Platelet Lipid Rafts Research Articles

Integrin-Dependent Transient Density Increase in Detergent-Resistant Membrane Rafts in Platelets Activated by Thrombin.

Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction via sucrose density gradient centrifugation. The platelet detergent-resistant membrane shifted to a higher density on the sucrose density gradient upon thrombin stimulation. The shift peaked at 1 min and returned to the control level at 60 min. During this time, platelets underwent clot retraction and spreading on a fibronectin-coated glass strip. Thrombin induced the transient tyrosine phosphorylation of several proteins in the detergent-resistant membrane raft fraction and the transient translocation of fibrin and myosin to the detergent-resistant membrane raft fraction. The level of phosphatidylserine (36:1) was increased and the level of phosphatidylserine (38:4) was decreased in the detergent-resistant membrane raft fraction via the thrombin stimulation. Furthermore, Glanzmann's thrombasthenia integrin αIIbβ3-deficient platelets underwent no detergent-resistant membrane shift to a higher density upon thrombin stimulation. As the phosphorylation of the myosin regulatory light chain on Ser19 was at a high level in Glanzmann's thrombasthenia resting platelets, thrombin caused no further phosphorylation of the myosin regulatory light chain on Ser19 or clot retraction. These observations suggest that the fibrin-integrin αIIbβ3-myosin axis and compositional change of phosphatidylserine species may be required for the platelet detergent-resistant membrane shift to a higher density upon stimulation with thrombin.

An agent-based approach for modelling and simulation of glycoprotein VI receptor diffusion, localisation and dimerisation in platelet lipid rafts

Receptor diffusion plays an essential role in cellular signalling via the plasma membrane microenvironment and receptor interactions, but the regulation is not well understood. To aid in understanding of the key determinants of receptor diffusion and signalling, we developed agent-based models (ABMs) to explore the extent of dimerisation of the platelet- and megakaryocyte-specific receptor for collagen glycoprotein VI (GPVI). This approach assessed the importance of glycolipid enriched raft-like domains within the plasma membrane that lower receptor diffusivity. Our model simulations demonstrated that GPVI dimers preferentially concentrate in confined domains and, if diffusivity within domains is decreased relative to outside of domains, dimerisation rates are increased. While an increased amount of confined domains resulted in further dimerisation, merging of domains, which may occur upon membrane rearrangements, was without effect. Modelling of the proportion of the cell membrane which constitutes lipid rafts indicated that dimerisation levels could not be explained by these alone. Crowding of receptors by other membrane proteins was also an important determinant of GPVI dimerisation. Together, these results demonstrate the value of ABM approaches in exploring the interactions on a cell surface, guiding the experimentation for new therapeutic avenues.

Flow cytometry identifies an early stage of platelet apoptosis produced by agonists of the P2X1 and P2X7 receptors

Platelets express P2X1 receptors and our data also show the expression of P2X7 receptors. We studied the role of both receptors in platelet apoptosis by incubation of PRP with P2X agonists, then centrifuged to remove viable platelets, and analyzed the supernatant by flow cytometry to identify a sparse platelet-derived population that stained with MitoTracker dyes and CD41. BzATP, a potent agonist of P2X receptors, and ABT737, an activator of intrinsic apoptosis, produced altered platelets that stained moderately for annexin V and corresponded to an early stage apoptotic platelet (ESAP). Over a range of BzATP concentrations, we observed a dose-dependent formation of ESAPs between 5 and 500 uM BzATP, together with a variable formation of ESAPs at nanomolar ATP or BzATP (50–200 nM). Production of ESAPs occurred with αβ-meATP, while responses with either BzATP or αβ-meATP showed desensitization at a higher agonist concentration. Formation of ESAPs by either 100 nM or 0.5 mM BzATP was inhibited by preincubation of platelets with latrunculin A, an inhibitor of the actin cytoskeleton that prevents apoptosis. ESAP production was totally inhibited by preincubation of platelets with methyl-beta-cyclodextrin, which removes cholesterol from lipid rafts. Our data show that both P2X1 and P2X7 receptors are localized in platelet lipid rafts where P2X-agonists act to produce early stage apoptotic platelets.

