Von Willebrand Factor Binding Function Research Articles

Essential role of glycoprotein Ibα in platelet activation

AbstractGlycoprotein Ibα (GPIbα), the ligand-binding subunit of platelet GPIb-IX complex, interacts with von Willebrand factor (VWF) exposed at the injured vessel wall, initiating platelet adhesion, activation, hemostasis, and thrombus formation. The cytoplasmic tail of GPIbα interacts with 14-3-3ζ, regulating the VWF-GPIbα–elicited signal transduction and VWF binding function of GPIbα. However, we unexpectedly found that the GPIbα–14-3-3ζ association, beyond VWF-dependent function, is essential for general platelet activation. We found that the myristoylated peptide of GPIbα C-terminus MPαC, a potential GPIbα inhibitor, by itself induced platelet aggregation, integrin αIIbβ3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIbα in mouse platelets (10aa−/−) decreased platelet aggregation, integrin αIIbβ3 activation, granule secretion, and PS exposure induced by various physiological agonists. Phosphoproteome-based kinase activity profiling revealed significantly upregulated protein kinase C (PKC) activity in MPαC-treated platelets. MPαC-induced platelet activation was abolished by the pan-PKC inhibitor and PKCα deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa−/− platelets. GPIbα regulates PKCα activity by sequestering 14-3-3ζ from PKCα. In vivo, the deletion of the GPIbα cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIbα cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF–GPIbα axis.

Abstract 94: Randomized, Double-blind, Placebo-controlled Phase 1 Study Of Vwf-binding Aptamer, Bb-031: Safety, Tolerability, Pharmacokinetic And Pharmacodynamic Activity In Healthy Volunteers

Introduction: Von Willebrand Factor (VWF) is a ubiquitous component of thrombi extracted by endovascular thrombectomy from patients with large vessel occlusion (LVO) stroke. BB-031 is a modified RNA aptamer that targets von Willebrand Factor (VWF) that lyses occlusive thrombi and improves outcomes in pre-clinical models of acute ischemic stroke (AIS). Hypothesis: BB-031 is safe, tolerable, and inhibits both VWF binding and activity in a dose-dependent manner. Methods: Healthy participants were randomized to receive BB-031 or placebo (6:2) by intravenous bolus injection at single ascending doses (SAD) of 0.1, 0.3, 1.0, 2.0 and 4.0 mg/kg (total n=40). Serial Clinical assessment, and blood sample collection for pharmacokinetic (PK) and pharmacodynamic (PD) analysis were performed. Non-compartmental PK analysis was conducted using Phoenix WinNonlin. PD evaluation included measurement of VWF inhibition and whole blood thrombosis (Platelet Function Analyzer [PFA-200]). Results: BB-031 was safe and well-tolerated for 28 days following single IV doses up to 4.0 mg/kg. There were no significant adverse events (SAEs), or treatment-emergent adverse events (TEAEs) leading to dose discontinuation. Minor events included bleeding at an IV site in 1 participant, and minor bleeding of an ulcer on the tongue in 1 participant. None of the minor TEAEs were dose-dependent. BB-031 demonstrated nonlinear, dose-dependent plasma PK across the dose range tested, with an apparent mean terminal half-life (t 1/2 ) of 18 min at 0.1 mg/kg to 67 min at 4.0 mg/kg. Dose-dependent changes in VWF binding were observed; >95% maximal mean change from baseline was reached following a single IV dose of 4.0 mg/kg. Finally, PFA-200 results showed complete inhibition of clot formation (closing time ≥300 seconds) at all doses tested with a dose-dependent duration of inhibition and return to normal range. Conclusion: This first-in-human SAD study of BB-031 demonstrated safety following a single IV dose of 0.1 to 4 mg/kg, and dose-dependent patterns of VWF binding and platelet function changes. These results lay the foundation for future studies in patients suffering from thrombotic conditions including AIS.

