Identification of a Novel 14-3-3ζ Binding Site Within the Cytoplasmic Domain of Platelet Glycoprotein Ibα That Plays a Key Role in Regulating the von Willebrand Factor Binding Function of Glycoprotein Ib-IX
Rationale: The interaction between platelet glycoprotein (GP) Ib-IX and von Willebrand factor (VWF) is initiated by conformational changes in immobilized VWF and is also regulated by the intraplatelet proteins 14-3-3ζ and filamin A. Both 14-3-3ζ and filamin A associate with the cytoplasmic domain of GPIbα, whereas little is known about their relationship in regulating the VWF binding function of GPIb-IX.
Objective: To explore the mechanism underlying the roles of 14-3-3ζ and filamin A in regulating the VWF binding function of GPIb-IX.
Methods and Results: A truncation mutant of GPIbα (Δ565) deleting the C-terminal 14-3-3ζ binding sites retained 14-3-3ζ binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIbα totally abolished 14-3-3ζ binding, indicating that the residues 551 to 564 of GPIbα are important in the interaction between 14-3-3ζ and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbα suggesting phosphorylation of a population of GPIbα molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIbα eliminated the association of 14-3-3ζ with Δ565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbα with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions.
Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3ζ to central filamin A binding site of GPIbα, and the dimeric 14-3-3ζ binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.
The interaction of glycoprotein (GP) Ibα with subendothelial-bound von Willebrand factor (VWF) initiates circulated platelet transient adhesion to the injured vascular wall under flow conditions.1,2 GPIbα-VWF interaction also triggers intracellular signaling events, such as elevation of cytoplasmic calcium3 and cGMP levels,4 and activations of multiple protein kinase pathways,4,5 which result in the activation of the ligand binding function of integrin αIIbβ3, leading to platelet aggregation and thrombus formation.1,4,5 VWF conformational changes in response to high shear stress are thought to be critical for initiating platelet adhesion,2,6 whereas there is increasing evidence that the interactions of intraplatelet proteins with the cytoplasmic domain of GPIbα also play key roles in the regulation of VWF binding function of GPIb-IX.7–10
Several proteins interact with the intracellular domains of GPIb-IX complex and play important roles in the regulation of platelet function. Filamin A, which interacts with GPIbα at amino acids within the 557 to 579 sequence, attaches the GPIb-IX complex to the membrane skeleton.9,11 Association of filamin A with the cytoplasmic domain of GPIbα is essential for VWF-induced platelet activation and GPIb-IX anchorage on VWF surface under high shear conditions.8,9 Calmodulin binds directly to the juxtamembrane cytoplasmic sequences of GPIbβ and GPV.12,13 Inhibition of calmodulin leads to GPIbα and GPV ectodomain shedding.13–15 Our studies indicate that the interaction of 14-3-3ζ with the C-terminal domain of GPIbα is critical for the GPIb-IX-VWF interaction.7,8
The interaction of 14-3-3ζ with GPIb-IX was firstly discovered during GPIb-IX purification.16 The subsequent studies demonstrated that the C-terminal 5-aa residues (605 to 610) containing the phosphorylated serine 609 are critical for the association of 14-3-3ζ with GPIb-IX.17,18 Deletion or blocking of the 14-3-3ζ binding site within the C-terminal of GPIbα results in dissociation of 14-3-3ζ from GPIb-IX and inhibition of VWF binding function of GPIb-IX.7,17,18 The second 14-3-3ζ binding site locates in the cytoplasmic domain of GPIbβ in which Ser166 plays a key role for the binding.19,20 Unlike Ser609 of GPIbα, which is constitutively phosphorylated in resting platelets,18 the phosphorylation of Ser166 is dynamically regulated by protein kinase A,8,21 which provides an important mechanism for the negative regulation of VWF binding function of GPIb-IX.8 However, this 14-3-3ζ binding site by itself is not strong enough to sustain the attachment of 14-3-3ζ to GPIb-IX complex.7,22 Recently, amino acids within the 580 to 590 sequence of GPIbα were identified to be another 14-3-3ζ binding site.23 Phosphorylations of Ser587 and Ser590 are required for the interaction. A peptide named A4 corresponding to residues 557 to 575 of GPIbα has been shown to interact with 14-3-3ζ.20 However, it is not clear whether this site is involved in GPIb-IX interaction with 14-3-3ζ in intact GPIb-IX molecule, or whether 14-3-3ζ binding to this site is important for GPIb-IX function.