Lipid rafts are essential for release of phosphatidylserine-exposing extracellular vesicles from platelets

Platelets protect the vascular system during damage or inflammation, but platelet activation can result in pathological thrombosis. Activated platelets release a variety of extracellular vesicles (EVs). EVs shed from the plasma membrane often expose phosphatidylserine (PS). These EVs are pro-thrombotic and increased in number in many cardiovascular and metabolic diseases. The mechanisms by which PS-exposing EVs are shed from activated platelets are not well characterised. Cholesterol-rich lipid rafts provide a platform for coordinating signalling through receptors and Ca2+ channels in platelets. We show that cholesterol depletion with methyl-β-cyclodextrin or sequestration with filipin prevented the Ca2+-triggered release of PS-exposing EVs. Although calpain activity was required for release of PS-exposing, calpain-dependent cleavage of talin was not affected by cholesterol depletion. P2Y12 and TPα, receptors for ADP and thromboxane A2, respectively, have been reported to be in platelet lipid rafts. However, the P2Y12 antagonist, AR-C69931MX, or the cyclooxygenase inhibitor, aspirin, had no effect on A23187-induced release of PS-exposing EVs. Together, these data show that lipid rafts are required for release of PS-exposing EVs from platelets.

Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis.Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed.Lipid rafts were mainly observed in 1–4 fractions, and non-rafts were distributed on 5–12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains.Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned.

Abstract 12616: Interactive Protein Network of Factor XIII In Platelet Lipid Rafts

Background: Factor XIII plays a pivotal role in platelet aggregation. Factor XIII depends on lipid rafts to assemble the proteins required for clot retraction. Several proteins in this assembly remain unidentified. The aim of this study was to investigate which proteins localized in platelet lipid rafts are related to Factor XIII. Methods: Lipid rafts of platelets were isolated by using Lubrol WX 0.05% (w/v) and sucrose gradient. After ultracentrifugation, sucrose fractions were sampled. Lipidomic analysis was performed to identify the platelet lipid raft fractions enriched in cholesterol (Ch) and sphingomyelin (SM) and less so in phosphatidylcholine (PC). These fractions were selected for further proteomic analyses. The network of associated proteins was drawn by STRING database. Results: Sucrose fractions containing platelet lipid rafts were identified (Figure 1A). After proteomics analysis, an interactive network of proteins around FXIIIA was observed (Figure 1B). For these 19 proteins, their STRING score (range 0.576 to 0.900) and the type of their interaction with FXIIIA (activation, binding, catalysis and reaction) are summarized in Table 1. Conclusion: We identified 19 associated proteins with Factor XIII in human platelet lipid rafts. This could provide further insights into clinical targets of pathways through this molecule.

Alterations in cholesterol and ganglioside GM1 content of lipid rafts in platelets from patients with Alzheimer disease.

The aim of this study was to investigate the changes in the protein, cholesterol, and ganglioside GM1 content of lipid rafts in platelets from patients with Alzheimer disease (AD), and identify potential blood biomarkers of the disease. A total of 31 Chinese patients with AD and 31 aged-matched control subjects were selected. Lipid rafts were isolated from platelets using Optiprep gradient centrifugation. The protein content of lipid rafts was evaluated using Micro BCA assay, the cholesterol content using molecular probes, ganglioside GM1 content using colorimetry and dot-blotting analysis. The results showed that the cholesterol and ganglioside GM1 content of lipid rafts from platelets was significantly higher in patients with AD than aged-matched control subjects, whereas the protein content of lipid rafts did not show any differences between the 2 groups. These results indicate that the increases in the cholesterol and ganglioside GM1 content of lipid rafts from the platelets of patients with AD might serve as a biochemical adjunct to the clinical diagnosis of AD.

C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets

C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling.

The association of thromboxane A2 receptor with lipid rafts is a determinant for platelet functional responses

We have investigated the presence of thromboxane A2 (TXA2) receptor associated with lipid rafts in human platelets and the regulation of platelet function in response to TXA2 receptor agonists when lipid rafts are disrupted by cholesterol extraction. Platelet aggregation with TXA2 analogs U46619 and IBOP was almost blunted in cholesterol-depleted platelets, as well as αIIbβ3 integrin activation and P-selectin exposure. Raft disruption also inhibited TXA2-induced cytosolic calcium increase and nucleotide release, ruling out an implication of P2Y12 receptor. An important proportion of TXA2 receptor (40%) was colocalized at lipid rafts. The presence of the TXA2 receptor associated with lipid rafts in platelets is important for functional platelet responses to TXA2.

