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.

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

2010 ◽  
Vol 429 (2) ◽  
pp. 369-377 ◽  
Author(s):  
Analia Garcia ◽  
Soochong Kim ◽  
Kamala Bhavaraju ◽  
Simone M. Schoenwaelder ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Functional Responses ◽  
Induce Platelet Aggregation ◽  
Erk Phosphorylation

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

A critical role of lipid rafts in the organization of a key FcγRIIa-mediated signaling pathway in human platelets

2003 ◽  
Vol 89 (02) ◽  
pp. 318-330 ◽  
Author(s):  
Stéphane Bodin ◽  
Cécile Viala ◽  
Ashraf Ragab ◽  
Bernard Payrastre
Keyword(s):  
Lipid Rafts ◽  
Critical Role ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Temporal Organization ◽  
Membrane Microdomains ◽  
Cross Linking ◽  
Dependent Manner ◽  
Signaling Complex

SummaryThe involvement of platelet FcγRIIa in heparin-associated thrombocytopenia (HIT) is now well established. However, the precise sequence of molecular events initiated by FcγRIIa cross-linking in platelets remains partly characterized. We investigated here the role of lipid rafts in the spatio-temporal organization of the FcγRIIa-dependent signaling events. Upon cross-linking, FcγRIIa relocated in rafts where the kinase Lyn and the adapter LAT were among the major phosphotyrosyl proteins. Upon stimulation by HIT sera, the second messenger phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulated in rafts in a P2Y12 adenosine diphosphate (ADP) recep- tor-dependent manner. PtdIns(3,4,5)P3 was then essential to specifically recruit phospholipase Cγ2 (PLCγ2) to these membrane microdomains. Controlled disruption of rafts by methyl γ-cyclodextrin reversibly abolished PtdIns(3,4,5)P3 production, PLC activation and platelet responses induced by FcγRIIa cross-linking without affecting the tyrosine phosphorylation events. This work demonstrates that platelet rafts are essential for the integration of a key signaling complex leading to the rapid production of PtdIns(3,4,5)P3 and in turn PLCγ2 activation during HIT.

scholarly journals Role of Lipid Rafts in Hematopoietic Stem Cells Homing, Mobilization, Hibernation, and Differentiation

Cells ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 630 ◽  
Author(s):  
Munther Alomari ◽  
Dana Almohazey ◽  
Sarah Ameen Almofty ◽  
Firdos Alam Khan ◽  
Mohammad Al hamad ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Lipid Rafts ◽  
Transforming Growth Factor ◽  
Critical Role ◽  
Transforming Growth Factor Β ◽  
Lymphoid Cells ◽  
Membrane Microdomains ◽  
Hematopoietic Stem

Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells that can differentiate into myeloid or lymphoid cells. The mobilization and differentiation processes are affected by the external environment, such as extracellular matrix and soluble molecules in the niche, where the lipid rafts (LRs) of the HSCs act as the receptors and control platforms for these effectors. LRs are membrane microdomains that are enriched in cholesterol, sphingolipid, and proteins. They are involved in diverse cellular processes including morphogenesis, cytokinesis, signaling, endocytic events, and response to the environment. They are also involved in different types of diseases, such as cancer, Alzheimer’s, and prion disease. LR clustering and disruption contribute directly to the differentiation, homing, hibernation, or mobilization of HSCs. Thus, characterization of LR integrity may provide a promising approach to controlling the fate of stem cells for clinical applications. In this review, we show the critical role of LR modification (clustering, disruption, protein incorporation, and signal responding) in deciding the fate of HSCs, under the effect of soluble cytokines such as stem cell factor (SCF), transforming growth factor- β (TGF-β), hematopoietic-specific phospholipase Cβ2 (PLC-β2), and granulocyte colony-stimulating factor (G-CSF).

Phosphoinositide 3-kinases and the regulation of platelet function

2004 ◽  
Vol 32 (2) ◽  
pp. 387-392 ◽  
Author(s):  
S.P. Jackson ◽  
C.L. Yap ◽  
K.E. Anderson
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Critical Role ◽  
Receptor Activation ◽  
Clear Understanding ◽  
Integrin Αiibβ3 ◽  
Adhesion Receptor ◽  
Input Signals

A clear understanding of the role of PI (phosphoinositide) 3-kinases in supporting the haemostatic function of platelets has been slow to evolve. In fact, insight into the roles of individual PI 3-kinase isoforms in platelet function remains rudimentary. However, based on in vitro studies using wortmannin and LY294002, there is evidence for an important role for PI 3-kinases in regulating a broad range of functional platelet responses, including primary platelet adhesion, cytoskeletal remodelling and platelet aggregation. One of the critical platelet responses involves affinity regulation of the major platelet integrin αIIbβ3, the primary receptor mediating platelet aggregation and thrombus growth. The input signals regulating integrin αIIbβ3 can be divided into three main groups: (1) Gq-coupled receptors linked to the activation of PLCβ (phospholipase Cβ); (2) Gi-coupled receptors linked to the regulation of adenylate cyclase and Rap1b; and (3) adhesion receptor signalling involving Src kinase-dependent activation of PLCγ isoforms. PI 3-kinases have not been demonstrated to play a critical role in Gq-dependent platelet activation; however, one or more PI 3-kinase isoforms appears to be important for Gi-dependent activation of Rap1b and adhesion receptor activation of PLCγ isoforms. Thus distinct co-operative PI 3-kinase signalling mechanisms appear to play an important role in regulating the adhesive function of integrin αIIbβ3.