In this study, we show that 14-3-3ζ binding site within the 551 to 564 region of GPIbα serves as one of the binding sites for the dimeric 14-3-3ζ protein. We demonstrate that GPIbα truncated at amino acid 565 interacted with 14-3-3ζ. Mutated cells containing GPIbα with alanine substituted for Ser559 showed increased association of GPIb-IX with the membrane skeleton and reduced ristocetin-induced VWF binding and adhesion to VWF-coated surface under flow conditions. Furthermore, a cell-permeable peptide that which eliminates the association of 14-3-3ζ with GPIbα truncated at residue 565 potently inhibited ristocetin-induced platelet aggregation, VWF binding to platelets, and platelet adhesion to immobilized VWF under shear stress. These data suggest that the interaction of 14-3-3ζ with the central region of GPIbα may regulate GPIb-IX association with the membrane skeleton and thus regulate VWF binding.
An expanded version of Methods section is available in the Online Data Supplement at http://circres.ahajournals.org and includes detailed information regarding the following: antibodies and reagents, cell lines expressing recombinant GPIb-IX and mutants, coimmunoprecipitation of GPIb-IX with endogenous 14-3-3ζ and filamin A, coimmunoprecipitation of 14-3-3ζ with GPIb-IX, peptides inhibition assay, antibodies against the peptides corresponding to the cytoplasmic domain of GPIbα, enzyme immunoassays, association of GPIb-IX with membrane skeleton, platelet preparation and aggregation, flow cytometric analysis of VWF binding to GPIb-IX-expressing cells and platelets, and cell adhesion under flow conditions.
Amino Acids Within the 551 to 564 Sequence of GPIbα Interact With 14-3-3ζ
Two phosphoserine-containing sequences 580 to 59023 and 605 to 61017 in the cytoplasmic domain of GPIbα have been identified to interact with 14-3-3ζ (Figure 1A). In addition, purified 14-3-3ζ bound to A4 peptide based on the cytoplasmic domain of GPIbα (Arg557-Gly575), and a peptide overlapped with A4 in 557 to 561 sequence also showed lower affinity for 14-3-3ζ.20 To further identify whether this is a 14-3-3ζ binding site in GPIbα, several truncation and substitution mutants of GPIbα (Figure 1A) were constructed and stably transfected with wild-type GPIbβ and GPIX into Chinese hamster ovary (CHO) cells. CHO cells expressing wild-type (1b9) or mutant GPIb-IX with GPIbα truncated at residue 551 (Δ551) or 565 (Δ565) were verified by anti-GPIbα N- and C-terminal antibodies (Online Figure I). The 14-3-3ζ binding capacity of these mutants was evaluated by coimmunoprecipitated with anti-GPIbα N-terminal antibody (SZ2). Figure 1B indicates Δ565 mutant coimmunoprecipitated with endogenous 14-3-3ζ, although the amount was much lower than that coimmunoprecipitated with wild-type GPIb-IX. In contrast, Δ551 mutant failed to coimmunoprecipitate with 14-3-3ζ. Interestingly, neither wild-type nor mutant GPIbα bound endogenous 14-3-3ζ when GPIbα was expressed in CHO cells in the absence of wild-type GPIbβ and GPIX (data not shown). To further confirm these results, GPIb-IX–expressing cell lysates were incubated with 14-3-33ζ–coated beads. 14-3-3ζ precipitated with wild-type GPIb-IX and Δ565 mutant, whereas it did not precipitate with Δ551 mutant (Figure 1C). Furthermore, endogenous 14-3-3ζ and 14-3-3ζ–coated beads associated with truncations of GPIbα at residue 583 (Δ583) or 605 (Δ605) (data not shown).17 These data indicate that, in addition to the known C-terminal region 14-3-3ζ binding sites, the region between 551 and 564 is also important for 14-3-3ζ binding to GPIbα.
