Binding Mechanism between Platelet Glycoprotein and Cyclic Peptide Elucidated by McMD-Based Dynamic Docking.

The cyclic peptide OS1 (amino acid sequence: CTERMALHNLC), which has a disulfide bond between both termini cysteine residues, inhibits complex formation between the platelet glycoprotein Ibα (GPIbα) and the von Willebrand factor (vWF) by forming a complex with GPIbα. To study the binding mechanism between GPIbα and OS1 and, therefore, the inhibition mechanism of the protein-protein GPIbα-vWF complex, we have applied our multicanonical molecular dynamics (McMD)-based dynamic docking protocol starting from the unbound state of the peptide. Our simulations have reproduced the experimental complex structure, although the top-ranking structure was an intermediary one, where the peptide was bound in the same location as in the experimental structure; however, the ß-switch of GPIbα attained a different conformation. Our analysis showed that subsequent refolding of the ß-switch results in a more stable binding configuration, although the transition to the native configuration appears to take some time, during which OS1 could dissociate. Our results show that conformational changes in the ß-switch are crucial for successful binding of OS1. Furthermore, we identified several allosteric binding sites of GPIbα that might also interfere with vWF binding, and optimization of the peptide to target these allosteric sites might lead to a more effective inhibitor, as these are not dependent on the ß-switch conformation.

Structure-Based Cyclic Glycoprotein Ibα-Derived Peptides Interfering with von Willebrand Factor-Binding, Affecting Platelet Aggregation under Shear.

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26-34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.

Adhesion G protein-coupled receptors-Candidate metabotropic mechanosensors and novel drug targets.

While a wide range of G protein-coupled receptors (GPCR) have emerged as prime targets for pharmacological intervention long ago, a distinct group of GPCR has only recently been identified and become a research subject to fundamental and clinical scientists. Adhesion-type GPCR (aGPCR) are exceptional members of the GPCR superfamily in many aspects: structurally, they appear as chimeric surface molecules that possess signature domains of heptahelical (7TM) and adhesion proteins, many aGPCR are autoproteolytically processed, and several homologues have lately been shown to operate as mechanosensors. Bound together by the recent discovery of tethered agonism in aGPCR, these molecular and functional features have entered first models on how aGPCR are activated. Here, I briefly review recent discoveries pertaining to the role of aGPCR as metabotropic mechanosensors that control a large variety of processes in all major tissue types.

Cryopreserved platelets augment the inflammatory response: role of phosphatidylserine- and P-selectin-mediated platelet phagocytosis in macrophages.

BACKGROUND: Cryopreservation in dimethyl sulfoxide and storage at -80 °C extends the shelf life of platelets to at least 2 years, allowing greater availability in rural and military areas. While cryopreserved platelets (CPPs) have been extensively characterized for coagulation and thrombin generation, reports on the mechanism of adverse reactions to CPPs transfusion are scarce. Here, we tested the hypothesis that CPPs facilitate phagocytosis by Kupffer cells and subsequently promote the inflammatory response in Kupffer cells. STUDY DESIGN AND METHODS: P-selectin expression, glycoprotein Ibα clustering and phosphatidylserine (PS) surface exposure on platelets stored at 22 °C, 4 °C and - 80 °C for 3 days were examined by flow cytometry. The phagocytosis of mepacrine-labeled platelets coincubated with THP-1 cells was examined by flow cytometry and confocal microscopy, and the release of cytokines from THP-1 cells was measured by enzyme-linked immunosorbent assay. RESULTS: CPPs showed a marked enhancement of exposed PS but dramatically reduced glycoprotein Ibα expression and clustering compared with platelets stored at 4 °C. Activation of THP-1 cells was stronger by CPPs than by platelets stored at 22 °C and 4 °C. CPP interference tests using annexin V and anti-P-selectin showed that CPPs induced increases in PS- and P-selectin-mediated phagocytosis, as well as secretion of the proinflammatory cytokine tumor necrosis factor-α, and interleukins IL-1ß and IL-6, but a decrease in transforming growth factor-ß production in THP-1 cells. Surface-exposed PS was more effective than P-selectin for the activation of THP-1 cells. CONCLUSION: CPPs triggered PS and P-selectin-mediated phagocytosis by macrophages and stimulated the inflammatory response of macrophages.

Refrigeration-Induced Binding of von Willebrand Factor Facilitates Fast Clearance of Refrigerated Platelets.

