Glycoprotein Ib beta is the only phosphorylated major membrane glycoprotein in human platelets.
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ABSTRACT:
SUBMITTER: Wyler B
PROVIDER: S-EPMC1146575 | biostudies-other | 1986 Mar
REPOSITORIES: biostudies-other
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Project description:Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3-38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation.
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Project description:Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.
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Project description:Platelet adhesion to the sub-endothelial matrix and damaged endothelium occurs through a multi-step process mediated in the initial phase by glycoprotein Ib binding to von Willebrand factor (vWF), which leads to the subsequent formation of a platelet plug. The plant-derived ?-3 fatty acid ?-linolenic acid is an abundant alternative to fish-derived n-3 fatty acids and has anti-inflammatory and antithrombotic properties. In this study, we investigated the impact of ?-linolenic acid on human platelet binding to vWF under high-shear flow conditions (mimicking blood flow in stenosed arteries). Pre-incubation of fresh human blood from healthy donors with ?-linolenic acid at dietary relevant concentrations reduced platelet binding and rolling on vWF-coated microchannels at a shear rate of 100 dyn/cm2 Depletion of membrane cholesterol by incubation of platelet-rich plasma with methyl-? cyclodextrin abrogated platelet rolling on vWF. Analysis of glycoprotein Ib by applying cryo-electron tomography to intact platelets revealed local clusters of glycoprotein Ib complexes upon exposure to shear force: the formation of these complexes could be prevented by treatment with ?-linolenic acid. This study provides novel findings on the rapid local rearrangement of glycoprotein Ib complexes in response to high-shear flow and highlights the mechanism of in vitro inhibition of platelet binding to and rolling on vWF by ?-linolenic acid.
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Project description:The impact of fluid flow on structure and dynamics of biomolecules has recently gained much attention. In this article, we present a molecular-dynamics algorithm that serves to generate stable water flow under constant temperature, for the study of flow-induced protein behavior. Flow simulations were performed on the 16-residue beta-switch region of platelet glycoprotein Ibalpha, for which crystal structures of its N-terminal domain alone and in complex with the A1 domain of von Willebrand factor have been solved. Comparison of the two structures reveals a conformational change in this region, which, upon complex formation, switches from an unstructured loop to a beta-hairpin. Interaction between glycoprotein Ibalpha and von Willebrand factor initiates platelet adhesion to injured vessel walls, and the adhesion is enhanced by blood flow. It has been hypothesized that the loop to beta-hairpin transition in glycoprotein Ib alpha is induced by flow before binding to von Willebrand factor. The simulations revealed clearly a flow-induced loop-->beta-hairpin transition. The transition is dominated by the entropy of the protein, and is seen to occur in two steps, namely a dihedral rotation step followed by a side-group packing step.
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Project description:Since the hemorrhage in severe dengue seems to be primarily related to the defect of the platelet, the possibility that dengue virus (DENV) is selectively tropic for one of its surface receptors was investigated. Flow cytometric data of DENV-infected megakaryocytic cell line superficially expressing human glycoprotein Ib (CD42b) and glycoprotein IIb/IIIa (CD41 and CD41a) were analyzed by our custom-written software in MATLAB. In two-dimensional analyses, intracellular DENV was detected in CD42b+, CD41+ and CD41a+ cells. In three-dimensional analyses, the DENV was exclusively detected in CD42b+ cells but not in CD42b- cells regardless of the other expressions. In single-cell virus-protein analyses, the amount of DENV was directly correlated with those of CD42b at the Pearson correlation coefficient of 0.9. Moreover, RT- PCR and apoptosis assays showed that DENV was able to replicate itself and release its new progeny from the infected CD42b+ cells and eventually killed those cells. These results provide evidence for the involvement of CD42b in DENV infection.
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Project description:Interaction of glycoprotein Ib alpha (GPIb alpha) with von Willebrand factor (VWF) initiates platelet adhesion to injured vascular wall to stop bleeding. A major contact between GPIb alpha and VWF involves the beta-switch region, which is a loop in the unliganded GPIb alpha but switches to a beta-hairpin in the complex structure. Paradoxically, flow enhances rather than impedes GPIb alpha-VWF binding. Gain-of-function mutations (e.g., M239V) in the beta-switch reduce the flow requirement for VWF binding, whereas loss-of-function mutations (e.g., A238V) increase the flow requirement. These phenomena cannot be explained by crystal structures or energy calculations. Herein we demonstrate that the beta-hairpin is unstable without contacting VWF, in that it switches to a loop in free molecular dynamics simulations. Simulations with a novel flow molecular dynamics algorithm show that the loop conformation is unstable in the presence of flow, as it switches to beta-hairpin even without contacting VWF. Compared with the wild-type, it is easier for the M239V mutant but harder for the A238V mutant to switch to beta-hairpin in the presence of flow. These results elucidate the structural basis for the two mutants and suggest a regulatory mechanism by which flow activates GPIb alpha via inducing a loop-to-beta-hairpin conformational transition on the beta-switch, thereby promoting VWF binding.
