scholarly journals Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cγ2 regulation in platelets

2002 ◽  
Vol 364 (3) ◽  
pp. 755-765 ◽  
Author(s):  
Peter WONEROW ◽  
Achim OBERGFELL ◽  
Jonathan I. WILDE ◽  
Régis BOBE ◽  
Naoki ASAZUMA ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Membrane Domains ◽  
Adapter Protein ◽  
Glycoprotein Vi ◽  
Receptor Signalling ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Collagen Receptor ◽  
Lines Of Evidence

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin αIIbβ3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cγ2 (PLCγ2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCγ2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates αIIbβ3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCγ2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and αIIbβ3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCγ2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.

scholarly journals LAT Is Required for Tyrosine Phosphorylation of Phospholipase Cγ2 and Platelet Activation by the Collagen Receptor GPVI

1999 ◽  
Vol 19 (12) ◽  
pp. 8326-8334 ◽  
Author(s):  
Jean-Max Pasquet ◽  
Barbara Gross ◽  
Lynn Quek ◽  
Naoki Asazuma ◽  
Weiguo Zhang ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Human Platelets ◽  
Major Pathway ◽  
Glycoprotein Vi ◽  
Fibrinogen Receptor ◽  
Protein Receptor ◽  
Thrombin Activation ◽  
Granule Secretion ◽  
A Minor ◽  
Collagen Receptor

ABSTRACT In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cγ2 (PLCγ2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcγRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCγ2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCγ2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin αIIbβ3 in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCγ2, leading to downstream responses such as α-granule secretion and activation of integrin αIIbβ3. The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCγ2. We propose a model in which LAT and SLP-76 are required for PLCγ2 phosphorylation but are regulated through independent pathways downstream of Syk.

scholarly journals Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

2004 ◽  
Vol 383 (3) ◽  
pp. 581-588 ◽  
Author(s):  
Gavin E. JARVIS ◽  
Denise BEST ◽  
Steve P. WATSON
Keyword(s):  
Tyrosine Phosphorylation ◽  
Chain Complex ◽  
Fc Receptor ◽  
Adapter Protein ◽  
Wild Type ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Src Homology 2 ◽  
Src Homology ◽  
Collagen Receptor

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP-76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cγ2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (∼10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRγ chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti-(α2 integrin) antibodies Ha1/29 and HMα2, but not by blockade of αIIbβ3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI–FcRγ chain complex, and is facilitated by binding of collagen to integrin α2β1.

The Role of ITAM- and ITIM-coupled Receptors in Platelet Activation by Collagen

2001 ◽  
Vol 86 (07) ◽  
pp. 276-288 ◽  
Author(s):  
Naoki Asazuma ◽  
Ben Atkinson ◽  
Oscar Berlanga ◽  
Denise Best ◽  
Regis Bobe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Platelet Activation ◽  
T Lymphocyte ◽  
Pivotal Role ◽  
Membrane Domains ◽  
Glycoprotein Vi ◽  
Ig Superfamily ◽  
Collagen Receptor ◽  
Lymphocyte Receptors

SummaryThe major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned within the last two years. It is a member of the Ig superfamily of proteins and is constitutively associated with the ITAM-bearing Fc receptor γ-chain (FcR γ-chain). GPVI signals through a pathway that involves several of the proteins used by Fc, B- and T-lymphocyte receptors and which takes place in glycolipid-enriched membrane domains in the plasma membrane known as GEMs. Responses to GPVI are regulated by PECAM-1 (CD31) and possibly other ITIM-bearing receptors. Despite a pivotal role for GPVI, there are important differences between signalling events to collagen and GPVI-specific ligands. This may reflect a role for co-receptors in the response to collagen.

The Regulatory Role Of Camp In Induction Of Human Platelet Receptor For Fibrinogen

1981 ◽  
Author(s):  
S E Graber ◽  
J Hawiger
Keyword(s):  
Human Platelets ◽  
Membrane Receptor ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Platelet Receptor ◽  
The Relationship ◽  
Camp Levels ◽  
Dose Dependent ◽  
Stimulation Of