Abstract 4023: Aspirin “Resistance”: Role of Pre-existent Platelet Reactivity and Correlation Between Tests

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Andrew L Frelinger ◽  
Youfu Li ◽  
Matthew D Linden ◽  
Inge Tarnow ◽  
Marc R Barnard ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Normal Subjects ◽  
Aspirin Resistance ◽  
The Other ◽  
List Type ◽  
Platelet Function Test ◽  
Function Test

Background: Aspirin “resistance” (i.e. hyporesponsiveness to aspirin in a platelet function test) has been widely reported, but the underlying mechanism is unclear. We examined the role of pre-existent platelet hyperreactivity in aspirin “resistance”. We also determined the correlation between aspirin resistance defined by serum thromboxane (TX) B 2 (the most specific test of aspirin’s effect) and other assays of platelet function. Methods: Platelet function measured before and after aspirin 81 mg daily for 7 days was analyzed by Spearman’s rank correlation. Normal subjects (n=165) were studied because virtually all clinically relevant patients are already taking aspirin. An additional advantage of the use of normal subjects is that the platelet response to stimuli is not influenced (with resultant increased scatter of the data) by an underlying disease, e.g. coronary artery disease, which causes platelet hyperreactivity. Results: The proportion of the post-aspirin platelet function predicted by the pre-aspirin platelet function was 28.3 ± 7.5% (mean ± asymptotic standard error) for serum TXB 2 , 39.3 ± 6.8% for urinary 11-dehydro TXB 2 , 4.4 ± 7.7% for arachidonic acid-induced platelet aggregation, 40.4 ± 7.1% for ADP-induced platelet aggregation, 26.3 ± 9.2% for the VerifyNow Aspirin Assay®, and 45.0 ± 10.9% for the TEG® PlateletMapping ™ System with arachidonic acid. Spearman rank order correlations were highly significant for comparisons between assays when both pre-aspirin and post-aspirin results were included in the analysis. However, residual serum TXB 2 levels post-aspirin treatment were not significantly associated with post-treatment results of any of the other assays. Platelet count correlated with pre-aspirin serum TXB 2 and VerifyNow Aspirin Assay, but not with any post-aspirin platelet function test. Conclusions: Aspirin “resistance” (i.e. hyporesponsiveness to aspirin in a laboratory test) is in part unrelated to aspirin but is the result of underlying platelet hyperreactivity prior to the institution of aspirin therapy. Individuals identified as aspirin “resistant” defined by serum TXB 2 are not the same individuals identified by the other tests.

IMPORTANCE OF LARGE VON WILLEBRAND FACTOR (vWF) MULTIMERS IN vWF INTERACTION WITH PLATELET GLYCOPROTEIN IIb/IIIa

1987 ◽  
Author(s):  
M Yamamoto ◽  
Y Ando ◽  
K Watanabe ◽  
H Iri ◽  
Y Araki ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Platelet Glycoprotein ◽  
Induce Platelet Aggregation ◽  
Maximum Extent ◽  
Physiological Relevance ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

Recently it has been reported that, in addition to binding to glycoprotein (GP) lb, vWF also interacts with GPIIb/IIIa, although the physiological relevance of this interaction is not completeley clear. In this paper, we have investigated the role of different size of vWF multimers in vWF-mediated platelet aggregation. Different size of vWF multimers were purified from human plasma through Sephacryl S-1000 column according to the method of Fowler et al. Fractions were analysed by SDS-agarose gel electrophoresis by the method of Ruggeri et al. When each fraction was examined for ristocetin cofactor activity (RCo), only larger multimers exhibited significant RCo. The maximum extent of ristocetin-induced platelet agglutination by larger multimers (10 μg/ml) was 80%, while that of intermediate and lower multimers at the same concentration was 20% and 0%, respectively. Each fraction was then added to washed platelet suspensions in the presence of 10 μM ADP and 0.3 mM CaCl2. Only larger multimers induced platelet aggregation, while intermediate and lower multimers failed to induce platelet aggregation. The maximum extent of aggregation in the presence of larger multimers (10 μg/ml) was 70% of that in the presence of fibrinogen instead. Similar experiments were peformed using platelet-rich plasma from a patient with afibrinogenemia in stead of washed normal platelets. ADP caused significant aggregation only when purified vWF larger multimers or fibrinogen was added. This vWF-mediated aggregation was completely inhibited by monoclonal antibody to GPIIb/IIIa (1 μg/ml) and synthetic peptide, Arg-Gly-Asp-Ser, (1 mM).Our results indicate that larger multimers of vWF play major roles in vWF interaction with GPIIb/IIIa.