OBJECTIVE: Apheresis platelets for transfusion treatment are currently stored at room temperature because after refrigeration platelets are rapidly cleared on transfusion. In this study, the role of von Willebrand factor (VWF) in the clearance of refrigerated platelets is addressed. APPROACH AND RESULTS: Human and murine platelets were refrigerated in gas-permeable bags at 4°C for 24 hours. VWF binding, platelet signaling events, and platelet post-transfusion recovery and survival were measured. After refrigeration, the binding of plasma VWF to platelets was drastically increased, confirming earlier studies. The binding was blocked by peptide OS1 that bound specifically to platelet glycoprotein (GP)Ibα and was absent in VWF-/- plasma. Although surface expression of GPIbα was reduced after refrigeration, refrigeration-induced VWF binding under physiological shear induced unfolding of the GPIbα mechanosensory domain on the platelet, as evidenced by increased exposure of a linear epitope therein. Refrigeration and shear treatment also induced small elevation of intracellular Ca2+, phosphatidylserine exposure, and desialylation of platelets, which were absent in VWF-/- platelets or inhibited by OS1, which is a monomeric 11-residue peptide (CTERMALHNLC). Furthermore, refrigerated VWF-/- platelets displayed increased post-transfusion recovery and survival than wild-type ones. Similarly, adding OS1 to transgenic murine platelets expressing only human GPIbα during refrigeration improved their post-transfusion recovery and survival. CONCLUSIONS: Refrigeration-induced binding of VWF to platelets facilitates their rapid clearance by inducing GPIbα-mediated signaling. Our results suggest that inhibition of the VWF-GPIbα interaction may be a potential strategy to enable refrigeration of platelets for transfusion treatment.

Molecular basis for unique specificity of human TRAF4 for platelets GPIbß and GPVI.

Tumor necrosis factor (TNF)-receptor associated factor 4 (TRAF4), an adaptor protein with E3-ligase activity, is involved in embryogenesis, cancer initiation and progression, and platelet receptor (GPIb-IX-V complex and GPVI)-mediated signaling for reactive oxygen species (ROS) production that initiates thrombosis at arterial shears. Disruption of platelet receptors and the TRAF4 interaction is a potential target for therapeutic intervention by antithrombotic drugs. Here, we report a crystal structure of TRAF4 (amino acid residues 290∼470) in complex with a peptide from the GPIbß receptor (amino acid residues 177∼181). The GPIbß peptide binds to a unique shallow surface composed of two hydrophobic pockets on TRAF4. Further studies revealed the TRAF4-binding motif Arg-Leu-X-Ala. The TRAF4-binding motif was present not only in platelet receptors but also in the TGF-ß receptor. The current structure will provide a template for furthering our understanding of the receptor-binding specificity of TRAF4, TRAF4-mediated signaling, and related diseases.

The glycocalyx promotes cooperative binding and clustering of adhesion receptors.

Cell adhesion plays a pivotal role in various biological processes, e.g., immune responses, cancer metastasis, and stem cell differentiation. The adhesion behaviors depend subtly on the binding kinetics of receptors and ligands restricted at the cell-substrate interfaces. Although much effort has been directed toward investigating the kinetics of adhesion molecules, the role of the glycocalyx, anchored on cell surfaces as an exterior layer, is still unclear. In this paper, we propose a theoretical approach to study the collective binding kinetics of a few and a large number of binders in the presence of the glycocalyx, representing the cases of initial and mature adhesions of cells, respectively. The analytical results are validated by finding good agreement with our Monte Carlo simulations. In the force loading case, the on-rate and affinity increase as more bonds form, whereas this cooperative effect is not observed in the displacement loading case. The increased thickness and stiffness of the glycocalyx tend to decrease the affinity for a few bonds, while they have less influence on the affinity for a large number of bonds. Moreover, for a flexible membrane with thermally-excited shape fluctuations, the glycocalyx is exhibited to promote the formation of bond clusters, mainly due to the cooperative binding of binders. This study helps to understand the cooperative kinetics of adhesion receptors under physiologically relevant loading conditions and sheds light on the novel role of the glycocalyx in cell adhesion.

Assessment of neonatal platelet adhesion, activation, and aggregation.

