Function of Platelet Glycosphingolipid Microdomains/Lipid Rafts

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.


Platelet Lipid Rafts
The fluid mosaic model has supported our understanding of cellular membranes for a long time. Recent studies suggest that plasma membrane lipids are not homogeneously distributed and that the membranes may contain microdomains or compartments. Glycosphingolipids form microdomains containing cholesterol in the cell membrane. Glycosphingolipid-and cholesterol-rich microdomains are referred to as lipid rafts. Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and proteins which are stabilized into platforms involved in the regulation of a number of cellular processes [1]. Lipid rafts are isolated as a detergent-resistant membrane (DRM) fraction by sucrose density gradient centrifugation. Recent studies have demonstrated that lipid rafts are spatially and compositionally heterogeneous in the cell membrane. In migrating T cells, GM3 ganglioside-rich rafts containing a chemokine receptor are present at their leading edge, whereas GM1-rich rafts containing integrin β1 are present at their uropod [2].
In 1996, platelet DRM was shown to be rich in glycoprotein CD36, Src, and Lyn [3]. Platelet rafts are important membrane microdomains in responses such as adhesion and aggregation. The localization of the adhesion receptor glycoprotein (GP)Ib-IX-V complex to lipid rafts is required for platelet adhesion to the vessel wall by binding the von Willebrand factor (vWF) [4,5]. In resting platelets, phosphatidylserine (PS) is asymmetrically restricted to the inner leaflet of the plasma membrane. An increase in intracellular Ca 2+ concentration during platelet activation can lead to the exposure of PS in the outer leaflet. PS forms a procoagulant binding site for tenase and prothrombinase coagulation Resting platelets (left) and platelets stimulated for 3 min with 1 U/mL thrombin (right) were lysed in Triton X-100 and then adjusted to 40% sucrose. A sucrose gradient (5-30%) in a volume of 6 mL was layered over the lysate (4 mL) and was centrifuged. Ten fractions were collected from top to bottom after centrifugation and subjected to immunoblotting with an anti-fibrinogen polyclonal antibody. In resting platelets, fibrinogens Aα (67 kDa), Bβ (52 kDa), and γ (47 kDa) were detected in the non-raft fraction (lanes 7-10). In contrast, fibrins α (65 kDa), β (50 kDa), and γ (47 kDa) were detected in the raft fraction (lanes 5,6) of thrombin-stimulated platelets. (B) Localization of fibrin (left panel) and BCθ-positive cholesterol-rich rafts (right panel) of thrombin-stimulated spreading platelets on fibronectin by scanning immunoelectron microscopy. Spreading platelets were incubated with 15 μg/mL BCθ for 30 min followed by glutaraldehyde fixation and immunolabeling with anti-biotin IgG gold. Goldpositive fibrins were localized in the central region of spreading platelet (left). In contrast, goldpositive cholesterol-rich rafts were localized uniformly on the membrane (right). The study was approved by the institutional ethics committee.
Lysenin, the earthworm toxin, is a specific probe of sphingomyelin (SM)-rich rafts in living cells [22,23]. SM is a major component of raft lipids in platelets [9]. Therefore, we investigated the subcellular distribution of SM-rich rafts in spreading platelets. Lysenin-positive SM-rich rafts were localized in the central area of adhering platelets stimulated with thrombin ( Figure 2A, left panel). Lysenin-positive SM-rich rafts and fibrin mostly colocalized as a patch in the double-stained the central area of spreading platelets stimulated with thrombin ( Figure 2A, middle panel). Next, we investigated the spreading of platelets by time-lapse differential interference contrast (DIC) imaging Resting platelets (left) and platelets stimulated for 3 min with 1 U/mL thrombin (right) were lysed in Triton X-100 and then adjusted to 40% sucrose. A sucrose gradient (5-30%) in a volume of 6 mL was layered over the lysate (4 mL) and was centrifuged. Ten fractions were collected from top to bottom after centrifugation and subjected to immunoblotting with an anti-fibrinogen polyclonal antibody. In resting platelets, fibrinogens Aα (67 kDa), Bβ (52 kDa), and γ (47 kDa) were detected in the non-raft fraction (lanes 7-10). In contrast, fibrins α (65 kDa), β (50 kDa), and γ (47 kDa) were detected in the raft fraction (lanes 5,6) of thrombin-stimulated platelets. (B) Localization of fibrin (left panel) and BCθ-positive cholesterol-rich rafts (right panel) of thrombin-stimulated spreading platelets on fibronectin by scanning immunoelectron microscopy. Spreading platelets were incubated with 15 µg/mL BCθ for 30 min followed by glutaraldehyde fixation and immunolabeling with anti-biotin IgG gold. Gold-positive fibrins were localized in the central region of spreading platelet (left). In contrast, gold-positive cholesterol-rich rafts were localized uniformly on the membrane (right). The study was approved by the institutional ethics committee. delayed significantly. As a result, we demonstrated that fibrin converted by thrombin translocates immediately into platelet DRM rafts in a coagulation factor XIII (FXIII)-dependent manner. Therefore, we proposed that fibrin is translocated to SM-rich rafts in the presence of FXIII crosslinking activity and that platelet SM-rich rafts act as platforms where extracellular fibrin and intracellular actomyosin join to promote clot retraction [21,24,25]. A spatial distinction between SM-rich rafts and cholesterol-rich rafts in platelets is illustrated (Figure 3).