Membrane receptor for fibrinogen plays an essential role in adhesion and aggregation of human platelets by allowing fibrinogen to bridge two or more platelets together. Whereas in normal, unstimulated platelets fibrinogen receptor is not available, it becomes mobilized upon stimulation of platelets with thrombin, ADP, and other stimuli. The mechanism(s) regulating availability of membrane receptor for fibrinogen remains unknown. Following our recent demonstration that prostacyclin (PGI2) prevents mobilization of fibrinogen receptor by thrombin and ADP (Nature 1980, 283,195), we investigated the relationship between cAMP levels and fibrinogen receptor availability. Platelets separated from plasma proteins were briefly exposed to a low thrombin concentration (0.05 U/ml) followed by hirudin to inactivate free thrombin. Binding of 125I-fi- brinogen and cAMP levels were determined in parallel samples. A dose-dependent rise in platelet cAMP levels from 3.3 pM to 10.3 pM/108 platelets in response to PGI2 (3×10-9M - 3×108M) was accompanied by a corresponding inhibition of 125I-fibrinogen binding. The degree of the cAMP increment correlated with binding inhibition (r=0.96). The inhibition of 125I-fibrinogen binding by PGI2 was sustained up to 120 min and was paralleled by a persistent rise in cAMP level. Stimulation of platelet cAMP synthesis “from within” by a ribosylation of the nucleotide regulatory component with subunit A1 of cholera toxin also increased cAMP levels and inhibited fibrinogen receptor mobilization.These results provide evidence that “up and down” regulation of fibrinogen receptor in platelets is linked to changes in cAMP levels induced by different types of adenyl cyclase antagonists and agonists.

Fyn and Lyn phosphorylate the Fc receptor γ chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4246-4253 ◽  
Author(s):  
Lynn S. Quek ◽  
Jean-Max Pasquet ◽  
Ingeborg Hers ◽  
Richard Cornall ◽  
Graham Knight ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Fc Receptor ◽  
Inhibitory Pathway ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Granule Secretion ◽  
Activation Motif ◽  
Additional Role ◽  
Feedback Pathway

Abstract Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor γ (FcRγ chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn−/−platelets, tyrosine phosphorylation of FcRγ chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn−/−platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and α-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn−/−lyn−/−double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLCγ2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway.

A new monoclonal antibody, mAb 204-11, that influences the binding of platelet GPVI to fibrous collagen

2003 ◽  
Vol 89 (06) ◽  
pp. 996-1003 ◽  
Author(s):  
Jun Mizuguchi ◽  
Sachiko Kawashima ◽  
Michiko Nagamatsu ◽  
Yoshiki Miura ◽  
Tomohiro Nakagaki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Binding Site ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Collagen Binding ◽  
Glycoprotein Vi ◽  
Phosphorylation Of Proteins ◽  
Dimeric Form ◽  
Collagen Receptor

SummaryThe newly identified platelet collagen receptor glycoprotein VI binds to fibrous collagen, inducing platelet activation. Several antibodies against GPVI have been reported, including a patient’s auto-antibodies, that activates platelets through their ability to crosslink this glycoprotein. We have developed a monoclonal antibody (mAb) against GPVI using the recombinant extracellular domain of GPVI as an antigen. This antibody, mAb 204-11, induced platelet aggregation and tyrosine phosphorylation of proteins similar to those induced by GPVI-reactive proteins, collagen and convulxin. Its interaction with GPVI was analyzed by measuring the effect of the antibody on GPVI binding to collagen using a dimeric form of recombinant GPVI, GPVI-Fc2. MAb 204-11 inhibited the binding of GPVI-Fc2 to fibrous collagen particles, but enhanced the GPVI binding to immobilized collagen, suggesting that the antibody binds to a region near the collagen binding site of GPVI. MAb 204-11 also inhibited the GPVI binding to convulxin at a low concentration, but not completely. Since mAb 204-11 reacts specifically with GPVI and is applicable for immunoblotting and immunoprecipitation, this antibody would be useful for studies on GPVI.