BACKGROUND: Acquired and inherited bleeding disorders may present in the neonatal period with devastating lifelong effects. Diagnosing bleeding disorders in the neonatal population could aid in preventing and treating the associated complications. However, currently available platelet function testing is limited in neonates, owing to difficulties in obtaining an adequate blood volume, a lack of normal reference ranges, and an incomplete understanding of the neonatal platelet functional phenotype. OBJECTIVE: To develop small-volume, whole blood platelet function assays in order to quantify and compare neonatal and adult platelet function. METHODS AND RESULTS: Peripheral blood was obtained from healthy, full-term neonates at 24 h of life. Platelet activation, secretion and aggregation were measured via flow cytometry. Platelet adhesion and aggregation were assessed under static and flow conditions. As compared with adult platelets, peripheral neonatal platelet P-selectin expression and integrin glycoprotein IIbIIIa activation were significantly reduced in response to the G-protein-coupled receptor (GPCR) agonists thrombin receptor activator peptide-6 (TRAP-6), ADP, and U46619, and the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway agonists collagen-related peptide (CRP) and rhodocytin. Neonatal platelet aggregation was markedly reduced in response to TRAP-6, ADP, U46619, CRP and rhodocytin as compared with adult platelets. The extents of neonatal and adult platelet adhesion and aggregate formation under static and shear conditions on collagen and von Willebrand factor were similar. CONCLUSIONS: As compared with adult platelets, we found that neonatal platelet activation and secretion were blunted in response to GPCR or ITAM agonists, whereas the extent of neonatal platelet adhesion and aggregate formation was similar to that of adult platelets.

Secretory component mediates Candida albicans binding to epithelial cells.

OBJECTIVES: Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. MATERIALS AND METHODS: Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. RESULTS: Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. CONCLUSION: These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers.

Structure and function of platelet receptors initiating blood clotting.

At the clinical level, recent studies reveal the link between coagulation and other pathophysiological processes, including platelet activation, inflammation, cancer, the immune response, and/or infectious diseases. These links are likely to underpin the coagulopathy associated with risk factors for venous thromboembolic (VTE) and deep vein thrombosis (DVT). At the molecular level, the interactions between platelet-specific receptors and coagulation factors could help explain coagulopathy associated with aberrant platelet function, as well as revealing new approaches targeting platelet receptors in diagnosis or treatment of VTE or DVT. Glycoprotein (GP)Ibα, the major ligand-binding subunit of the platelet GPIb-IX-V complex, that binds the adhesive ligand, von Willebrand factor (VWF), is co-associated with the platelet-specific collagen receptor, GPVI. The GPIb-IX-V/GPVI adheso-signaling complex not only initiates platelet activation and aggregation (thrombus formation) in response to vascular injury or disease but GPIbα also regulates coagulation through a specific interaction with thrombin and other coagulation factors. Here, we discuss the structure and function of key platelet receptors involved in thrombus formation and coagulation in health and disease, with a particular focus on platelet GPIbα.

A factor VIII-derived peptide enables von Willebrand factor (VWF)-binding of artificial platelet nanoconstructs without interfering with VWF-adhesion of natural platelets.

There is substantial clinical interest in synthetic platelet analogs for potential application in transfusion medicine. To this end, our research is focused on self-assembled peptide-lipid nanoconstructs that can undergo injury site-selective adhesion and subsequently promote site-directed active platelet aggregation, thus mimicking platelet's primary hemostatic actions. For injury site-selective adhesion, we have utilized a coagulation factor FVIII-derived VWF-binding peptide (VBP). FVIII binds to VWF's D'-D3 domain while natural platelet GPIbα binds to VWF's A1 domain. Therefore, we hypothesized that the VBP-decorated nanoconstructs will adhere to VWF without mutual competition with natural platelets. We further hypothesized that the adherent VBP-decorated constructs can enhance platelet aggregation when co-decorated with a fibrinogen-mimetic peptide (FMP). To test these hypotheses, we used glycocalicin to selectively block VWF's A1 domain and, using fluorescence microscopy, studied the binding of fluorescently labeled VBP-decorated nanoconstructs versus platelets to ristocetin-treated VWF. Subsequently, we co-decorated the nanoconstructs with VBP and FMP and incubated them with human platelets to study construct-mediated enhancement of platelet aggregation. Decoration with VBP resulted in substantial construct adhesion to ristocetin-treated VWF even if the A1-domain was blocked by glycocalicin. In comparison, such A1-blocking resulted in significant reduction of platelet adhesion. Without A1-blocking, the VBP-decorated constructs and natural platelets could adhere to VWF concomitantly. Furthermore, the constructs co-decorated with VBP and FMP enhanced active platelet aggregation. The results indicate significant promise in utilizing the FVIII-derived VBP in developing synthetic platelet analogs that do not interfere with VWF-binding of natural platelets but allow site-directed enhancement of platelet aggregation when combined with FMP.

MMGBSA as a tool to understand the binding affinities of filamin-peptide interactions.