Raft Heterogeneity
Platelet DRM shifts to a higher density in sucrose gradients upon platelet activation, suggesting that platelet lipid rafts are dynamic membrane microdomains. Not only actin and fibrin but also small GTPases (Rac, cdc42) and cytoskeleton regulatory proteins (moesin, Arp3, VASP) were detected in the DRM fraction of activated platelets [9]. The possible mechanism of the DRM shift to a higher density in sucrose gradients upon platelet activation presumably involves the high protein-to-lipid ratio [26].
In porcine lung membranes, two distinct types of DRM were obtained after sucrose density gradient centrifugation using Triton X-100. Light DRM contained cerebroside, whereas dense DRM contained Ca 2+ ATPase and the IP3 receptor [27]. In the adult mouse cerebellum, two distinct types of DRM were also obtained after sucrose density gradient centrifugation using Triton X-100. Light DRM contained cerebroside and sulfatide [28]. In B-lymphocytes, two distinct types of DRM were obtained after sucrose density gradient centrifugation using Brij 98. Light DRM contained ganglioside GM1 and MHC II, whereas dense DRM contained ganglioside GM2 and MHC I [29]. These results suggest endocytosis of MHC molecules by distinct lipid rafts. In HEK293T cells, two distinct types of DRM were also obtained after sucrose density gradient centrifugation using sodium carbonate (pH 11). Light DRM contained ganglioside GM1, whereas dense DRM contained cholesterol and flotillins [30]. Therefore, the platelet DRM shifts in sucrose gradients might be due to changes in lipid composition. Lactosylceramide and ganglioside GM3 are the major glycosphingolipids of human platelets [31]. Resting platelets do not express ganglioside GD3. The stimulation of platelets with ADP resulted in the formation of ganglioside GD3 by GD3 synthesis from the GM3 pool [31,32]. The GD3 synthase is CMP-NeuAc:NeuAc α2-3Gal β1-4Glc β1-1′Cer α2,8-sialyltransferase [33]. The stimulation of platelets with thrombin showed an increase in the amount of ganglioside GM3 [34]. The stimulation of platelets with ADP showed a decrease in the amount of cholesterol in the DRM raft fraction [10]. The precise mechanism of DRM shifts to a higher density in sucrose gradients upon platelet activation remains to be elucidated.

Raft Heterogeneity
Platelet DRM shifts to a higher density in sucrose gradients upon platelet activation, suggesting that platelet lipid rafts are dynamic membrane microdomains. Not only actin and fibrin but also small GTPases (Rac, cdc42) and cytoskeleton regulatory proteins (moesin, Arp3, VASP) were detected in the DRM fraction of activated platelets [9]. The possible mechanism of the DRM shift to a higher density in sucrose gradients upon platelet activation presumably involves the high protein-to-lipid ratio [26].