A Membrane-Permeable Peptide Based on the 557 to 561 Sequence of GPIbα Competes With GPIbα Truncated at Residue 565 for 14-3-3ζ Binding
It is known that the 14-3-3ζ ligand proteins generally have a relatively conservative phosphoserine-containing motif, such as KRLpSLP in PCTAIRE-1 protein kinase.24 Serine 559 is the only serine in the 551 to 564 sequence of GPIbα, and RGSLP(557–561) is similar to the reported 14-3-3ζ binding motifs. Thus, to explore the possibility that Ser559 is phosphorylated and involved in 14-3-3ζ–GPIbα interaction, we synthesized a membrane-permeable (and a nonpermeable) peptide named MP-P (C13H27CONH-RGpSLP) corresponding to the cytoplasmic domain of GPIbα (557 to 561) with Ser559 phosphorylated. We also synthesized a membrane-permeable (and a nonpermeable) nonphosphorylated peptide (MP) with the identical sequence as MP-P and a membrane-permeable scrambled phospho-peptide (SMP-P) as controls. The nonpermeable peptides were immobilized and assessed for the ability to bind purified 14-3-3ζ. The RGpSLP peptide was found to bind purified 14-3-3ζ, whereas there was no appreciable specific binding of 14-3-3ζ to the nonphosphorylated RGSLP peptide or scrambled A4 peptide (Online Figure II). To investigate whether the membrane-permeable peptides could compete with GPIbα truncated at residue 565 for endogenous 14-3-3ζ binding, Δ565 cells was preincubated with MP-P or control peptides and then coimmunoprecipitated with SZ2. The results showed that addition of MP-P dose-dependently displaced 14-3-3ζ from Δ565 mutant of GPIbα, whereas MP and SMP-P had no obvious effect (Figure 1D). These results further indicate 14-3-3ζ interacts with amino acids within the 551 to 564 sequence of GPIbα and also suggest that Ser559 is phosphorylated when it binds to 14-3-3ζ.
Specific Binding of Antibodies to Ser559 Contained Peptides Corresponding to the Cytoplasmic Domain of GPIbα
To determine whether Ser559 in the cytoplasmic domain of GPIbα is phosphorylated in platelets, antibodies were developed using synthetic peptides corresponding to the 557 to 561 sequence of GPIbα with Ser559 phosphorylated or nonphosphorylated. Anti-Ser559 phosphorylated antibody (anti-pS559) reacted specifically with the phosphorylated peptide but not with the nonphosphorylated peptide, and the anti-Ser559 nonphosphorylated antibody (anti-S559) reacted specifically with the nonphosphorylated peptide in dot blot experiments (Figure 2A). The specificity and reactivity of the antibodies were further determined by ELISA with the peptides coated on the microtiter plates (Figure 2B). The following studies indicate these antibodies react with the intact GPIbα protein in ELISA, but, unfortunately, they do not recognize GPIbα protein in Western blot. We also developed antibodies with relatively longer peptides corresponding to 555 to 563 sequence of GPIbα. Again, the antibodies reacted specifically with the peptides used for immunizing, but did not react with the intact GPIbα protein in Western blot (data not shown). Thus, we had to examine the phosphorylation state of Ser559 by ELISA.
To further determine the specific binding of anti-pS559 and anti-S559 antibodies to Ser559 epitope but not to other serine-containing epitopes in GPIbα protein, a stable cell line which expressed wild-type GPIbβ and GPIX complexed with a GPIbα mutant bearing a mutation of serine 559 to an alanine (S559A) was established. Mutant and wild-type GPIbα proteins were purified by gel filtration from the S559A mutant and wild-type GPIb-IX cells. Anti-GPIbα antibody (SZ2) was coated on the microtiter plates, purified wild-type GPIbα and the S559A mutant proteins were immobilized to the coated SZ2 to further purify the proteins. With anti–Ser609-phosphorylated GPIbα antibody (anti-pS609)18 and anti–Ser166-phosphorylated GPIbβ antibody (anti–GPIbβ-P)8 as positive and negative controls, respectively, anti-S559 (Figure 2C) or anti-pS559 (Figure 2D) antibody was incubated with wild-type or S559A mutant GPIbα protein. Both of the antibodies reacted with wild-type GPIbα, but the reactivities of the 2 antibodies to the S559A mutated GPIbα protein diminished significantly, indicating that both anti-pS559 and anti-S559 specifically recognize the Ser559 epitope and also suggesting that there are Ser559-phosphorylated and -nonphosphorylated forms of GPIbα in CHO cells (Figure 2C and 2D).