Unique surface‐exposed hydrophobic residues in the C1 domain of factor VIII contribute to cofactor function and von Willebrand factor binding

BackgroundThe identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 domain, this implies that the FVIII C1 domain comprises unique surface‐exposed elements involved in VWF and FIXa interaction. ObjectiveThe aim of this study is to identify residues in the FVIII C1 domain that contribute to VWF and FIXa binding. MethodsStructures and primary sequences of FVIII and FV were compared to identify surface‐exposed residues unique to the FVIII C1 domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. ResultsFive surface‐exposed hydrophobic residues unique to the FVIII C1 domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface‐exposed V2125, which is spatially next to F2127, affected only VWF binding. ConclusionThe surface‐exposed hydrophobic residues in C1 domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 domain in FVIII life cycle.

Serine 559 Of Glycoprotein Ibα Is Phosphorylated By Protein Kinase A Which Plays Key Roles In Regulating Von Willebrand Factor Binding, Surface Expression, and Signaling Of Glycoprotein Ib-IX

Conformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate glycoprotein (GP) Ib-IX-dependent platelet adhesion, and simultaneously trigger signaling cascades leading to integrin αIIbβIII activation, platelet spreading, and thrombus formation. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and play key roles in regulating the VWF binding function of GPIb-IX. Several phosphoserine containing motifs in GPIb-IX complex, such as Ser609 of GPIbα and Ser166 of GPIbβ, have been confirmed to bind to 14-3-3ζ and involved in the regulation of VWF binding function. We have recently identified a novel 14-3-3ζ binding site located in the filamin A binding domain of GPIbα at Ser559. Phosphorylation of GPIbα at Ser559 not only is important for 14-3-3ζ binding, but also plays a key role in the regulation of VWF binding function of GPIb-IX. However, the protein kinase responsible for Ser559 phosphorylation remains unknown. Here we show that a peptide corresponding to the Arg557-Pro561 region of GPIbα and purified GPIbα could be phosphorylated at Ser559 by the catalytic subunit of protein kinase A (PKA). Ser559 phosphorylation was enhanced by PKA activators and reduced by PKA inhibitors in platelets and Chinese hamster ovary (CHO) cells expressing GPIb-IX. Furthermore, PKA inhibitor enhanced VWF binding to the CHO cells expressing GPIb-IX mutant replacing Ser559 of GPIbα (S559A) with alanine, while reduced in the CHO cells of Ser166 of GPIbβ (S166A) with alanine. GPIb-IX association with filamin A and membrane expression were enhanced in S559A cells, whereas reduced in S166A cells. The peptide disrupting the interaction of 14-3-3ζ with Ser559 of GPIbα inhibited platelet spreading on VWF matrix and GPIb-IX-VWF interaction-induced association of Src with GPIb-IX. These data indicate that Ser559 of GPIbα is phosphorylated by PKA which plays key roles in regulating VWF binding, surface expression, and signaling of GPIb-IX. Disclosures:No relevant conflicts of interest to declare.

Reversible Association of 14-3-3 with Platelet Glycoprotein Ibα Regulates the Ability of Cells to Adhere, Roll and Spread On VWF

