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.

Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3561-3569 ◽  
Author(s):  
Andrew C. Pearce ◽  
Jonathan I. Wilde ◽  
Gina M. Doody ◽  
Denise Best ◽  
Osamu Inoue ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
G Protein ◽  
Cell Types ◽  
Human Platelets ◽  
Glycoprotein Vi ◽  
Activation Motif ◽  
G Protein Coupled ◽  
Collagen Receptor

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)–coupled collagen receptor GPVI and by the G protein–coupled receptor agonist thrombin. The glycoprotein VI (GPVI)–specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

Glycoprotein VI is associated with GPIb-IX-V on the membrane of resting and activated platelets

2005 ◽  
Vol 93 (04) ◽  
pp. 716-723 ◽  
Author(s):  
Elizabeth Gardiner ◽  
Maria Matzaris ◽  
Simon Taylor ◽  
Lakshmi Wijeyewickrema ◽  
Yukio Ozaki ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Activated Platelets ◽  
Low Shear Stress ◽  
Pre Treatment ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Collagen Receptor ◽  
Low Shear

SummaryThe platelet collagen receptor, glycoprotein (GP)VI, initiates platelet aggregation at low shear stress while GPIb-IX-V, which binds von Willebrand factor, elicits platelet aggregation under high shear conditions. To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface, we first ascertained that aggregation induced by a GPVI-specific agonist, collagen-related peptide, like collagen, is markedly cross-blocked by a GPIbα-specific monoclonal antibody, SZ2. Immunoprecipitation of GPIb-IX with anti-GPIbα from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblot-ting with anti-GPVI demonstrated association between GPIb-IX and GPVI. This association was maintained when platelets were activated by thrombin. Pre-treatment of platelets with methyl-β-cyclodextrin to disrupt lipid rafts did not affect association in resting platelets under these conditions of detergent lysis. The association is also independent of cytoskeletal attachment, since it was unaffected by treatment with N-ethylmaleimide or DNaseI, which dissociate GPIb-IX from filamin and the actin-containing cytoskeleton, respectively. Finally, the association involves an interaction between the ectodomains of GPIbα and GPVI, since soluble fragments of GPIbα (glycocalicin) and GPVI are co-precipitated from the platelet supernatant under conditions where GPVI is shed. A contribution of GPIb-IX-V to GPVI-induced platelet responses, and vice versa, therefore warrants further investigation.

Testosterone Potentiation of Ionophore and ADP Induced Platelet Aggregation : Relationship to Arachidonic Acid Metabolism

1981 ◽  
Vol 46 (02) ◽  
pp. 538-542 ◽  
Author(s):  
R Pilo ◽  
D Aharony ◽  
A Raz
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Unsaturated Fatty Acids ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Arachidonic Acid Metabolism ◽  
Ionophore A23187 ◽  
Low Concentrations ◽  
Induced Aggregation

SummaryThe role of arachidonic acid oxygenated products in human platelet aggregation induced by the ionophore A23187 was investigated. The ionophore produced an increased release of both saturated and unsaturated fatty acids and a concomitant increased formation of TxA2 and other arachidonate products. TxA2 (and possibly other cyclo oxygenase products) appears to have a significant role in ionophore-induced aggregation only when low concentrations (<1 μM) of the ionophore are employed.Testosterone added to rat or human platelet-rich plasma (PRP) was shown previously to potentiate platelet aggregation induced by ADP, adrenaline, collagen and arachidonic acid (1, 2). We show that testosterone also potentiates ionophore induced aggregation in washed platelets and in PRP. This potentiation was dose and time dependent and resulted from increased lipolysis and concomitant generation of TxA2 and other prostaglandin products. The testosterone potentiating effect was abolished by preincubation of the platelets with indomethacin.