Phosphorylation of a Population of GPIbα Molecules at Ser559 in Resting Platelets
Next, we examined the phosphorylation of Ser559 of GPIbα in resting platelets. To exclude the possible interference of other proteins, GPIbα was purified by gel filtration from resting platelets and then coated onto the microtiter plates. With the anti–GPIbβ-P as the negative control, and anti-pS609 as the positive control, both anti-pS559 and anti-S559 antibodies reacted with GPIbα (Figure 3A), suggesting that a population of GPIbα is phosphorylated at Ser559. To further verify that Ser559 of GPIbα is phosphorylated, purified GPIbα from resting platelets was treated with potato acid phosphatase to dephosphorylate the GPIbα protein, then reacted with anti-pS559. As shown in Figure 3B, treatment of purified GPIbα with potato acid phosphatase dramatically inhibited the binding of anti-pS559 to GPIbα. In addition, both anti-pS559 and anti-S559 antibodies immunoprecipitated GPIb-IX complex from platelet lysate (Online Figure III). Taken together, these data suggest that there are 2 populations of GPIbα in resting platelets: a Ser559-phosphorylated form and a Ser559-nonphosphorylated form.
The S559A Mutation and MP-P Enhances the Association of GPIb-IX With the Cytoskeleton
Filamin A interacting with amino acids 557 to 579 or 536 to 568 within cytoplasmic domain of GPIbα anchors the GPIb-IX complex to the membrane skeleton (one component of the platelet cytoskeleton).9,11 Thus, association of 14-3-3ζ with the novel 14-3-3ζ binding site at phosphorylated Ser559 of GPIbα, which is just located within the 557 to 579 sequence, should interfere with the interaction of filamin A with the cytoplasmic domain of GPIbα and the attachment of GPIb-IX complex to the membrane skeleton. To test this hypothesis, CHO cells expressing either wild-type or mutant GPIb-IX (S559A), which contains a serine-to-alanine substitution at Ser559 of GPIbα to mimic constitutive dephosphorylation, were analyzed for the association of GPIb-IX with the cytoskeleton. As illustrated in Figure 4A, the amount of soluble S559A mutant was significantly decreased compared with that of wild-type GPIb-IX, indicating that dephosphorylation of Ser559, which disrupts the novel 14-3-3ζ binding site of GPIbα, promotes the association of GPIb-IX with the membrane skeleton.
To confirm the role of the novel 14-3-3ζ binding site in the association of GPIb-IX with the membrane skeleton in platelets, washed platelets were incubated with MP-P, control peptides, or vehicle and then subjected to GPIb-IX distribution analysis. Consistent with S559A data, the soluble GPIbα was obviously reduced in platelets treated with MP-P (Figure 4B), indicating that inhibition of the interaction of 14-3-3ζ with the novel 14-3-3ζ binding site of GPIbα by MP-P enhances the association of GPIb-IX with the membrane skeleton in platelets.
To further investigate the mechanism through which MP-P enhances the association of GPIb-IX with the membrane skeleton, filamin A was coimmunoprecipitated with GPIbα from platelets treated with increasing concentrations of MP-P or control peptides. The amount of filamin A coimmunoprecipitated with GPIbα was dose-dependently increased by MP-P (Figure 4C), indicating that dissociation of 14-3-3ζ from the novel 14-3-3ζ binding site of GPIbα enhances the association of GPIbα with filamin A. Taken together, these data suggest that Ser559-phosphorylated form of GPIbα interacts with 14-3-3ζ, leading to the dissociation of filamin A from GPIbα and the detachment of GPIb-IX from the membrane skeleton; on the other hand, Ser559-nonphosphorylated form of GPIbα incurs opposite responses.
Effects of S559A Mutation on the VWF Binding Function of GPIb-IX and Cell Adhesion to VWF Under Flow
Association of GPIb-IX with the membrane skeleton negatively regulates VWF binding function of GPIb-IX.25 Furthermore, a membrane-permeable peptide corresponding to 557 to 569 domain of GPIbα inhibited VWF-induced platelet agglutination.26 To investigate the role of Ser559 in the VWF binding function of GPIb-IX, CHO cells expressing either wild-type or S559A mutated GPIb-IX were analyzed for ristocetin-induced VWF binding. Cells expressing S559A mutant showed a significantly reduced VWF binding function compared with cells expressing wild-type GPIb-IX (Figure 5A and 5B). These data are consistent with previous findings25,26 and further indicate that Ser559 is involved in the regulation of VWF binding function of GPIb-IX.