Abstract 1061Platelet adhesion to sites of vascular injury is required for the arrest of bleeding. Initial platelet adhesion is mediated by binding of von Willebrand factor (VWF) to the platelet glycoprotein (GP) Ib-IX-V complex, leading to the activation of integrin α IIbβ3 and other molecules that mediate firm adhesion, spreading and thrombus formation. The GPIb-IX-V complex comprises 4 polypeptides: GPIbα, GPIbβ, GPIX and GPV, in a 2:4:2:1 stoichiometry. Only the first three polypeptides are required for full VWF binding function. GPIbα is a 610 amino acid polypeptide that binds every known complex ligand within its N-terminal 300 amino acids. The cytoplasmic domain comprises 96 amino acids and contains binding sites for filamin, PI 3-kinase and the scaffolding protein 14-3-3. The association of 14-3-3 with the GPIbα cytoplasmic domain regulates the affinity for VWF. Typically, 14-3-3 requires phosphoserine- or phosphothreonine-containing motifs to bind target proteins. One such motif is in the GPIbα cytoplasmic domain surrounding Ser609, which is phosphorylated and known to bind 14-3-3. Mutation of Ser609 to Ala abrogates 14-3-3 association, which has been proposed to reduce the ability of GPIbα to bind VWF. Platelet aggregation results in the dissociation of 14-3-3 from a subpopulation of GPIbα. Ser609 also becomes dephosphorylated upon platelet spreading. To dissect further the functional roles of 14-3-3 association with GPIbα, we expressed in Chinese hamster ovary (CHO) cells GPIb-IX complexes (GPIbα, GPIbβ, and GPIX) containing either wild type GPIbα, or GPIbα mutants S609A or S609E. In other proteins, mutation of Ser to Glu at the 14-3-3 binding site mimics phosphoserine, recapitulates 14-3-3 binding and often prevents 14-3-3 dissociation. We first assessed the ability of the WT and mutant GPIbα to associate with 14-3-3. As expected, we detected little 14-3-3 binding to GPIbα S609A. GPIbα S609E bound 14-3-3 to the same extent as did WT GPIbα, indicating that the Glu substitution was able to mimic Ser phosphorylation at residue 609. We then assessed the ability of the CHO cells to attach to and roll on VWF under flow over a wide range of shear rates. At 3.26 and 10 dyne/cm2 the α 609A and α 609E cells rolled twice as fast as the WT cells. Both CHO cells and platelets display a characteristic velocity nadir as shear rates increase. The α 609A and α 609E cell showed defective shear-enhanced adhesion; their slowest velocity was ∼3-fold faster than the WT cells. Because GPIbα is dephosphorylated upon platelet spreading, we also assessed the effect of the mutations on cell spreading on VWF. All three cell lines adhered similarly to VWF but a higher percentage of α 609A cells spread (67% vs 58% for WT and α 609E). Of the spread cells, the α S609E cells spread less well; their spread area was 15% less than the WT and α S609A cells. The morphology of the adherent, spread cells was dramatically different among the different cell lines. WT cells displayed a few filopodial extensions along with punctate phalloidin staining indicative of focal adhesions. In some cases the cells displayed stress fibers. The α S609A cells extended more and longer filopodia than the WT cells but had fewer focal adhesions and more stress fibers. The CHO α S609E cells extended thin filopodia that tended to be polarized at two sides of the cell body, and had fewer focal adhesions and no stress fibers. We also examined the effect of the mutations on localization of the GPIb-IX complex to lipid raft membrane microdomains, which is required for platelet adhesion to VWF. Raft GPIbα was reduced by 40% in the S609A cells but increased 1.6-fold in the S609E cells.In summary, lack of 14-3-3 association decreased raft localization of the complex, reduced shear-induced cell adhesion, but increased cell spreading. Stable 14-3-3 association increased raft localization, but decreased shear-induced cell adhesion and decreased the ability of cells to fully spread. Together, our results demonstrate that regulated 14-3-3 association mediated by the phosphorylation status of S609 is required for coordinated adhesion, and cell spreading. Together, our results demonstrate that the functions of the GPIb-IX complex are regulated by the ability of GPIbα Ser609 to both bind and release 14-3-3 and suggest that it is not 14-3-3 binding per se that regulates GPIbα function. Disclosures:No relevant conflicts of interest to declare.

Role of 14-3-3ζ in Platelet Glycoprotein Ibα-von Willebrand Factor Interaction-Induced Signaling

The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture initiates platelet transient adhesion to the injured vessel wall, which triggers intracellular signaling cascades leading to platelet activation and thrombus formation. 14-3-3ζ has been verified to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX. However, the data regarding the role of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling still remain controversial. In the present study, the data indicate that the S609A mutation replacing Ser609 of GPIbα with alanine (S609A) significantly prevented the association of 14-3-3ζ with GPIbα before and after the VWF binding to GPIbα. GPIb-IX-VWF interaction-induced activations of Src family kinases and protein kinase C were clearly reduced in S609A mutation. Furthermore, S609A mutation significantly inhibited GPIb-IX-VWF interaction-induced elevation of cytoplasmic Ca2+ levels in flow cytometry analysis. Taken together, these data indicate that the association of 14-3-3ζ with the cytoplasmic domain of GPIbα plays an important role in GPIb-IX-VWF interaction-induced signaling.