Functional Involvement of Thrombospondin in Platelet Aggregation Induced by Low Versus High Concentrations of Thrombin

1986 ◽  
Vol 55 (01) ◽  
pp. 136-142 ◽  
Author(s):  
K J Kao ◽  
David M Shaut ◽  
Paul A Klein
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Inhibitory Effect ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Platelet Binding ◽  
Functional Involvement ◽  
Thrombin Activation ◽  
Platelet Aggregates ◽  
High Concentrations

SummaryThrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (≤0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (≥60 μg/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (≥60 μg/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin. In conclusion, our findings demonstrate that TSP is functionally important for platelet aggregation induced by low (≤0.05 U/ml) but not high (≥0.1 U/ml) concentrations of thrombin. High concentrations of exogenous TSP may univalently saturate all its platelet binding sites consequently interfering with TSP-crosslinking of thrombin-activated platelets.

scholarly journals Impaired platelet responses to thrombin and collagen in AKT-1–deficient mice

Blood ◽  
2004 ◽  
Vol 104 (6) ◽  
pp. 1703-1710 ◽  
Author(s):  
Juhua Chen ◽  
Sarmishtha De ◽  
Derek S. Damron ◽  
William S. Chen ◽  
Nissim Hay ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Dense Granules ◽  
Fibrinogen Binding ◽  
Downstream Effectors ◽  
Low Concentrations ◽  
Phosphoinositide 3 Kinase ◽  
Deficient Mice ◽  
Granule Release

Abstract We investigated the role of Akt-1, one of the major downstream effectors of phosphoinositide 3-kinase (PI3K), in platelet function using mice in which the gene for Akt-1 had been inactivated. Using ex vivo techniques, we showed that Akt-1-deficient mice exhibited impaired platelet aggregation and spreading in response to various agonists. These differences were most apparent in platelets activated with low concentrations of thrombin. Although Akt-1 is not the predominant Akt isoform in mouse platelets, its absence diminished the amount of total phospho-Akt and inhibited increases in intracellular Ca2+ concentration in response to thrombin. Moreover, thrombin-induced platelet α-granule release as well as release of adenosine triphosphate from dense granules was also defective in Akt-1-null platelets. Although the absence of Akt-1 did not influence expression of the major platelet receptors for thrombin and collagen, fibrinogen binding in response to these agonists was significantly reduced. As a consequence of impaired αIIbβ3 activation and platelet aggregation, Akt-1 null mice showed significantly longer bleeding times than wild-type mice. (Blood. 2004;104:1703-1710)

scholarly journals Function of Platelet Glycosphingolipid Microdomains/Lipid Rafts

2020 ◽  
Vol 21 (15) ◽  
pp. 5539
Author(s):  
Keisuke Komatsuya ◽  
Kei Kaneko ◽  
Kohji Kasahara
Keyword(s):  
Lipid Rafts ◽  
Platelet Adhesion ◽  
G Protein Coupled Receptors ◽  
Clot Retraction ◽  
Functional Roles ◽  
Glycoprotein Vi ◽  
Cellular Processes ◽  
Specific Proteins ◽  
G Protein Coupled ◽  
Collagen Receptor

Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. The rafts at the cell surface play important functions in signal transduction. Recent reports have demonstrated that lipid rafts are spatially and compositionally heterogeneous in the single-cell membrane. In this review, we summarize our recent data on living platelets using two specific probes of raft components: lysenin as a probe of sphingomyelin-rich rafts and BCθ as a probe of cholesterol-rich rafts. Sphingomyelin-rich rafts that are spatially and functionally distinct from the cholesterol-rich rafts were found at spreading platelets. Fibrin is translocated to sphingomyelin-rich rafts and platelet sphingomyelin-rich rafts act as platforms where extracellular fibrin and intracellular actomyosin join to promote clot retraction. On the other hand, the collagen receptor glycoprotein VI is known to be translocated to cholesterol-rich rafts during platelet adhesion to collagen. Furthermore, the functional roles of platelet glycosphingolipids and platelet raft-binding proteins including G protein-coupled receptors, stomatin, prohibitin, flotillin, and HflK/C-domain protein family, tetraspanin family, and calcium channels are discussed.

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.

scholarly journals CD44 Engagement Promotes Matrix-Derived Survival through the CD44-SRC-Integrin Axis in Lipid Rafts

2008 ◽  
Vol 28 (18) ◽  
pp. 5710-5723 ◽  
Author(s):  
Jia-Lin Lee ◽  
Mei-Jung Wang ◽  
Putty-Reddy Sudhir ◽  
Jeou-Yuan Chen
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Integrin Activation ◽  
Variant Isoforms ◽  
Survival Signal ◽  
The Matrix ◽  
Signaling Axis ◽  
Tightly Coupled ◽  
Carboxyl Terminal

ABSTRACT CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin β1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.