We also examined whether substitution of alanine for serine 559 in GPIbα affects GPIb-IX–dependent cell adhesion to VWF under flow conditions. As shown in Figure 5C, in contrast to the cells expressing wild-type GPIb-IX, when an equal amount of S559A cells were perfused through the VWF-coated surface under the same flow conditions, the number of adherent cells was significantly decreased. Taken together, these results indicate that Ser559 is critical for the VWF binding function of GPIb-IX.
MP-P Specifically Inhibits Ristocetin-Induced Platelet Aggregation/Agglutination
We have shown that MP-P competes with Δ565 mutant for 14-3-3ζ binding (Figure 1D), and Ser559 is critical for the VWF binding function of GPIb-IX (Figure 5). To further determine whether Ser559 plays a role in the VWF binding function of GPIb-IX in human platelets, the effects of MP-P on ristocetin-induced platelet aggregation/agglutination were examined. MP-P dose-dependently inhibited ristocetin-induced platelet aggregation (Figure 6A); in contrast, the control peptides had no obvious effect (Figure 6B). Ristocetin-induced platelet aggregation involves GPIb-IX–dependent platelet agglutination and subsequent integrin αIIbβ3-mediated platelet aggregation. To determine whether the inhibitory effect of MP-P specifically results from inhibition of GPIb-IX–dependent platelet agglutination, RGDS peptide which blocks integrin-dependent platelet aggregation, was preincubated with platelets. MP-P obviously inhibited ristocetin-induced platelet agglutination in the presence of RGDS (Figure 6C). Furthermore, we also examined the effect of MP-P on platelet aggregation induced by ADP or α-thrombin. MP-P or control peptides did not have any effect on ADP- or α-thrombin–induced platelet aggregation (Figure 6D and 6E). Taken together, these data indicate that the blockage of Ser559 with MP-P leads to the inhibition of VWF-dependent platelet agglutination.
MP-P Inhibits VWF Binding to Platelets
To further determine the effect of MP-P on ristocetin-induced GPIb-IX-VWF interaction, we directly examined the binding of VWF to MP-P-treated platelets. Washed platelets were pretreated with MP-P or control peptides (in the presence of RGDS or EDTA) and then incubated with ristocetin in the presence or absence of VWF. MP-P but not the control peptides obviously inhibited ristocetin-induced VWF binding to platelets (P=0.026) (Figure 7A and 7B). These data directly indicate that MP-P inhibits the VWF binding function of GPIb-IX.
The Effect of MP-P on Platelet Adhesion to VWF
In vivo, GPIb-IX-VWF interaction initiates platelet adhesion to the injured vessel wall under flow conditions. Thus, we examined the effect of MP-P on platelet adhesion to immobilized VWF under flow. Washed platelets were preincubated with MP-P, control peptides, or vehicle in the presence of RGDS and then perfused through the VWF-coated glass capillaries. Compared with the control peptides or vehicle control, the number of adherent platelets pretreated with MP-P was significantly decreased (Figure 7C). Thus, these results demonstrate that MP-P inhibits platelet adhesion to VWF under flow conditions.
In the present study, we provide evidence indicating: (1) amino acids between Ala551 and Arg564 in the cytoplasmic domain of GPIbα serve as one of the 14-3-3ζ binding sites; (2) Ser559 of GPIbα, which is present in resting platelets in both phosphorylated and nonphosphorylated forms, is critical for the dissociation of GPIb-IX from membrane skeleton and the VWF binding function of GPIb-IX; and (3) a membrane-permeable peptide corresponding to the 557 to 561 sequence of GPIbα inhibits the VWF binding function of GPIb-IX.
The conclusion that amino acids within the 551 to 564 domain of GPIbα interact with 14-3-3ζ is supported by several lines of evidence: (1) GPIbα truncated at amino acid 565 but not 551 coimmunoprecipitated with endogenous 14-3-3ζ; (2) 14-3-3ζ–coated beads precipitated GPIbα truncated at amino acid 565 but not the truncation at residue 551; (3) purified 14-3-3ζ bound to A4 peptide (residues Arg557-Gly575), and another peptide based on cytoplasmic domain of GPIbα (residues Arg543-Gly561) overlapped with A4 at amino acids Gly543-Pro561, also interacted with 14-3-3ζ with relatively lower affinity20; (4) the 557 to 561 sequence (RGSLP) in the cytoplasmic domain of GPIbα is similar to the reported 14-3-3ζ binding motifs, such as RX(Ar)(+)pSXP27; in particular, the ELISA data suggest that a part of Ser559 is phosphorylated in resting platelets; and (5) the phosphoserine peptide corresponding to the 557 to 561 sequence of GPIbα dose-dependently inhibited association of 14-3-3ζ with GPIbα truncated at amino acid 565.