The effect of EPA and DHA on platelets treated with multiple agonists found in the thrombus core (671.3)

The omega‐3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been consistently associated with reduced platelet function, and more recently, with impaired thrombin generation. However, almost all studies to date have focused on how EPA and DHA alter platelet physiology in response to monotypic agonist stimulation. This reductionist approach does not recapitulate the complex chemical environment found in a developing thrombus. Therefore, we asked if platelets treated with multiple agonists like those found in the thrombus core would also display EPA and DHA‐mediated inhibition. We found that optical aggregometry and dense granule secretion was diminished after EPA and DHA incorporation in platelets stimulated with the core clot agonists. We also noted a reduction in both the protein content of platelet lipid rafts and thrombin generation after core agonist stimulation, consistent with our earlier observations. Transmission electron micrographic imaging also established that EPA and DHA‐treated platelets showed moderate ultrastructural malformations after core agonist treatment. These results all suggest that EPA and DHA can abrogate platelet signaling responses within the core of a developing thrombus, further illustrating the potential health benefits of increased EPA and DHA intake.Grant Funding Source: Supported by an Institutional Development Award (IDeA) from the NIH/NIGMS, #P20GM103443

フィブリンの血小板脂質ラフト移行と血餅退縮

フィブリンの血小板脂質ラフト移行と血餅退縮

The Neuronal Protein GAP-43 Associates with Microtubules and Lipid Rafts in Platelets and Megakaryocytes and Is Involved in Platelet Genesis

Abstract 2201GAP-43 (neuromodulin) is a membrane-associated protein involved in neurite outgrowth, pathfinding and branching during development where it regulates neurotransmitter release and endocytosis, and plays a role in cytoskeletal signal transduction at the nerve ending. We have found GAP-43 to be expressed in platelets and megakaryocytes (MK) as evidenced by immunoblot and flow cytometry analysis. Immunofluorescence shows that GAP-43 localizes intracellularly as discrete punctuate structures mostly near the marginal microtubule (MT) coil of platelets and along the MT bundles in MKs and proplatelets. In contrast to neuronal cells where GAP-43 is associated with their detergent insoluble F-actin cytoskeleton, it is mostly soluble in platelets unless the platelets are first treated with taxol, a drug that stabilizes MTs. MT disruption with nocodazole or by chilling decreases the amount of GAP-43 in the detergent-insoluble cytoskeleton and disturbs its localization in proplatelets. GAP-43 colocalizes in platelets and MKs with the glycosylphosphatidylinositol-linked lipid raft marker Thy-1 and Cherry-PH PLC-delta1 (pleckstrin homology domain of phospholipase C-delta1) that stains membrane rafts. Since GAP-43 overexpression in MKs diminishes proplatelet formation, it appears to play a negative role in thrombopoiesis. Together, the data shows that GAP-43 is associated with MT and lipid rafts and suggests a role in signaling reactions between these two components to modulate proplatelet elaboration. Disclosures:No relevant conflicts of interest to declare.

Cellular prion protein in blood platelets associates with both lipid rafts and the cytoskeleton

The recently shown transmissibility of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion emphasises the need for better understanding of the cellular prion protein (PrPc) in blood. A substantial amount of cell-associated PrPc in blood resides in platelets. Platelet activation leads to up-regulation of PrPc on the platelet surface and its release on exosomes and microparticles. The sub-cellular localisation and function of platelet PrPc, however, is poorly understood. In the present study, we investigated the association of PrPc with platelet lipid rafts and the platelet cytoskeleton. Immuno-fluorescence microscopy showed that the signals of PrPc and P-selectin, both of which occupy intracellular alpha granules, were separated on the membrane, suggesting organisation in different membrane domains. A flotation assay of platelet lysates demonstrated that a relatively small portion of platelet PrPc floats with lipid rafts, regardless of platelet activation status. This was reversed by depolymerisation of the platelet cytoskeleton, which led to flotation of most platelet PrPc, suggesting that interactions with the cytoskeleton prevent flotation of PrPc rafts. This association of PrPc with the platelet cytoskeleton was confirmed by its presence in both the isolated membrane skeleton and actin cytoskeleton. Platelet activation significantly increased the amount of PrPc associated with the cytoskeleton. Our results indicate that the localisation of PrPc in platelets is complex, with the majority of PrPc present within platelet lipid rafts linked to the platelet cytoskeleton. This localisation places PrPc in a position where it can interact with proteins involved in platelet signalling and eventually with vCJD prions.

