, our study here have validated an approach through which more elaborate studies can be designed and carried out to probe directly the GPIbα/GPIbβ interaction and to elucidate the regulatory mechanism of the GPIb-IX complex.

Acknowledgments

We thank Dr. Yuandong Peng for help with the rolling assay on VWF surface. This work was supported by grants from the National Institutes of Health (HL082808), the American Heart Association (0565078Y), and the Welch Foundation (AU-1581). X.M. was a recipient of the Harry S. and Isabel C. Cameron Foundation Fellowship. R.L. was supported in part by the Howard Temin Award (CA096706).

Footnotes

Addendum

X.M., J.F.D. and R.L. designed research, X.M., S.Z.L., and W.S. performed research, A.D.M., M.C.B. and J.A.L. contributed vital reagents, X.M., S.Z.L., J.F.D. and R.L. analyzed data, X.M. and R.L. wrote the paper.

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