Previous studies showed that GPIbα truncated at amino acid 569, 576, 594, 595, 591, or 605 causes loss of association with 14-3-3ζ, as detected by Western blot.17,22,23 These results are consistent with the present observations that GPIbα truncated at residue 565 caused markedly reduced binding of 14-3-3ζ compared with wild-type GPIbα. In this study, we observed the weak 14-3-3ζ band coimmunoprecipitated with Δ565 mutant GPIbα but not with Δ551 in Western blot analysis, suggesting that the affinity of 14-3-3ζ binding site in the central region is obviously lower than that of intact GPIbα. These data are supported by the finding that purified 14-3-3ζ bound with higher affinity to GPIbα C-terminal peptide (Gly592-Leu610) than central peptide A4.20 Because 14-3-3ζ is dimeric, it is possible that high-affinity interaction of 14-3-3ζ with GPIbα involves the binding of dimeric 14-3-3ζ to both C-terminal and central 14-3-3ζ binding sites.
14-3-3ζ binding proteins generally have a phosphoserine containing motif.27 The 557 to 561 sequence of GPIbα is similar to the reported 14-3-3ζ binding motifs. Thus an anti–phospho-Ser559 antibody was developed to detect the phosphorylation state of Ser559 in resting platelets. Unfortunately, perhaps because of the special conformation around Ser559 that prevents the antibodies to recognize the epitope in the protein, which often happens in antibody production, neither the antibodies raised by 5-aa peptides corresponding to the 557 to 561 sequence, nor the antibodies raised by 9-aa peptides based on the 555 to 563 sequence reacted with the GPIbα protein in Western blot (data not shown). Thus, we had to examine the phosphorylation state of Ser559 by ELISA. The data suggest that both phosphorylated and nonphosphorylated forms of Ser559 exist in resting platelets. Moreover, phosphorylation of Ser559 is supported by the following evidence: (1) the peptide containing phosphoserine of Ser559 (MP-P) but not the nonphosphorylated peptide diminished the association of 14-3-3ζ with GPIbα truncated at amino acid 565; (2) substitution of alanine for Ser559 resulted in the promotion of GPIbα-filamin A association and inhibition of GPIbα-dependent platelet function; and (3) both anti-pS559 and anti-S559 antibodies immunoprecipitated GPIb-IX complex from platelet lysate (Online Figure III). In addition, we also tried to determine Ser559 phosphorylation by mass spectrometry. Because of technical problems, no result was obtained. Therefore, direct evidence is still needed to confirm the phosphorylation of Ser559, particularly the phosphorylation state of Ser559 in response to platelet activation.
We had coimmunoprecipitated 14-3-3ζ with the S559A mutant, whereas there was no difference in 14-3-3ζ binding between the S559A and wild-type GPIb-IX. There was also no obvious difference in the GPIbα-14-3-3ζ interaction between platelets treated with MP-P and control peptides (data not shown). Furthermore, deletion of GPIbα central 535 to 569, 557 to 568, or 542 to 570 region did not diminish 14-3-3ζ binding to GPIb-IX complex.22,23 However, these data do not inevitably lead to the denial of the existence of the novel 14-3-3 binding site. The data with A4 peptide and GPIbα truncated at amino acid 565 have indicated that the novel 14-3-3ζ binding site interacted with 14-3-3ζ with low affinity.20 On the other hand, 2 identified C-terminal 14-3-3ζ binding sites, which are both indispensable for 14-3-3ζ attaching to GPIb-IX complex, associate with 14-3-33ζ with high affinity.17,22,23 Thus, it is reasonable that the deletion of the weak 14-3-3ζ binding site does not disrupt the 14-3-3ζ-GPIb-IX association. This condition is similar to that of the 14-3-3ζ binding site in GPIbβ, which also interacts with 14-3-3ζ with low affinity. As has been verified, deletion of the 14-3-3ζ binding site in GPIbβ does not diminish 14-3-3ζ attaching to GPIb-IX complex.7,22
A4 has been reported to inhibit ristocetin and shear-induced platelet aggregation after being delivered into platelets.10 However, the mechanism should be different from the present observation, because A4 disrupts the GPIbα-filamin A association and also should interfere with the interaction of 14-3-3ζ with GPIbα.10,20,28 Furthermore, similar to our finding, David et al reported recently that a membrane-permeable peptide based on the 557 to 569 region of GPIbα inhibited VWF-induced platelet agglutination, although the mechanism is still unclear.26