Glycoprotein (GP) VI Is Associated with GPIb-IX-V on the Membrane of Resting and Activated Platelets.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1553-1553
Author(s):  
Michael C. Berndt ◽  
Jane F. Arthur ◽  
Elizabeth E. Gardiner ◽  
Yukio Ozaki ◽  
Mark L. Kahn ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Soluble Fraction ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Low Shear Stress ◽  
Pre Treatment ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Collagen Receptor ◽  
Low Shear

Abstract The platelet collagen receptor, glycoprotein (GP)VI, initiates platelet aggregation at low shear stress while GPIb-IX-V, which binds von Willebrand factor, elicits platelet aggregation under high shear conditions. To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface, we first ascertained that aggregation induced by a GPVI-specific agonist, collagen-related peptide, like collagen, is markedly cross-blocked by a GPIbα-specific monoclonal antibody, SZ2. Immunoprecipitation of GPIb-IX with anti-GPIbα from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblotting with anti-GPVI demonstrated association between GPIb-IX and GPVI. This association was maintained when platelets were activated by thrombin. Pre-treatment of platelets with methyl-β-cyclodextrin to disrupt lipid rafts did not affect association in resting or activated platelets under these conditions of detergent lysis. The association is also independent of cytoskeletal attachment, since it was unaffected by treatment with N-ethylmaleimide or DNaseI, which dissociate GPIb-IX from filamin and the actin-containing cytoskeleton, respectively. Finally, the association involves an interaction between the ectodomains of GPIbα and GPVI, since soluble fragments of GPIbα (glycocalicin) and GPVI are co-precipitated from the platelet supernatant under conditions where GPVI is shed. A contribution of GPIb-IX-V to GPVI-induced platelet responses, and vice versa, therefore warrants further investigation.

A Critical Role for Membrane Sulfatide in Platelet Aggregation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 626-626
Author(s):  
Prasenjit Guchhait ◽  
Corie Shrimpton ◽  
Kochi Honke ◽  
Perumal Thiagarajan ◽  
Jose A. Lopez
Keyword(s):  
Platelet Aggregation ◽  
Lipid Rafts ◽  
Platelet Function ◽  
Critical Role ◽  
Membrane Microdomains ◽  
Systemic Lupus Erythematosis ◽  
Single Chain ◽  
Induce Platelet Aggregation ◽  
Adhesive Interactions

Abstract Sulfatide (galactocylceramide 3′-sulfate) is a sulfated glycosphingolipid expressed on the surfaces of erythrocytes, leukocytes, platelets and a variety other cells, that is known to interact with several cell adhesion molecules involved in hemostasis, including von Willebrand factor (VWF), laminin, thrombospondin, P-selectin and β2-glycoprotein I. Because these ligands are involved in many platelet adhesive interactions, we hypothesize that membrane sulfatide plays an important role in these processes. To examine this, we have cloned and purified a sulfatide-specific single-chain variable fragment (scFv) antibody from a phage-display library constructed from mRNA taken from the lymphocytes of patients with systemic lupus erythematosis. This scFv, PA38, specifically bound sulfatide, and did not react with the related sphingolipids cerebroside, ceramide, or sphingomyelin, or the phospholipids phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine. Using this tool, we examined the role of sulfatide in platelet function. We observed that PA38 dose-dependently (at 5 and 10 μg/ml) inhibited the aggregation of human platelets induced by either collagen or ADP. A control scFv produced in a similar manner had no effect. Furthermore, PA38 delayed platelet plug formation by 23 sec (with collagen-ADP agonist) and 46 sec (with collagen-epinephrine) in whole blood from normal human donors, as measured in a platelet function analyzer, PFA-100 (Dade Behring). Further, to verify that this was a sulfatide-specific effect, we compared collagen-induced platelet aggregation in normal mice to that of mice deficient in cerebroside sulfotransferase (CST)—a critical enzyme in the sulfatide synthetic pathway. The CST−/− mice fail to express sulfatide on the cell surface, and displayed defective platelet aggregation. Consistent with this, the PA38 also significantly inhibited collagen-induce platelet aggregation in wild-type mice. Given the importance of lipid rafts in signaling and adhesive processes, we looked for the localization of sulfatide in these membrane microdomains. Indeed, we found that sulfatide is enriched in lipid rafts suggesting a role for sulfatide in lipid-raft mediated events. Thus, we provide evidence for a key role of a membrane lipid, sulfatide in the adhesive interactions involved in platelet function. With one notable exception, the key adhesive roles in platelet-platelet interaction have all previously been assigned to proteins.

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