Role of Lipid Rafts in GPVI Agonist-Induced Platelet Signaling.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3576-3576
Author(s):  
Patricia G. Quinter ◽  
Todd M. Quinton ◽  
Carol A. Dangelmaier ◽  
Satya P. Kunapuli ◽  
James L. Daniel
Keyword(s):  
Platelet Aggregation ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Glycoprotein Vi ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Multiple Receptors ◽  
Platelet Signaling ◽  
Collagen Receptor

Abstract The collagen receptor glycoprotein VI (GPVI), plays an essential role in platelet activation and the regulation of hemostasis. Microdomains within the plasma membrane, called lipid rafts, have been implicated in GPVI signaling. The GPVI receptor has been shown to associate with the lipid rafts in both resting and activated platelets. It has been reported that there is a reduction in GPVI signaling in raft-disrupted platelets following activation with various GPVI agonists, especially at low to moderate agonist concentrations. Since platelet aggregation is potentiated by secreted adenosine 5′-diphosphate (ADP) at low concentrations of convulxin and at all concentrations of collagen and collagen-related peptide (CRP), we wanted to determine whether the decrease in GPVI signaling found in platelets with disrupted rafts was due to the loss of agonist potentiation by ADP. We compared platelet aggregation, protein phosphorylation, and calcium mobilization in platelets with intact and disrupted lipid rafts following activation with the GPVI agonists, collagen, convulxin and CRP. We show that lipid raft disruption inhibits aggregation induced by collagen and convulxin, but this inhibition is no longer apparent in the presence of ADP feedback inhibitors. Furthermore, raft-disrupted platelets had the same level of phosphorylation of proteins involved in GPVI signaling (i.e. Syk, LAT, and PLCγ2) and the same ability to mobilize calcium following activation with collagen or convulxin. Therefore, the effects of lipid raft disruption on aggregation can be attributed to the loss of ADP feedback. Interestingly, however, raft disruption directly inhibited aggregation and Syk phosphorylation induced by CRP in the presence and absence of ADP feedback. We propose that these differences are due to the fact that CRP is a relatively small, synthesized peptide of 37 amino acids, while collagen and convulxin are large ligands. These agonists are all able to bind the GPVI receptor, but they may not have the same ability to simultaneously cluster multiple receptors due to their size differential. The lipid rafts may be important for CRP stimulation, but not for collagen or convulxin, because they may have a higher density of the GPVI receptor than nonraft membrane regions, allowing CRP to cluster multiple receptors and activate the GPVI signaling cascade. When we disrupt the lipid rafts, we are reducing the effective concentration of GPVI available for activation by CRP but not by collagen or convulxin.

Role of the APS adapter protein in insulin receptor signalling

2000 ◽  
Vol 28 (5) ◽  
pp. A270-A270
Author(s):  
Z. Ahmed ◽  
B. J. Smith ◽  
P. Wylie ◽  
T. S. Pillay
Keyword(s):  
Insulin Receptor ◽  
Adapter Protein ◽  
Receptor Signalling ◽  
Insulin Receptor Signalling

Regulation and Function of WASp in Platelets by the Collagen Receptor, Glycoprotein VI

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4166-4176 ◽  
Author(s):  
Barbara S. Gross ◽  
Jonathan I. Wilde ◽  
Lynn Quek ◽  
Helen Chapel ◽  
David L. Nelson ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Kinase Inhibitors ◽  
Critical Role ◽  
Functional Expression ◽  
Apparent Difference ◽  
Gene Encoding ◽  
Glycoprotein Vi ◽  
Dense Granules ◽  
Collagen Receptor

Abstract Wiskott Aldrich syndrome (WAS) is an X-linked recessive disorder associated with abnormalities in platelets and lymphocytes giving rise to thrombocytopenia and immunodeficiency. WAS is caused by a mutation in the gene encoding the cytoskeletal protein (WASp). Despite its importance, the role of WASp in platelet function is not established. WASp was recently shown to undergo tyrosine phosphorylation in platelets after activation by collagen, suggesting that it may play a selective role in activation by the adhesion molecule. In the present study, we show that WASp is heavily tyrosine phosphorylated by a collagen-related peptide (CRP) that binds to the collagen receptor glycoprotein (GP) VI, but not to the integrin 2β1. Tyrosine phosphorylation of WASp was blocked by Src family kinase inhibitors and reduced by treatment with wortmannin and in patients with X-linked agammaglobulinemia (XLA), a condition caused by a lack of functional expression of Btk. This indicates that Src kinases, phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to the regulation of tyrosine phosphorylation of WASp. The functional importance of WASp was investigated in 2 WAS brothers who show no detectable expression of WASp. Platelet aggregation and secretion from dense granules induced by CRP and thrombin was slightly enhanced in the WAS platelets relative to controls. Furthermore, there was no apparent difference in morphology in WAS platelets after stimulation by these agonists. These observations suggest that WASp does not play a critical role in intracellular signaling downstream of tyrosine kinase-linked and G protein-coupled receptors in platelets.

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