We have proposed a “toggle switch” model as a mechanism for the involvement of 14-3-3ζ in regulating the VWF binding function of GPIb-IX in our previous report.7 However, how disruption of 14-3-3ζ-GPIbβ association induced by protein kinase A inhibition leads to promotion of the VWF binding function of GPIb-IX is still unclear.7,8 Here, we show that alanine substitution at Ser559 of GPIbα (S559A), which should disrupt the 14-3-3ζ binding at this site results in the association of GPIb-IX with the membrane skeleton and inhibition of VWF binding function of GPIb-IX. Our unpublished data further indicate that there is a competitive binding between filamin A and 14-3-3ζ on A4 peptide. Thus, there might be a phosphorylation and dephosphorylation of Ser559-dependent competitive binding between filamin A and 14-3-3ζ at Ser559 of GPIbα, which regulates the association of GPIb-IX with the membrane skeleton and subsequently regulates the VWF binding function of GPIb-IX (Figure 8). This hypothesis is supported by the findings that CHO cells expressing a GPIb-IX mutant replacing Ser166 of GPIbβ with alanine showed a significantly reduced association of GPIb-IX with the cytoskeleton (Online Figure IV), and the ratio of phosphorylation/nonphosphorylation of Ser559 obviously increased following platelet activation with VWF (Online Figure V). In particular, GPIb-IX complex immunoprecipitated by anti-pS559 antibody did not contain filamin A and phosphorylated GPIbβ at Ser166, on the other hand, GPIb-IX complex immunoprecipitated by anti-GPIbβ-P antibody contained filamin A, and a majority of Ser559 was not phosphorylated in the complex (Online Figure III). According to “toggle switch” model, the 14-3-3ζ monomer dissociated from Ser166 of GPIbβ incurred by protein kinase A inhibition would switch to Ser559-phosphorylated form of GPIbα to compete with filamin A for Ser559 binding, which results in dissociation of GPIb-IX from the membrane skeleton and subsequent promotion of the VWF binding function of GPIb-IX. Therefore, the present findings further support the “toggle switch” model, which is also supported by the observation that disruption of GPIbα–filamin A association activates VWF binding function of GPIb-IX.25 It has been reported that there are 2 GPIbβ for every GPIbα29 and there are two 14-3-3ζ binding sites17,23 at GPIbα C-terminal, whether there are also two 14-3-3ζ molecules involving in the “toggle switch” regulatory model is still unclear. In addition, the mechanism underlying the regulation of Ser559 phosphorylation remains to be investigated.
Compared with wild-type GPIb-IX cells, the S559A mutant cells showed a significant decrease in ristocetin-induced VWF binding and adhesion to immobilized VWF under flow conditions. The MP-P peptide, which disrupts the association of 14-3-3ζ with GPIbα truncated at amino acid 565, potently inhibits ristocetin-induced platelet agglutination, VWF binding to platelets, and platelet adhesion on VWF-coated surface under flow. The GPIbα-VWF interaction initiates circulated platelet adhesion, activation and subsequent thrombus formation. Thus, the identification of the novel 14-3-3ζ binding site and the MP-P peptide, should not only help to illuminate the mechanism underlying the interaction between 14-3-3ζ and filamin A in regulating the VWF binding function of GPIb-IX but also suggest a strategy to develop new class of antiplatelet agents for arterial thrombosis.
Sources of Funding
This work was supported by grants from National Natural Science Foundation of China (NSFC 30770795 to K.D), Program for New Century Excellent Talents in University (NCET-06-0167 to K.D), the Foundation for the Author of National Excellent Doctoral Dissertation of People’s Republic of China (FANEDD 200560 to K.D.), and grants from National Heart, Lung and Blood Institute (HL 062350 and HL 068819 to X.D.).
↵*Both authors contributed equally to this work.
Original received July 8, 2009; revision received October 14, 2009; accepted October 20, 2009.
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