Force-Regulated Refolding of the Mechanosensory Domain in the Platelet Glycoprotein Ib-IX Complex.
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ABSTRACT:
SUBMITTER: Zhang XF
PROVIDER: S-EPMC6531785 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:How glycoprotein (GP)Ib-IX complex on the platelet surface senses the blood flow through its binding to the plasma protein von Willebrand factor (VWF) and transmits a signal into the platelet remains unclear. Here we show that optical tweezer-controlled pulling of the A1 domain of VWF (VWF-A1) on GPIb-IX captured by its cytoplasmic domain induced unfolding of a hitherto unidentified structural domain before the dissociation of VWF-A1 from GPIb-IX. Additional studies using recombinant proteins and mutant complexes confirmed its existence in GPIb-IX and enabled localization of this quasi-stable mechanosensitive domain of ∼60 residues between the macroglycopeptide region and the transmembrane helix of the GPIbα subunit. These results suggest that VWF-mediated pulling under fluid shear induces unfolding of the mechanosensitive domain in GPIb-IX, which may possibly contribute to platelet mechanosensing and/or shear resistance of VWF-platelet interaction. The identification of the mechanosensitive domain in GPIb-IX has significant implications for the pathogenesis and treatment of related blood diseases.
| S-EPMC4296016 | biostudies-literature
Project description:The glycoprotein Ib-IX (GPIb-IX) complex expressed on platelet plasma membrane is involved in thrombosis and hemostasis via the initiation of adhesion of platelets to von Willebrand factor (VWF) exposed at the injured vessel wall. While most of the knowledge of the GPIb-IX complex was obtained from studies on platelets and transfected mammalian cells expressing the GPIb-IX complex, there is not an in vitro membrane system that allows systematic analysis of this receptor. The phospholipid bilayer Nanodisc composed of a patch of phospholipid surrounded by membrane scaffold protein is an attractive tool for membrane protein study. We show here that the GPIb-IX complex purified from human platelets has been reconstituted into the Nanodisc. The Nanodisc-reconstituted GPIb-IX complex was able to bind various conformation-sensitive monoclonal antibodies. Furthermore, it bound to VWF in the presence of botrocetin with an apparent K(d) of 0.73 ± 0.07 nM. The binding to VWF was inhibited by anti-GPIb? antibodies with epitopes overlapping with the VWF-binding site, but not by anti-GPIb? monoclonal antibody RAM.1. Finally, the Nanodisc-reconstituted GPIb-IX complex exhibited ligand binding activity similar to that of the isolated extracellular domain of GPIb?. In conclusion, the GPIb-IX complex in Nanodiscs adopts a native-like conformation and possesses the ability to bind its natural ligands, thus making a Nanodisc a suitable in vitro platform for further investigation of this hemostatically important receptor complex.
| S-EPMC3552449 | biostudies-literature
Project description:Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbα and the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbα and filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbα are involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.
| S-EPMC4982925 | biostudies-literature
Project description:The glycoprotein (GP) Ib-IX-V complex, the von Willebrand factor receptor on the platelet surface, is critically involved in hemostasis and thrombosis. The GPV subunit interacts with GPIb-IX to form the GPIb-IX-V complex, but the underlying molecular basis remains unclear. It was observed earlier that efficient expression of GPV in the plasma membrane requires co-expression of GPIb-IX.Hypothesizing that GPIb-IX stabilizes GPV through direct interaction and consequently enhances GPV surface expression, we aim in this study to identify structural elements in the complex that mediate the interaction between GPV and GPIb-IX by analyzing mutational effects on GPV surface expression in transfected Chinese hamster ovary cells.Enhancement of GPV surface expression by GPIb-IX requires transmembrane domains of both GPV and GPIb?, as replacing the GPV transmembrane domain with an unrelated poly-leucine-alanine sequence abolished the enhancing effect of GPIb-IX. Additional mutagenesis analysis of the GPV transmembrane helix identified three helical sides containing conserved polar residues as critical to efficient GPV surface expression. Similarly, replacing residues in three sides (Gly495/Ala502/Leu509, Phe491/Trp498/Val505, and Y492/L499/L506) of the GPIb? transmembrane domain with leucines preserved the surface expression level of GPIb-IX but significantly altered that of GPV.Our results demonstrate for the first time the importance of transmembrane domains for efficient surface expression of GPV and suggest that GPV and GPIb? transmembrane domains interact with each other, contributing to assembly of the GPIb-IX-V complex.