Abstract 499: Rac1 Plays a Stimulatory Role in Glycoprotein Ib-IX-Dependent Platelet Activation

The platelet receptor for von Willebrand factor (VWF), the glycoprotein Ib-IX (GPIb-IX) complex, mediates platelet adhesion at sites of vascular injury and transmits signals leading to platelet activation. Ligand occupancy of GPIb-IX stimulates the sequential activation of the Src family kinase (SFK) Lyn, phosphoinositide 3-kinase (PI3K), and Akt, leading to activation of integrin aIIbβ3, synthesis of thromboxane, granule secretion, and integrin-dependent stable platelet adhesion and aggregation. It remains unclear, however, as to how the PI3K/Akt pathway is activated following ligand binding to GPIb-IX. Using platelet-specific conditional Rac1 -/- mice and the Rac1 selective inhibitor NSC23766 , we examined the role of Rac1 in GPIb-IX-dependent platelet activation. Rac1-deficient platelets are defective in VWF/GPIb-induced aggregation, thromboxane synthesis and granule secretion, and stable adhesion to VWF under shear stress. Importantly, GPIb-IX-induced activation of integrin aIIbβ3 was also significantly impaired in Rac1 -/- or Rac1-inhibitor treated platelets, indicating that Rac1 is critical for GPIb-mediated integrin activation. However, Rac1 deficiency does not alter the VWF binding function of GPIb-IX, demonstrating that the functional defects observed in Rac1-deficient platelets derives from a defect in VWF-induced GPIb-IX signaling. To determine why Rac1 is important in GPIb-IX signaling, we further show that VWF/GPIb-induced activation of Rac1 was abolished in Lyn -/- mouse platelets or SFK-inhibitor treated human platelets but remained unaffected by the PI3K inhibitor LY-294002, suggesting that Rac1 is activated downstream of Lyn independent of PI3K. Moreover, VWF/GPIb-induced PI3K-dependent activation of Akt and P38 MAPK was abolished in Rac1-deficient platelets. Thus, our study reveals that Rac1 is an important new link in the GPIb-IX signaling pathway that mediates Lyn-dependent activation of the PI3K/Akt pathway leading to integrin activation.

The Association of 14-3-3ζ and Filamin A with the Central Cytoplasmic Domain of GPIbα Involves in Regulation of the Von Willebrand Factor Binding and Signaling Functions of Platelet Glycoprotein Ib-IX.

Abstract 5073 IntroductionConformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate platelet tethering and simultaneously trigger signaling cascades leading to platelet adhesion, spreading, and thrombus formation. Although the intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and involve in the regulation of VWF binding function, whereas, the mechanisms still remain unclear. We have recently identified a novel 14-3-3ζ binding site with partial phosphorylation of Ser559 in resting platelets located in the cytoplasmic filamin A binding domain of GPIbα. Disruption of the central cytoplasmic 14-3-3ζ binding site of GPIbα did not abolish the interaction between 14-3-3ζ and GPIb-IX, whereas obviously diminished VWF binding function. Here we show that disruption of 14-3-3ζ binding to GPIbα by GPIbα mutants or cell permeable peptides enhances the association of filamin A with GPIb-IX and the expression of GPIb-IX in the plasma membrane. In contrast, disruption of 14-3-3ζ binding to GPIbβ, which would enhance the opportunity for 14-3-3ζ monomer to bind to GPIbα, reduces filamin A-GPIb-IX interaction and GPIb-IX membrane expression. Compared with a GPIb-IX mutant truncating the whole cytoplasmic domain of GPIbα (Δ551), GPIb-IX mutant retaining only the central 14-3-3ζ binding site of the cytoplasmic domain of GPIbα (Δ565) showed an enhanced cell adhesion to VWF under flow and ristocetin-induced VWF binding. Furthermore, the two cell permeable peptides, MP-P and MPαC, which had been confirmed to block the central 14-3-3ζ binding site of GPIbα (MP-P) or diminished the association of 14-3-3ζ with GPIb-IX complex (MPαC), obviously inhibited ristocetin induced wild-type GPIb-IX cell aggregation. In addition, MP-P and MPαC inhibited botrocetin-induced platelet spreading and lamellipodia formation on VWF matrix, and GPIb-IX-VWF interaction induced co-immunoprecipitation of Src with GPIb-IX. Taken together, these data indicate that the binding of 14-3-3ζ and filamin A to the central cytoplasmic domain of GPIbα involves in regulation of surface expression, VWF binding and signaling functions of glycoprotein Ib-IX. DisclosuresNo relevant conflicts of interest to declare.