Roles of Cyclic Nucleotides in the Inhibition of Platelet Function by Physiolocic and Pharmacological Agents

1979 ◽  
Author(s):  
R.J. Haslam
Keyword(s):  
Platelet Aggregation ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Platelet Function ◽  
Inhibitory Effects ◽  
Pharmacological Agents ◽  
Arterial Blood ◽  
Cyclic Cmp ◽  
Amp Inhibition

Cyclic AMP mediates the inhibitions of platelet aggregation caused by PCI2, PGE1 and PGD2. Thus, these compounds activate platelet adenylate cyclase and Increase platelet cyclic AMP; their inhibitory effects are blockod by inhibitor? of adenylate cyclase, are potentiated by inhibitors of cyclic AKP phosphodiesterase and are mimicked hy N6 ,2'-0-dibutyryl cyclic AMP. Inhibition of adenylate cyclase does not potentiate platelet aggregation in the absence of inhibitory prostaglandins, indicating that platelet cyclic AMP is too low to affect aggregation under these conditions. To determine whether platelets in the circulation are exposed to agents that increase platelet cyclic AMP, washed rabbi platelets labelled with [3H] adenine were incubated with rabbit arterial blood under various conditions; any increases in cyclic [3H]AMP were measured. These experiments showed that freshly taken rabbit arterial blood does not normally contain any factors that can increase platelet cyclic AMP sufficiently to affect platelet function; specifically, circulating PGI2 was less than 0.1 pmol/ml of blood. It follows that increases in cyclic AMP in circulating rabbit platelets must occur only locally or under special conditions. The role of the moderate increases in platelet cyclic CMP caused by aggregating agents remains uncertain, but the inhibition of aggregation by compounds such as sodium nitroprusside that increase cyclic CMP up to 100-fold suggests that cyclic CMP may, like cyclic AMP, be an inhibitory mediator.

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.

Modulation of Tissue Factor-Factor VIIa Signaling by Lipid Rafts and Caveolae.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1744-1744
Author(s):  
Vineet Awasthi ◽  
Samir Mandal ◽  
Veena Papanna ◽  
L. Vijaya Mohan Rao ◽  
Usha Pendurthi
Keyword(s):  
Cell Signaling ◽  
Lipid Rafts ◽  
G Proteins ◽  
Intracellular Signaling ◽  
Factor Viia ◽  
Membrane Microdomains ◽  
Caveolin 1 ◽  
Intracellular Signaling Pathways ◽  
Plasma Membrane Microdomains

Abstract Tissue factor (TF) is a cellular receptor for clotting factor VIIa (VIIa) and the formation of TF-VIIa complexes on cell surfaces not only triggers the coagulation cascade but also transduces cell signaling via activation of protease-activated receptors (PARs), particularly PAR2. Although a number of recent studies provide valuable information on intracellular signaling pathways that are activated by TF-VIIa, the role of various cell surface components in mediating the interaction of TF-VIIa with PARs, and the subsequent signal transmittance are unknown. Unlike thrombin and trypsin, VIIa has to bind to its cellular receptor (TF) to activate PARs. The inability of TF-VIIa to effectively activate Ca2+ signaling and failure to desensitize the signaling to subsequently added trypsin suggest that the TF-VIIa is a poor activator of PAR2. Despite this, a number of studies have shown that VIIa is as effective as trypsin or PAR2 agonist peptide in activating intracellular signaling pathways and gene expression in cells expressing TF. Although the potential mechanism for this phenomenon is unknown, compartmentalization of TF, PAR2, and G-proteins in plasma membrane microdomains could facilitate a robust TF-VIIa-induced PAR2-mediated cell signaling. Although certain G-protein coupled receptors and G-proteins are known to be segregated into specialized membrane microdomains, lipid rafts and caveolae, little is known whether PARs are segregated into lipid rafts and caveolae, and how such segregation might influence their activation by TF-VIIa and the subsequent coupling to G-proteins. To obtain answers to some of these questions, in the present study, we have characterized TF and PAR2 distribution on tumor cell surfaces and investigated the role of lipid raft/caveolae in modulating the TF-VIIa signaling in tumor cells. Detergent extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF and PAR2 were distributed both in lipid rafts (low-density) and soluble fractions. Immunofluorescence confocal microscopy revealed that TF at the cell surface is localized in discrete plasma membrane microdomains, and colocalized with caveolin-1, a structural integral protein of caveolae, indicating caveolar localization of TF. Similar to TF, PAR2 also displayed significant punctuate staining and colocalization with caveloin-1. Further, a substantial fraction of TF and PAR2 was colocalized in caveolae. Disruption of lipid rafts/caveolae by ß-methyl cyclodextrin or filipin treatments reduced TF association with PAR2 in lipid rafts and caveolar fractions and impaired the TF-VIIa-induced cell signaling (PI hydrolysis and IL-8 gene expression). Additional studies showed that both mßCD and filipin treatments specifically impaired TF-VIIa cleavage of PAR2 and but had no significant effect on trypsin cleavage of PAR2. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling. In summary, the data presented herein demonstrate that TF localization at the cell membrane could influence different functions of TF differently. While caveolar localization of TF had no influence in propagating the procoagulant activity of TF, it is essential in supporting the TF-VIIa-induced cell signaling.

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