Glycoprotein VI agonists have distinct dependences on the lipid raft environment.

It has been reported that the association of glycoprotein VI (GPVI) with lipid rafts regulates GPVI signaling in platelets. Secreted adenosine 5'-diphosphate (ADP) potentiates GPVI-induced platelet aggregation at particular agonist concentrations. We have investigated whether the decrease in GPVI signaling, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-beta-cyclodextrin and measured signaling events downstream of GPVI activation. Lipid raft disruption decreases aggregation induced by low concentrations of convulxin, but this decrease is almost eliminated in the presence of ADP antagonists. Signaling indicators, such as protein phosphorylation and calcium mobilization, were not affected by raft disruption in collagen or convulxin stimulated platelets. Interestingly, however, raft disruption directly reduced GPVI signaling induced by collagen-related peptide. Lipid rafts do not directly contribute to signaling by the physiologic agonist collagen. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates GPVI signaling.

Platelet tetraspanin complexes and their association with lipid rafts

Tetraspanins are a superfamily of integral membrane proteins that facilitate the organization of membrane and intracellular signaling molecules into dynamic signaling microdomains, tetraspanin-enriched microdomains (TEMs). Four tetraspanin family members have been identified in platelets: CD9, CD151 and TSSC6, which are constitutively associated with alphaIIbbeta3, and CD63, which is present on granule membranes in resting platelets and associates with alphaIIbbeta3-CD9 following platelet activation. CD63 and CD9 associate with a type II phosphatidylinositol 4-kinase, PI4K55, in both resting and activated platelets. Immunoelectron microscopic studies showed co-localization of CD63 and PI4K55 on internal membranes of resting platelets and on the filopodia of thrombin-activated platelets. Because TEMs in malignant cell lines appear to be distinct from prototypic lipid rafts, this study examined whether CD63-PI4K55 and CD9-PI4K55 complexes were resident in platelet-lipid rafts, or formed distinct microdomains. CD63, CD9 and PI4K55 were recovered from low-density membrane fractions (LDMFs) of sucrose gradients following platelet lysis in Brij 35, but unlike lipid-raft proteins were not insoluble in Triton X-100, being absent from LDMFs of platelets lysed with Triton. Incubation of platelets with methyl-beta-cyclodextrin, to deplete cholesterol and disrupt lipid rafts, shifted the complexes to higher density sucrose gradient fractions, but did not disrupt the tetraspanin-PI4K55 complexes. These results demonstrate that tetraspanin complexes in platelets form cholesterol-associated microdomains that are distinct from lipid rafts. It is probable that TEMs and lipid rafts associate under certain conditions, resulting in the close proximity of distinct sets of signaling molecules, facilitating signal transduction.

Platelet Tetraspanin Complexes and Their Relation to Lipid Rafts.

CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. CD63 is located in the membranes of lysosomes and dense granules in resting platelets. Following platelet activation and granule exocytosis, CD63 is expressed on the platelet plasma membrane and co-localizes with the αIIbβ3-CD9 complex. D545, a monoclonal antibody (MoAb) directed at the second extracellular loop of CD63, inhibits activated platelet spreading on immobilized fibrinogen and FAK phosphorylation in the adherent platelets. To identify CD63-associated signaling enzymes that could be involved in the signaling complex, lipid kinase assays were performed on D545 immunoprecipitates. CD63 co-immunoprecipitated with a lipid kinase with the enzymatic properties of PI4-kinase type II, confirmed by re-precipitation and immunoblotting with 4C5G (MoAb specific for the 55kDa PI4-kinase, PI4K55). The CD63-PI4K55 complex could be co-precipitated from both resting and activated platelets using anti-CD63 MoAb, and co-localized on the filopodia of thrombin-activated platelets using immuno-electron microscopy. Previous studies have demonstrated that tetraspanins associate with cholesterol-enriched membrane domains in a variety of cells including platelets. There is evidence, however, that these tetraspanin-enriched microdomains (TEMs) can be distinguished from prototypic lipid rafts on the basis of detergent solubility and protein composition. To investigate the association of the CD63-PI4K55 complex with lipid rafts in platelets, resting and thrombin-activated platelets were lysed in buffer containing either 1% Brij 35, or Triton X-100, the low- and high-density membrane fractions separated by isopycnic sucrose gradient centrifugation, and the identification of the low-density membrane fractions (LDMF) confirmed by the presence of LAT. CD63, CD9 and PI4K55 were present in the LDMF of platelets lysed in Brij 35 but not in Triton X-100; they were also present in the denser membrane fractions. CD63 and CD9 associated with cholesterol, as demonstrated by recovery of these proteins in the pellet following centrifugation of platelets lysed with 1% digitonin(a cholesterol-precipitating reagent), but not from lysates made with Brij 35/Triton X-100. Incubation of platelets with methyl-β-cyclodextrin(mβCD) to partially deplete cholesterol and disrupt the lipid rafts shifted LAT, CD63, CD9 and PI4K55 to denser fractions within the gradient. Immunoprecipitation of mβCD-treated platelets with anti-PI4K55 MoAb co-precipitated CD63 and CD9, demonstrating that the complexes were not dependent on residence within LDMFs, but remained intact in the denser fractions and pellet. Platelet tetraspanin complexes associate with cholesterol-enriched domains under conditions of mild detergent extraction. The maintenance of the complexes, however, was not dependent on their residence within lipid rafts, as the complexes remained intact following cholesterol depletion. Their presence in LDMF suggests that tetraspanin complexes may associate with platelet lipid rafts under some conditions, which could bring tetraspanin protein partners into proximity with raft residents, and facilitate the assembly and interaction of signaling complexes following platelet activation.

Human Platelet Tissue Factor Is Localized in Lipid Rafts and Is Associated with GPIb-IX-V Complex in the Membrane: Association between Adhesive and Procoagulant Activities of Platelets.

The binding of platelet membrane GPIb-IX-V to extracellular matrix proteins and von Willebrand factor initiates the adhesion, spreading and activation of platelets. On the other hand, tissue factor (TF) triggers the blood coagulation by binding FVII/VIIa. In resting platelets, TF is stored mainly as a non active (“encrypted”) form, possibly through a dimer- or heterodimer complex. The encryption seems not to affect the TF capacity to bind FVII and VIIa, in such form that the membrane-based complex would be “primed” to trigger the clot formation upon platelet activation. Accordingly, the platelet adhesion and activation would be tightly interwoven with thrombin generation. Given the described localization of GPIbα in platelet lipid rafts, which appears to modulate its activity, we investigated whether TF is also present in these cholesterol-rich domains of the platelet membrane. Continuous sucrose gradients generated by ultracentrifugation at 180.000g x 18h at 4°C were used to obtain cholesterol-rich fractions from platelet lysates (1% Triton X-100 at 4°C). Western blots of each gradient fraction using a polyclonal anti-TF antibody revealed a protein of ~60kDa only in the fractions rich in cholesterol (using flotillin-1 as a marker). TF-dependent procoagulant activity (PCA), assessed by FXa generation was measured in resting and 5μM TRAP-stimulated, leukocyte-free platelets. In both cases, PCA was reduced after membrane cholesterol depletion with methyl-β-cyclo-dextrin. We also found that PCA was significantly enhanced in washed platelets treated with VWF and ristocetin, suggesting some form of functional or structural relationship between GPIb-IX-V complex and TF. GPIb-IX was immunoprecipitated (IP) from human platelet membranes by anti-GPIbα MoAbs (AP-1 or SZ2), in both resting and stimulated platelets (TRAP or VWF-ristocetin). The IP product was revealed with MoAb anti-TF (American Diagnostica, clone 4509) or polyclonal anti-TF, detecting a protein of ~47kDa. When we used MoAb anti-TF for the IP and MoAb anti-GPIX (BL-H6, anti-CD42a) to reveal the western blot, two distinct bands of ~22 and ~47kDa were disclosed, probably corresponding to GPIX and a heterodimer of GPIX and TF, respectively. These observations indicate that, at least a fraction of the platelet TF is localized in lipid rafts and that TF is also in close association with the GPIb-IX-V complex. This co-localization probably has important functional implications as demonstrated by the defective thrombin generation in patients with Bernard-Soulier syndrome. Moreover, this functional association may be relevant in both normal hemostasis and thrombus formation and growth.

