Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.
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ABSTRACT:
SUBMITTER: Wonerow P
PROVIDER: S-EPMC1222625 | biostudies-other | 2002 Jun
REPOSITORIES: biostudies-other
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Project description:We have investigated the effects of decreased levels of the complex between glycoprotein VI (GPVI) and the Fc receptor gamma-chain (FcRgamma) on responses to collagen and GPVI-specific ligands in murine platelets. We show that levels of GPVI-FcRgamma of the order of 50% and 20% of wild-type levels caused 2- and 5-fold shifts to the right respectively in the dose-response curve for aggregation in response to collagen, the snake toxin convulxin and the monoclonal antibody JAQ1. In addition, there is a delay in the onset of aggregation in response to collagen. In contrast, the stimulation of protein tyrosine phosphorylation by collagen (as measured after 150 s) and adhesion to a collagen-coated surface under static conditions were unaffected in platelets with 50% and 20% of wild-type levels of GPVI. In contrast, responses to a collagen-related peptide (CRP), made up of repeat glycine-proline-hydroxyproline motifs, were markedly inhibited and abolished in platelets expressing 50% and 20% of wild-type levels of GPVI respectively. We suggest that the marked effect of a reduction in GPVI levels on the CRP-induced activation of platelets is due to the multivalent nature of CRP and the fact that GPVI is its sole receptor on platelets. Thus it appears that the interaction of CRP with GPVI is determined by a combination of affinity and avidity. The observation that collagen does not behave like CRP in platelets expressing reduced levels of GPVI, even in the combined presence of blocking antibodies against integrin alpha2beta1 and GPV, suggests that collagen has a greater affinity than CRP for GPVI, and/or that other receptors are involved in its binding to platelets. The clinical significance of these results is discussed.
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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:Integrin engagement induces a cascade of signaling pathways that include tyrosine phosphorylation of numerous proteins that lead to modulation of the actin cytoskeleton. Src is a major intracellular mediator of integrin-dependent functions, but the mechanism(s) by which Src is regulated in response to integrin signals is not fully understood. Here, we demonstrate an important role for phospholipase C gamma 2 (PLCgamma2) in Src activation in the osteoclast. Through analysis of primary cells from PLCgamma2(-/-) mice, PLCgamma2 was found to be an important regulator of alpha(v)beta(3) integrin-mediated bone osteoclast cell adhesion, migration, and bone resorption. Adhesion-induced PYK2 and Src phosphorylation is decreased in the absence of PLCgamma2, and the interaction of Src with beta(3) integrin and PYK2 is dramatically reduced. Importantly, PLCgamma2 was found to be required for proper localization of Src to the sealing actin ring, and this function required both its catalytic activity and adapter domains. Based on these results, we propose that PLCgamma2 influences Src activation by mediating the localization of Src to the integrin complex and thereby regulating integrin-mediated functions in the osteoclast.
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Project description:The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)(10)GCP*G](n); P*=hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P(2)] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca(2+) ([Ca(2+)](i)) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca(2+) by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4, 5)P(3), but not the R28C (Arg(28)-->Cys) mutant which binds to PI(3, 4,5)P(3) with low affinity, also inhibits elevation of [Ca(2+)](i) in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCgamma2. These results demonstrate that PI 3-kinase is required for full activation of PLCgamma2 by GPVI in platelets and megakaryocytes.
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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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Project description:The seven-amino-acid peptide LSARLAF has been reported to activate platelets via the integrin GPIIb-IIIa (glycoprotein IIb-IIIa). Activation by LSARLAF is reinforced by release of ADP and thromboxanes, but the initiating event in the signalling cascade is not known. In the present study, we demonstrate that LSARLAF stimulates Src kinase-dependent tyrosine phosphorylation of many of the proteins in the GPIIb-IIIa cascade, including the tyrosine kinase Syk, the adapter SLP-76 (SH2-containing leucocyte phosphoprotein of 76 kDa) and PLCgamma2 (phospholipase Cgamma2). A critical role for PLCgamma2 in signalling by LSARLAF was demonstrated by abolition of aggregation in PLCgamma2-/- murine platelets to low concentrations of the peptide, although a partial recovery was seen with higher concentrations. In sharp contrast with the GPIIb-IIIa-regulated signalling cascade, aggregation was inhibited in murine platelets deficient in the adapter LAT (linker for activation of T-cells) and the Fc receptor gamma-chain. Aggregation was also partially inhibited by the cholesterol-lowering reagent, beta-methyl-cyclodextrin, at concentrations that disrupt membrane rafts, but do not interfere with signalling by GPIIb-IIIa. Furthermore, LSARLAF also stimulated tyrosine phosphorylation in GPIIb-deficient murine platelets, confirming that the integrin is not critical for activation of intracellular signalling pathways. LSARLAF also stimulated Ca2+ elevation in RBL-2H3 cells, which lack the platelet glycoproteins GPIIb, GPVI and GPIb. These results demonstrate that LSARLAF activates platelets through a PLCgamma2-dependent pathway that lies downstream of Src kinases and which is partially dependent on the Fc receptor gamma-chain, LAT and lipid rafts. The mechanism of cell activation by LSARLAF remains to be established, although the present results indicate that more than one surface glycoprotein may mediate this response.
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Project description:The platelet receptors, glycoprotein VI (GPVI) and integrin α2β1 jointly control collagen-dependent thrombus formation via protein tyrosine kinases. It is unresolved to which extent the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptor PECAM1 and its downstream acting protein tyrosine phosphatase PTPN11 interfere in this process. Here, we hypothesized that integrin α2β1 has a co-regulatory role in the PECAM1- and PTPN11-dependent restraint of thrombus formation. We investigated platelet activation under flow on collagens with a different GPVI dependency and using integrin α2β1 blockage. Blood was obtained from healthy subjects and from patients with Noonan syndrome with a gain-of-function mutation of PTPN11 and variable bleeding phenotype. On collagens with decreasing GPVI activity (types I, III, IV), the surface-dependent inhibition of PECAM1 did not alter thrombus parameters using control blood. Blockage of α2β1 generally reduced thrombus parameters, most effectively on collagen IV. Strikingly, simultaneous inhibition of PECAM1 and α2β1 led to a restoration of thrombus formation, indicating that the suppressing signaling effect of PECAM1 is masked by the platelet-adhesive receptor α2β1. Blood from 4 out of 6 Noonan patients showed subnormal thrombus formation on collagen IV. In these patients, effects of α2β1 blockage were counterbalanced by PECAM1 inhibition to a normal phenotype. In summary, we conclude that the suppression of GPVI-dependent thrombus formation by either PECAM1 or a gain-of-function of PTPN11 can be overruled by α2β1 engagement.
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