Filamins (FLN) are large dimeric proteins that cross-link actin and work as important scaffolds in human cells. FLNs consist of an N-terminal actin-binding domain followed by 24 immunoglobulin-like domains (FLN1-24). FLN domains are divided into four subgroups based on their amino acid sequences. One of these subgroups, including domains 4, 9, 12, 17, 19, 21, and 23, shares a similar ligand-binding site between the ß strands C and D. Several proteins, such as integrins ß2 and ß7, glycoprotein Ibα (GPIbα), and migfilin, have been shown to bind to this site. Here, we computationally estimated the binding free energies of filamin A (FLNa) subunits with bound peptides using the molecular mechanics-generalized Born surface area (MMGBSA) method. The obtained computational results correlated well with the experimental data, and they ranked efficiently both the binding of one ligand to all used FLNa-domains and the binding of all used ligands to FLNa21. Furthermore, the steered molecular dynamics (SMD) simulations pinpointed the binding hot spots for these complexes. These results demonstrate that molecular dynamics combined with free energy calculations are applicable to estimating the energetics of protein-protein interactions and can be used to direct the development of novel FLN function modulators.

Polyphosphate binds to human von Willebrand factor in vivo and modulates its interaction with glycoprotein Ib.

BACKGROUND: Polyphosphate, a phosphate polymer released by activated platelets, has recently been described as a potent modulator of blood coagulation and fibrinolysis. In blood plasma, polyphosphate binds to and alters the biological functions of factor XII, fibrin(ogen), thrombin and factor VII activating protease. OBJECTIVES: The aim of the present study is to investigate whether polyphosphate also binds to von Willebrand factor (VWF) and alters some of its activities. METHODS/RESULTS: When studying patients with type 1 von Willebrand disease (VWD) and their healthy relatives, we discovered a significant correlation between von Willebrand factor (VWF) and platelet polyphosphate levels. We have also found polyphosphate in preparations of VWF isolated from normal platelets and plasma. Surface plasmon resonance and electrophoretic mobility assays indicated that polyphosphate interacts with VWF in a dose- and time-dependent manner. Treatment of normal plasma with active exopolyphosphatase decreased the VWF ristocetin cofactor (VWF:RCo) activity, a functional measure of VWF binding to platelet glycoprotein receptor Ib. VWF collagen binding and multimerization were unaltered after polyphosphate depletion. Moreover, addition of polyphosphate increased the deficient VWF:RCo activity presented by plasma from patients with type 1 VWD. CONCLUSIONS: Our results reveal that a new role is played by polyphosphate in hemostasis by its interaction with VWF, and suggest that this polymer may be effective in the treatment of some types of VWD.

Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha.

Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIbα, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIbα sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIbα, GPIbß, and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIbß is critical for ADAM17-dependent shedding of GPIbα induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIbα and GPIbß subunits further lessened the restraint of GPIbß on GPIbα shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIbß cytoplasmic domain disrupted calmodulin binding to GPIbß but had little effect on GPIbα shedding. Analysis of induction of GPIbα shedding by membrane-permeable GPIbß-derived peptides implicated the association of GPIbß with an unidentified intracellular protein in mediating regulation of GPIbα shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.

Functional association of phosphoinositide-3-kinase with platelet glycoprotein Ibalpha, the major ligand-binding subunit of the glycoprotein Ib-IX-V complex.

BACKGROUND: The adhesion receptor glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet-mediated tumor metastasis. The cytoplasmic C-terminal domain of the ligand-binding GPIbalpha subunit contains binding sites for filamin (residues 561-572, critically Phe568/Trp570), 14-3-3zeta (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide-3-kinase (PI3-kinase) regulatory subunit, p85. OBJECTIVES: We previously showed that, as compared with wild-type receptor, deleting the contiguous sequence 580-590 or 591-610, but not upstream sequences, of GPIbalpha expressed as a GPIb-IX complex in Chinese hamster ovary cells inhibited VWF-dependent Akt phosphorylation, which is used as a read-out for PI3-kinase activity. Pulldown experiments using glutathione-S-transferase (GST)-p85 or GST-14-3-3zeta constructs, and competitive inhibitors of 14-3-3zeta binding, suggested an independent association of 14-3-3zeta and PI3-kinase with GPIbalpha. The objective of this study was to analyze a further panel of GPIbalpha deletion mutations within residues 580-610. RESULTS: We identified a novel deletion mutant, Delta591-595, that uniquely disrupts 14-3-3zeta binding but retains the functional p85/PI3-kinase association. Deletion of other sequences within the 580-610 region were less discriminatory, and either partially affected p85/PI3-kinase and 14-3-3zeta binding (Delta580-585, Delta586-590, Delta596-600, Delta601-605), or strongly inhibited binding of both proteins (Delta606-610). CONCLUSIONS: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14-3-3zeta in GPIb-dependent signaling or platelet functional studies involving truncation of the C-terminal residues in cell-based assays and mouse models. The Delta591-595 mutation provides another strategy for determining the function of GPIbalpha-associated 14-3-3zeta by selective disruption of 14-3-3zeta but not p85/PI3-kinase binding.