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and alpha2beta1 integrin) for efficient platelet activation.

Integrin-dependent interaction of lipid rafts with the actin cytoskeleton in activated human platelets

Dynamic connections between actin filaments and the plasma membrane are crucial for the regulation of blood platelet functions. Protein complexes associated with alphaIIbbeta3 integrin-based cytoskeleton structures are known to play a role in these processes. However, mechanisms involving lateral organizations of the plasma membrane remain to be investigated. Here, we demonstrate that a large fraction of platelet lipid rafts specifically associates with the actin cytoskeleton upon activation. This association was inhibited by antagonists of fibrinogen-alphaIIbbeta3 binding and did not occur in type I Glanzman's thrombasthenic platelets. The raft-cytoskeleton interaction is a reversible process correlating with the intensity and stability of platelet aggregation. Although only a minor fraction of alphaIIbbeta3 was recovered in rafts upon activation, this integrin specifically upregulated the level of PtdIns(4,5)P2 in membrane microdomains and induced the recruitment of several actin-modulating proteins known to directly or indirectly interact with this lipid. Controlled disruption of rafts did not affect alphaIIbbeta3-mediated platelet aggregation in response to high concentrations of thrombin but significantly inhibited fibrin clot retraction. We propose that rafts participate in the organization of membrane-cytoskeleton interactions where alphaIIbbeta3-mediated tension forces apply during the late phase of platelet activation.

Characterization of Platelet Lipid Rafts Leads to the Identification of RGS-19, Which Forms a Phosphorylation-Dependent Complex with 14-3-3ζ.

Lipid rafts are specialised membrane regions, rich in dynamic multi-protein complexes that are implicated in the regulation of platelet activation processes, particularly those mediated by GPVI and GPIb/IX/V. To elucidate the role of rafts in platelet function, we carried out a comprehensive characterization of the detergent-insoluble proteins associated with rafts from control and VWF-activated platelets using liquid chromatography coupled to tandem mass spectrometry. Over 160 proteins were identified including 72 proteins unique to platelet rafts and several novel platelet signaling and vesicle transport proteins e.g., rab 5, rab 8 and syntaxin 11. Indeed, the high level of signaling and trafficking proteins identified implicates rafts as concentrating platforms for the critical platelet functions of activation and secretion. Moreover, activation through GPIb-IX-V resulted in the translocation of many proteins into or out of rafts. Proteins recruited into rafts upon VWF-activation included GPIbαand regulator of G-protein signalling-19 (RGS-19). We confirmed that the novel platelet protein, RGS-19, upon VWF activation resides exclusively within the raft fraction and using confocal microscopy, RGS-19 co-localised with GPIbα and the raft-marker flottilin, to both the plasma and vesicle membranes of VWF-activated platelets. The phosphorylation-dependent interaction of RGS proteins with 14-3-3 leads to the inhibition of their GTPase-activating protein (GAP) activity. Aligning RGS-19 to other members of the RGS family, we identified that it contained a novel 14-3-3 binding motif. Immunoprecipitation with an antibody to 14-3-3ζ demonstrated that RGS-19 and GPIbα co-immunoprecipitated with 14-3-3ζ in VWF-activated rafts. Furthermore, we demonstrated that acid phosphatase treatment could significantly reduce the co-immunoprecipitation of RGS-19 with 14-3-3ζ from activated rafts. Thus, within 15 sec of VWF activation we are suggesting maximal suppression of the GAP activity of RGS-19 in rafts through a phosphorylation-dependent association with 14-3-3ζ. Our results provide the basis for a new hypothesis concerning how RGS proteins might govern signaling within platelet rafts.