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Project description:BACKGROUND: Cold storage of platelets reduces bacterial growth and preserves their hemostatic properties better than current procedures do. However, storage at 0°C induces [14-3-3?-glycoprotein Ib?] association, 14-3-3? release from phospho-Bad, Bad activation and apoptosis. DESIGN AND METHODS: We investigated whether arachidonic acid, which also binds 14-3-3?, contributes to coldinduced apoptosis. RESULTS: Cold storage activated P38-mitogen-activated protein kinase and released arachidonic acid, which accumulated due to cold inactivation of cyclooxygenase-1/thromboxane synthase. Accumulated arachidonic acid released 14-3-3? from phospho-Bad and decreased the mitochondrial membrane potential, which are steps in the induction of apoptosis. Addition of arachidonic acid did the same and its depletion made platelets resistant to cold-induced apoptosis. Incubation with biotin-arachidonic acid revealed formation of an [arachidonic acid-14-3-3?-glycoprotein Ib?] complex. Indomethacin promoted complex formation by accumulating arachidonic acid and released 14-3-3? from cyclo-oxygenase-1. Arachidonic acid depletion prevented the cold-induced reduction of platelet survival in mice. CONCLUSIONS: We conclude that cold storage induced apoptosis through an [arachidonic acid-14-3-3?-glycoprotein Ib?] complex, which released 14-3-3? from Bad in an arachidonic acid-dependent manner. Although arachidonic acid depletion reduced agonist-induced thromboxane A(2) formation and aggregation, arachidonic acid repletion restored these functions, opening ways to reduce apoptosis during storage without compromising hemostatic functions post-transfusion.
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Project description: Not available
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Project description:Donor-independent platelet concentrates for transfusion can be produced in vitro from induced pluripotent stem cells (iPSCs). However, culture at 37°C induces ectodomain shedding on platelets of glycoprotein Ib? (GPIb?), the von Willebrand factor receptor critical for adhesive function and platelet lifetime in vivo, through temperature-dependent activation of a disintegrin and metalloproteinase 17 (ADAM17). The shedding can be suppressed by using inhibitors of panmetalloproteinases and possibly of the upstream regulator p38 mitogen-activated protein kinase (p38 MAPK), but residues of these inhibitors in the final platelet products may be accompanied by harmful risks that prevent clinical application. Here, we optimized the culture conditions for generating human iPSC-derived GPIb?+ platelets, focusing on culture temperature and additives, by comparing a new and safe selective ADAM17 inhibitor, KP-457, with previous inhibitors. Because cultivation at 24°C (at which conventional platelet concentrates are stored) markedly diminished the yield of platelets with high expression of platelet receptors, 37°C was requisite for normal platelet production from iPSCs. KP-457 blocked GPIb? shedding from iPSC platelets at a lower half-maximal inhibitory concentration than panmetalloproteinase inhibitor GM-6001, whereas p38 MAPK inhibitors did not. iPSC platelets generated in the presence of KP-457 exhibited improved GPIb?-dependent aggregation not inferior to human fresh platelets. A thrombus formation model using immunodeficient mice after platelet transfusion revealed that iPSC platelets generated with KP-457 exerted better hemostatic function in vivo. Our findings suggest that KP-457, unlike GM-6001 or p38 MAPK inhibitors, effectively enhances the production of functional human iPSC-derived platelets at 37°C, which is an important step toward their clinical application. Stem Cells Translational Medicine 2017;6:720-730.
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Project description:Glycoprotein (GP) VI is a critical platelet collagen receptor. Phosphoinositide 3-kinase (PI3K) plays an important role in GPVI-mediated platelet activation, yet the major PI3K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3K. Thus, we investigated the contribution of PI3K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y(12) receptor antagonist AR-C69931MX partly reduced GPVI-induced Akt phosphorylation. Platelets from mice dosed with clopidogrel also showed partial Akt phosphorylation, indicating that GPVI-mediated Akt phosphorylation is regulated by both secretion-dependent and -independent pathways. In addition, GPVI-induced Akt phosphorylation in the presence of ADP antagonists was completely inhibited by PI3K inhibitor LY294002 and PI3Kbeta inhibitor TGX-221 indicating an essential role of PI3Kbeta in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca(2+) mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3Kalpha inhibitor PIK75, but were not affected by PI3Kgamma inhibitor AS252424 and PI3Kdelta inhibitor IC87114. Consistently, GPVI-induced integrin alpha(IIb)beta(3) activation of PI3Kgamma(-/-) and PI3Kdelta(-/-) platelets also showed no significant difference compared with wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on G(i) stimulation through P2Y(12) receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI3Kbeta plays an essential role in GPVI-mediated platelet aggregation and Akt activation.
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