Rational design of antithrombotic peptides to target the von Willebrand factor (vWf)--GPIb integrin interaction.

Conventional antithrombotic drug discovery requires testing of large numbers of drug candidates. We used computer-aided macromolecular interaction assessment (MIAX) to select antithrombotic molecules that mimic and therefore block platelet GPIb's binding to von Willebrand factor (vWf), an early step in thrombus formation. We screened a random array of 15-mer D-amino acid peptides for binding vWf. Structures of 4 candidate peptides were inferred by comparison to sequences in protein databases, conversion from the L to D conformations and molecular dynamics (MD) determinations of those most energetically stable. By MIAX, we deduced the amino acids and intermolecular hydrogen bonds contributing to the GPIb-vWf interaction interface. We docked the peptides onto vWf in silico to localize their binding sites and consequent potential for preventing GPIb-vWf binding. In vitro inhibition of ristocetin-initiated platelet agglutination confirmed peptide function and suitability for antithrombotic development, thereby validating this novel approach to drug discovery.

Specific heteromeric association of four transmembrane peptides derived from platelet glycoprotein Ib-IX complex.

As the receptor on the platelet surface for von Willebrand factor, glycoprotein (GP) Ib-IX complex is critically involved in hemostasis and thrombosis. How the complex is assembled from GP Ibalpha, GP Ib beta and GP IX subunits, all of which are type I transmembrane proteins, is not entirely clear. Genetic and mutational analyses have identified the transmembrane (TM) domains of these subunits as active participants in assembly of the complex. In this study, peptides containing the transmembrane domain of each subunit have been produced and their interaction with one another characterized. Only the Ib beta TM sequence, but not the Ibalpha and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ib beta-Ibalpha-Ib beta peptide complex (alphabeta(2)) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibalpha and Ib beta TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native alphabeta(2) complex, reproducing the same effect by the IX subunit in cells expressing the GP Ib-IX complex. Specific fluorescence resonance energy transfer was observed between donor-labeled alphabeta(2) peptide complex and acceptor-conjugated IX TM peptide in micelles. Finally, the mutation D135K in the IX TM peptide could hamper both the formation of the alphabeta(2) complex and the energy transfer, consistent with its reported effect in the full-length complex. Overall, our results have demonstrated directly the native-like heteromeric interaction among the isolated Ibalpha, Ib beta and IX TM peptides, which provides support for the four-helix bundle model of the TM domains in the GP Ib-IX complex and paves the way for further structural analysis. The methods developed in this study may be applicable to other studies of heteromeric interaction among multiple TM helices.

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

BACKGROUND: In the platelet glycoprotein (GP)Ib-IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted to the N-terminal domain of the GPIbalpha subunit. How the other subunits in the complex, GPIbbeta and GPIX, regulate the GPIbalpha-VWF interaction is not clear. OBJECTIVES AND METHODS: As GPIbalpha connects with two GPIbbeta subunits via disulfide bonds, we tested whether these intersubunit 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 GPIbalpha-GPIbbeta disulfide bonds. RESULTS: Mutating both Cys484 and Cys485 of GPIbalpha to serine prevents GPIbalpha from forming covalent disulfide bonds with GPIbbeta, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIbbeta subunits to form a disulfide bond between themselves. As 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 at a higher velocity. CONCLUSIONS: The disulfide bonds between GPIbalpha and GPIbbeta are necessary for optimal GPIbalpha binding to VWF. The structural plasticity around the disulfide bonds may also help to shed light on the inside-out mechanism underlying GPIbbeta modulation of VWF binding.

Glycoprotein Ibalpha forms disulfide bonds with 2 glycoprotein Ibbeta subunits in the resting platelet.

It is widely accepted that glycoprotein (GP) Ib contains one Ibalpha and one Ibbeta subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ibalpha, C484 or C485, forms the disulfide bond with Ibbeta. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibbeta. In the context of isolated peptides containing the Ibalpha or Ibbeta transmembrane domain and nearby Cys residue, C484 and C485 in the Ibalpha peptide were both capable of forming a disulfide bond with the Ibbeta peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibbeta subunits but only 1 Ibalpha and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ibalpha, not the number of Ibbeta subunits therein. Overall, these results demonstrate that Ibalpha is covalently connected to 2 Ibbeta subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The alphabeta2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.