| S-EPMC3499136 | biostudies-literature
Project description: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.
| S-EPMC2563799 | biostudies-literature
Project description:We have previously reported that the structural elements of the GP Ib-IX complex required for its localization to glycosphingolipid-enriched membranes (GEMs) reside in the Ibβ and IX subunits. To identify them, we generated a series of cell lines expressing mutant GP Ibβ and GP IX where 1) the cytoplasmic tails (CTs) of either or both GP Ibβ and IX are truncated, and 2) the transmembrane domains (TMDs) of GP Ibβ and GP IX were swapped with the TMD of a non-GEMs associating molecule, human transferrin receptor. Sucrose density fractionation analysis showed that the removal of either or both of the CTs from GP Ibβ and GP IX does not alter GP Ibα-GEMs association when compared with the wild type. In contrast, swapping of the TMDs of either GP Ibβ or GP IX with that of transferrin receptor results in a significant loss (∼ 50%) of GP Ibα from the low density GEMs fractions, with the largest effect seen in the dual TMD-replaced cells (> 80% loss) when compared with the wild type cells (100% of GP Ibα present in the GEMs fractions). Under high shear flow, the TMD-swapped cells adhere poorly to a von Willebrand factor-immobilized surface to a much lesser extent than the previously reported disulfide linkage dysfunctional GP Ibα-expressing cells. Thus, our data demonstrate that the bundle of GP Ibβ and GP IX TMDs instead of their individual CTs is the structural element that mediates the β/IX complex localization to the membrane GEMs, which through the α/β disulfide linkage brings GP Ibα into the GEMs.
| S-EPMC4571966 | biostudies-literature
Project description:OBJECTIVE:The platelet glycoprotein Ib-IX (GP Ib-IX) receptor is a well-characterized adhesion receptor supporting hemostasis and thrombosis via interactions with von Willebrand factor. We examine the GP Ib-IX/von Willebrand factor axis in murine polymicrobial sepsis, as modeled by cecal ligation and puncture (CLP). APPROACH AND RESULTS:Genetic absence of the GP Ib-IX ligand, von Willebrand factor, prolongs survival after CLP, but absence of the receptor, GP Ib-IX, does not. Because absence of either von Willebrand factor or GP Ib-IX significantly impairs hemostasis and thrombosis, we sought to define additional GP Ib-IX-dependent pathways impacting survival in the CLP model. We document that the absence of GP Ib-IX leads to reduced platelet-neutrophil and platelet-monocyte interactions. Twenty-four hours after CLP, absence of GP Ib-IX coincides with an alteration in cytokine levels, such as tumor necrosis factor-? secreted by monocytes, and increased macrophage-1 antigen expression by neutrophils. CONCLUSIONS:In contrast to the well-characterized proinflammatory properties of platelets, we describe in the CLP model an anti-inflammatory property associated with platelet GP Ib-IX. Thus, a single platelet receptor displays a dual modulatory role in both the thrombotic and inflammatory pathways associated with polymicrobial sepsis. In sharing leucine-rich motifs with toll-like receptors, platelet GP Ib-IX can be considered a multifunctional participant in hemostasis, thrombosis, and the inflammatory cascade. The results highlight a dynamic role for platelets in systemic inflammation and add to the complex pathophysiologic events that occur during the dysregulated coagulation and inflammation associated with sepsis.