A Phosphorylation-Dependent Binding of 14-3-3ζ to the Central Region of the GPIbalpha Cytoplasmic Domain.

Abstract 2995Poster Board II-973 Introduction:The interaction of glycoprotein (GP) Ib-IX with subendothelial-bound von Willebrand factor (VWF) initiates circulated platelet transient adhesion on the injured vascular wall under flow conditions. VWF conformational changes in response to high shear stress are thought to be critical for initiating platelet adhesion, there is increasing evidences indicate that the interactions of intraplatelet proteins 14-3-3ζ and filamin A with the cytoplasmic domain of GPIbalpha also play key roles in the regulation of VWF binding function of GPIb-IX, whereas it is unclear whether their structural linkage has functional implication. This study was to explore the mechanism underlying the roles of 14-3-3ζ and filamin A in the regulation of the VWF binding function of GPIb-IX. Methods and Results:A truncation mutant of GPIbalpha (at residue 565) deleting the C-terminal 14-3-3ζ binding sites retains 14-3-3ζ binding function, in contrast, deletion of the C-terminal residues 551-610 of GPIbalpha totally abolished 14-3-3ζ binding, indicating that the residues 551-564 of GPIbalpha is important in the interaction between 14-3-3ζ and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), M-R557GpSLP561 corresponding to residues 557-561 of GPIbalpha eliminated the association of 14-3-3ζ with the truncation mutant of GPIbalpha . 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 GPIbalpha 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 GPIbalpha, 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. Disclosures:No relevant conflicts of interest to declare.

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

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 14-3-3zeta and filamin A. Both 14-3-3zeta and filamin A associate with the cytoplasmic domain of GPIbalpha, whereas little is known about their relationship in regulating the VWF binding function of GPIb-IX. To explore the mechanism underlying the roles of 14-3-3zeta and filamin A in regulating the VWF binding function of GPIb-IX. A truncation mutant of GPIbalpha (Delta565) deleting the C-terminal 14-3-3zeta binding sites retained 14-3-3zeta binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIbalpha totally abolished 14-3-3zeta binding, indicating that the residues 551 to 564 of GPIbalpha are important in the interaction between 14-3-3zeta and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIbalpha eliminated the association of 14-3-3zeta with Delta565. 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 GPIbalpha 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. These data suggest a phosphorylation-dependent binding of 14-3-3zeta to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3zeta 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.

Signaling and regulation of the platelet glycoprotein Ib–IX–V complex

The platelet adhesion receptor, the glycoprotein Ib-IX-V complex, not only mediates platelet adhesion but also transmits signals leading to platelet activation, aggregation and secretion. Significant progress has been made recently on the signaling pathways and regulatory mechanisms involving glycoprotein Ib-IX-V function. The interaction of glycoprotein Ib-IX-V with its ligand, von Willebrand factor, is dually controlled by von Willebrand factor conformation and intracellular signal-mediated regulation of glycoprotein Ib-IX-V receptor function that requires the zeta isoform of the 14-3-3 protein family (14-3-3zeta). Glycoprotein Ib-IX-V signaling is mediated by the Src family of protein kinases, phospholipase C, calcium elevation, phosphoinositol 3-kinase, and multiple amplification mechanisms including the nitric oxide-cGMP pathway, the mitogen-activated protein kinase pathway, the immunoreceptor tyrosine-based activation motif pathway, and ADP and thromboxane A2 pathways. Progress in understanding the mechanism(s) regulating glycoprotein Ib-IX-V should help develop inhibitors and modifiers that interfere or augment its von Willebrand factor binding function and thus be useful for treating thrombosis and bleeding disorders. Characterization of intracellular molecules and pathways in glycoprotein Ib-IX-V signaling has implications in the development of new agents and for the use of existing drugs that affect glycoprotein Ib-IX-V signaling.