In porcine lung membranes, two distinct types of DRM were obtained after sucrose density gradient centrifugation using Triton X-100. Light DRM contained cerebroside, whereas dense DRM contained Ca 2+ ATPase and the IP3 receptor [27]. In the adult mouse cerebellum, two distinct types of DRM were also obtained after sucrose density gradient centrifugation using Triton X-100. Light DRM contained cerebroside and sulfatide [28]. In B-lymphocytes, two distinct types of DRM were obtained after sucrose density gradient centrifugation using Brij 98. Light DRM contained ganglioside GM1 and MHC II, whereas dense DRM contained ganglioside GM2 and MHC I [29]. These results suggest endocytosis of MHC molecules by distinct lipid rafts. In HEK293T cells, two distinct types of DRM were also obtained after sucrose density gradient centrifugation using sodium carbonate (pH 11). Light DRM contained ganglioside GM1, whereas dense DRM contained cholesterol and flotillins [30]. Therefore, the platelet DRM shifts in sucrose gradients might be due to changes in lipid composition. Lactosylceramide and ganglioside GM3 are the major glycosphingolipids of human platelets [31]. Resting platelets do not express ganglioside GD3. The stimulation of platelets with ADP resulted in the formation of ganglioside GD3 by GD3 synthesis from the GM3 pool [31,32]. The GD3 synthase is CMP-NeuAc:NeuAc α2-3Gal β1-4Glc β1-1 Cer α2,8-sialyltransferase [33]. The stimulation of platelets with thrombin showed an increase in the amount of ganglioside GM3 [34]. The stimulation of platelets with ADP showed a decrease in the amount of cholesterol in the DRM raft fraction [10]. The precise mechanism of DRM shifts to a higher density in sucrose gradients upon platelet activation remains to be elucidated.
The adaptor protein Disabled-2 (Dab2) as a key regulator of platelet signaling is a sulfatide-binding protein. Its interaction is mediated by two N-terminal conserved basic motifs (amino acid residues 24-29 and 49-54) with a dissociation constant Kd of 0.6 µM [47]. Dab2 is present in the cytoplasm and α-granules of platelets and is released from the platelets in response to platelet activation. Dab2 interacts with the cytoplasmic tail of the integrin αIIbβ3 and regulates inside-out signaling [48]. On the other hand, Dab2 released from α-granules inhibit platelet aggregation by competing with fibrinogen for binding to the integrin αIIbβ3, an interaction that is modulated by Dab2 binding to sulfatide at the outer leaflet of the plasma membrane. The Dab2 sulfatide-binding motif peptide can prevent sulfatide-induced platelet aggregation [49,50]. The bleeding time is prolonged and thrombus formation is impaired in Dab2-deficient mice. Dab2-deficient platelets elicited a selective defect in platelet aggregation and spreading on fibrinogen by thrombin stimulation [51].
Sulfatide on the platelet surface interacts with a blood coagulation factor, playing a major role in hemostasis. Blood coagulation cascade has two pathways: intrinsic pathway and extrinsic pathway. Coagulation factor XII is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic surfaces, such as sulfatide on the plasma membrane. Annexins (ANXs) are implicated in the regulation of blood coagulation reactions by binding to sulfatide [52]. ANXA3, ANXA4, and ANXA5 inhibit sulfatide-induced plasma coagulation. ANXA4 inhibits sulfatide-induced autoactivation of Factor XII to Factor XIIa and the conversion of its natural substrate Factor XI to Factor XIa [53].
Ganglioside GD3 is rapidly expressed on the platelet surface following platelet activation and internalized to the cytoskeleton where it transiently associates first with the Src family kinase Lyn then with the Fc receptor gamma chain [32]. The binding of bacterial cells to human platelets contributes to the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis strain SF100 is mediated by two surface proteins, PblA and PblB. α2-8-linked sialic acid residues on platelet membrane ganglioside GD3 are the primary targets for PblA/PblB-mediated binding to human platelets. [54].
Globotriaosylceramide Gb3 is a functional receptor of the Shiga toxin [40]. Shiga toxin is the principal virulence factor of enterohemorrhagic Escherichia coli. Thrombocytopenia caused by platelet consumption in thrombi is a primary symptom of hemolytic uremic syndrome associated with Shiga toxin. Shiga toxin1 and its B (binding) subunit bind to platelets, leading to fibrinogen binding and platelet aggregation [55]. The possible existence of glycosphingolipid-specific rafts, such as sulfatide-rich rafts, remains to be explored.