| S-EPMC3991762 | biostudies-literature
Project description:Members of the 14-3-3 family of proteins function as adapters/modulators that recognize phosphoserine/phosphothreonine-based binding motifs in many intracellular proteins and play fundamental roles in signal transduction pathways of eukaryotic cells. In platelets, 14-3-3 plays a wide range of regulatory roles in phosphorylation-dependent signaling pathways, including G-protein signaling, cAMP signaling, agonist-induced phosphatidylserine exposure, and regulation of mitochondrial function. In particular, 14-3-3 interacts with several phosphoserine-dependent binding sites in the major platelet adhesion receptor, the glycoprotein Ib-IX complex (GPIb-IX), regulating its interaction with von Willebrand factor (VWF) and mediating VWF/GPIb-IX-dependent mechanosignal transduction, leading to platelet activation. The interaction of 14-3-3 with GPIb-IX also plays a critical role in enabling the platelet response to low concentrations of thrombin through cooperative signaling mediated by protease-activated receptors and GPIb-IX. The various functions of 14-3-3 in platelets suggest that it is a possible target for the treatment of thrombosis and inflammation.
| S-EPMC5981166 | biostudies-literature
Project description:The glycoprotein (GP)Ib-IX complex is critical to hemostasis and thrombosis. Its proper assembly is closely correlated with its surface expression level and requires cooperative interactions among extracellular and transmembrane domains of Ib?, Ib? and IX subunits. Two interfaces have been previously identified between the extracellular domains of Ib? and IX.To understand how extracellular domains interact in GPIb-IX.The Ib? extracellular domain (Ib?E ) or the IX counterpart (IXE ) in GPIb-IX was replaced with a well-folded Ib?E /IXE chimera called Ib?Eabc , and the effect of domain replacement on assembly and expression of the receptor complex in transiently transfected Chinese hamster ovary cells was analyzed.Replacing IXE with Ib?Eabc in GPIb-IX retained interface 1 but not interface 2 between the extracellular domains. While this domain replacement preserved complex integrity, the expression levels of Ib? and Ib? were significantly reduced. Additional domain replacement with Ib?Eabc or Ib?E in GPIb-IX produced the complex at disparate expression levels that cannot be simply explained by two separate interfaces. In particular, when Ib?E in GPIb-IX was replaced by Ib?Eabc , Ib? and IX were expressed at approximately 70% of the wild-type level. Their levels were not reduced when IXE was changed further to Ib?E .Our results demonstrate the importance of the association between Ib? and IX extracellular domains for complex assembly and efficient expression, and provide evidence for the structural malleability of these domains that may accommodate and propagate conformational changes therein.
| S-EPMC4137403 | biostudies-literature
Project description:Platelets are generated from the cytoplasm of megakaryocytes (MKs) via actin cytoskeleton reorganization. Zyxin is a focal adhesion protein and wildly expressed in eukaryotes to regulate actin remodeling. Zyxin is upregulated during megakaryocytic differentiation; however, the role of zyxin in thrombopoiesis is unknown. Here we show that zyxin ablation results in profound macrothrombocytopenia. Platelet lifespan and thrombopoietin level were comparable between wild-type and zyxin-deficient mice, but MK maturation, demarcation membrane system formation, and proplatelet generation were obviously impaired in the absence of zyxin. Differential proteomic analysis of proteins associated with macrothrombocytopenia revealed that glycoprotein (GP) Ib-IX was significantly reduced in zyxin-deficient platelets. Moreover, GPIb-IX surface level was decreased in zyxin-deficient MKs. Knockdown of zyxin in a human megakaryocytic cell line resulted in GPIbα degradation by lysosomes leading to the reduction of GPIb-IX surface level. We further found that zyxin was colocalized with vasodilator-stimulated phosphoprotein (VASP), and loss of zyxin caused diffuse distribution of VASP and actin cytoskeleton disorganization in both platelets and MKs. Reconstitution of zyxin with VASP binding site in zyxin-deficient hematopoietic progenitor cell-derived MKs restored GPIb-IX surface expression and proplatelet generation. Taken together, our findings identify zyxin as a regulator of platelet biogenesis and GPIb-IX surface expression through VASP-mediated cytoskeleton reorganization, suggesting possible pathogenesis of macrothrombocytopenia.
| S-EPMC8520529 | biostudies-literature