Inhibition of the Binding of von Willebrand Factor to the Platelet Glycoprotein Ib-IX with an Inhibitor of 14-3-3 and Glycoprotein Ib-IX Interaction.

Platelet adhesion and activation are essential for thrombosis and hemostasis. In arteries and capillaries where blood flow shear rate is high, initial platelet adhesion is dependent on the binding of von Willebrand factor (VWF) to its platelet receptor, the glycoprotein (GP) Ib-IX-V complex (GPIb-IX), which consists of four subunits, GPIbα, GPIbβ, GPIX and GPV. We have shown previously that a phosphoserine-dependent intracellular signaling molecule, 14-3-3, interacts with the C-terminal SIRYSGHSL610 sequence in the cytoplasmic domain of GPIbα, and this interaction is dependent upon phosphorylation at Serine 609 of GPIbα. Here we show that a short cell-permeable myristoylated phospho-peptide corresponding to the 14-3-3 binding sequence of GPIbα inhibits VWF binding to platelets and VWF-mediated platelet adhesion. This peptide also specifically inhibits VWF-dependent platelet agglutination induced by ristocetin but has no significant effect on platelet aggregation induced by platelet agonists such as ADP and collagen. Furthermore, intravenous injection of this phospho-peptide caused significantly prolonged bleeding time in mice. Thus, 14-3-3 interaction with GPIbα plays critical roles in VWF binding function of GPIb-IX and hemostatic function of platelets. These results also suggest a new type of anti-platelet drugs that may potentially be useful in treating thrombosis.

Gain of von Willebrand factor–binding function by mutagenesis of a species-conserved residue within the leucine-rich repeat region of platelet glycoprotein Ibα

AbstractGlycoprotein (GP) Ibα, a member of the leucine-rich repeat (LRR) protein family, mediates platelet adhesion to immobilized von Willebrand factor (VWF). We investigated the role in VWF binding of charged residues in the LRR region of GP Ibα that are conserved in human, canine, and murine proteins. Substitution of His86 with either Ala or Glu resulted in a gain of VWF-binding function as judged by increased VWF binding in the presence of the modulators ristocetin and botrocetin and by enhanced adhesion of Chinese hamster ovary (CHO) cells expressing the mutant GP Ibα to immobilized VWF under conditions of flow. This is the first report of a gain-of-function phenotype resulting from mutations in the LRR region of GP Ibα. Because His86 is 2 nm away from the region of GP Ibα with the largest surface of contact with VWF, the data suggest that the LRRs regulate GP Ibα affinity for VWF allosterically.

A critical role for 14-3-3ζ protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications

AbstractThe platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates platelet adhesion and activation. The cytoplasmic domains of the GPIb α and β subunits contain binding sites for the phosphorylation-dependent signaling molecule, 14-3-3ζ. Here we show that a novel membrane-permeable inhibitor of 14-3-3ζ-GPIbα interaction, MPαC, potently inhibited VWF binding to platelets and VWF-mediated platelet adhesion under flow conditions. MPαC also inhibited VWF-dependent platelet agglutination induced by ristocetin. Furthermore, activation of the VWF binding function of GPIb-IX induced by GPIbβ dephosphorylation is diminished by mutagenic disruption of the 14-3-3ζ binding site in the C-terminal domain of GPIbα, mimicking MPαC-induced inhibition, indicating that the inhibitory effect of MPαC is likely to be caused by disruption of 14-3-3ζ binding to GPIbα. These data suggest a novel 14-3-3ζ-dependent regulatory mechanism that controls the VWF binding function of GPIb-IX, and also suggest a new type of antiplatelet agent that may be potentially useful in preventing or treating thrombosis.

Gain of von Willebrand Factor Binding Function Induced by a Mutation within the Leucine-Rich Repeat Region of Platelet Glycoprotein Ibα.

The first step of platelet adhesion to areas of vessel injury is carried out by the platelet glycoprotein (GP) Ib-IX-V complex, which binds von Willebrand factor affixed to the subendothelial matrix. The GP Ib-IX-V complex comprises four polypeptides, each belonging to the leucine-rich repeat (LRR) family of proteins. Glycoprotein Ibα is the largest of these and contains the VWF binding site within a ~300-amino acid domain at its N-terminus that contains 8 tandem LRRs. In this study, we investigated the role in VWF binding of charged residues in this region of GP Ibα that are conserved across three species: humans, dogs and mice. Each of eleven charged residues was converted to Ala: His37, Glu40, Asp83, His86, Asp106, Glu128, Lys137, Lys152, Glu162, Asp175 and Lys189. All of the GP Ibα mutants expressed normally on the surface of CHO bIX cells, which stably express GP Ibb and GP IX. GP Iba expression was detected by the monoclonal antibody WM23, which binds a region uninvolved by the mutations. In three mutants, His86Ala, Glu128Ala, and Asp175Ala, the mutations appeared to induce large conformational changes, as they markedly altered the binding of the antibodies AN51 and SZ2, with epitopes located N-terminal and C-terminal to the LRRs, respectively. As a result, cells expressing the mutants Glu128Ala and Asp175Ala were unable to adhere to the surface when perfused over immobilized VWF. By contrast, cells expressing mutant His86Ala adhered to the surface and rolled on it much more slowly than did cells expressing wild-type GP Ibα. The His86Ala cells also displayed a left-shifted VWF binding curve in the presence of ristocetin compared to the wild-type cells. We investigated this residue further by converting the His to Glu, a residue of opposite charge. Cells expressing the His86Glu mutant rolled even more slowly over a VWF surface than the His86Ala cells at 20 dynes/cm2, but not at 2.5 dynes/cm2. Previously, mutations of GP Ibα causing gain of VWF-binding function have been confined to the disulfide loop formed by a bond between Cys209 and Cys248, which lies immediately C-terminal to the LRR region of GP Ibα. This is the first report of a gain-of-function phenotype due to a mutation in the LRR region of GP Ibα. Since His86 is more than 20 Å away from the major region of GP Ibα in direct contact with VWF defined by published crystal structures, our data suggests that the leucine-rich repeats regulate the affinity for VWF through long-range allosteric alterations of GP Ibα structure.

The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments.

The glycoprotein Ib (GPIb), a two-chain integral platelet membrane protein, acts as a receptor for von Willebrand factor. In order to obtain information on the domain involved in this function, as well as on the structural organization of GPIb, the protein has been purified and submitted to limited proteolysis using three different enzymes. The resulting fragments were topographically oriented by means of partial NH2-terminal sequence analysis and immunological identification using monoclonal antibodies. One of these antibodies (LJ-Ib1) inhibited the von Willebrand factor-GPIb interaction completely, one (LJ-P3) partially, and one (LJ-Ib10) had no inhibitory effect. Three distinct fragments, the 38-kDa fragment produced by Serratia marcescens protease as well as the 45- and 35-kDa fragments produced by trypsin, had the same NH2 terminus as the intact GPIb alpha-chain (apparent molecular mass = 140 kDa). These fragments and the alpha-chain reacted with the inhibitory antibodies. On the other hand, three fragments produced by Staphylococcus aureus V8 protease, one of 92 kDa similar to the previously described "macroglycopeptide" and two others of 52 and 45 kDa, had NH2-terminal sequences different from that of the GPIb alpha-chain and reacted only with the noninhibitor monoclonal antibody LJIb10. Thus, the binding domain for von Willebrand factor resides near the NH2 terminus of the GPIb alpha-chain, whereas the carbohydrate-rich region is part of the innermost portion of GPIb and does not appear to be involved in the von Willebrand factor binding function.