Protein S-Palmitoylation: Lipid Raft Targeting Modification
S-palmitoylation is a posttranslational modification catalyzed by palmitoyl acyltransferases from the zincfinger and Asp-His-His-Cys domain-containing (DHHC) enzyme family. It is involved in the attachment of the saturated palmitoyl acyl chain (C16:0) delivered by palmitoyl-CoA to a cysteine residue [79][80][81]. DHHC4 and DHHC5 facilitate fatty acid uptake by palmitoylating and targeting CD36 to the plasma membrane [82]. DHHC5 palmitoylates flotillin-2 in neuronal cells [83]. DHHC2 affects palmitoylation, and functions of tetraspanins CD9 and CD151 [84]. The enzymatic removal of S-acyl modifications in mammalian cells is catalyzed by acyl protein thioesterase (APT) and APT can remove palmitate groups from palmitoylated proteins [80]. Two protein palmitoyl thioesterases (PPTs) have been described as being capable of catalyzing the removal of fatty acids from proteins, in other words, acyl protein thioesterase 1 (APT1), and palmitoyl protein thioesterase 1 (PPT1). APT1 is reported to depalmitoylate the alpha subunit of G proteins and LAT in vitro. APT1 is itself palmitoylated and contain a hydrophobic pocket to accept palmitoylated substrates.
Protein palmitoylation is a dynamic modification that regulates the lipid raft targeting of proteins [85]. The basic forces driving raft formation are lipid interactions. The saturated acyl chains and high acyl chain melting temperatures of glycosphingolipids mediate glycosphingolipid clustering in combination with cholesterol, which has the properties of a "liquid-ordered phase." In contrast, most phospholipids have unsaturated acyl chains, low melting temperatures, and the properties of a liquid-disordered phase. Lipid rafts are considered to exist as phase-separated domains. The linkage of membrane proteins to saturated acyl chains by palmitoylation is considered to facilitate the translocation of these proteins to lipid rafts.

G protein-Coupled Receptors (P2Y1, P2Y12, CXCR4)
Platelet activation by several agonists such as collagen, ADP, and thrombin is followed by platelet granule release, integrin αIIbβ3 activation, aggregation, and thrombus formation. All these processes are triggered by an increase in cytosolic Ca 2+ concentration ((Ca 2+ )i). Ca 2+ , diacylglycerol-regulated guanine nucleotide exchange factor I, and protein kinase C have been shown to be critical elements that link increased (Ca 2+ ) to platelet secretion and integrin αIIbβ3 activation (inside-out signaling). ADP induces multiple platelet responses via seven transmembrane G protein-coupled receptors, P2Y1, and P2Y12. Lipid raft integrity is required for the P2Y1 and P2Y12 signaling pathways. P2Y1 is translocated to the DRM raft fraction by in vitro stimulation with ADP [10]. Importantly, in vivo oral administration to rats with clopidogrel, a P2Y12 antagonist, induces disruption of P2Y12 oligomers and their partition removal from lipid rafts [89].
Platelets are a source of chemokine stromal cell-derived factor-1α (SDF-1α), which is stored in α-granules. Platelet-derived SDF-1α modulates paracrine mechanisms such as chemotaxis [90]. Platelet-derived SDF-1α is also an autocrine activator of platelets through its receptor CXCR4 [91][92][93][94]. SDF-1α-induced platelet aggregation in inhibited by the pertussis toxin, suggesting that its effect is mediated by a pertussis-toxin-sensitive G protein such as Gαi. SDF-1α induces platelet aggregation via phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway [20]. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that CXCR4 (35%), the heterotrimeric G proteins Gαi-1 (93%), Gαi-2 (91%), and Gβ (50%) and PI3Kβ (4%), and Akt2 (4.5%) are localized in the DRM raft fraction. Gαi-1 and Gαi-2 are S-palmitoylated on a cysteine residue (Cys3). SDF-1α is highly expressed in atherosclerotic plaques [95], suggesting that platelet aggregation by SDF-1α/CXCR4 axis contributes to the pathologies such as atherosclerosis. Surface expression of SDF-1α on platelets is a biomarker in ischemic events [90]. The SDF-1α expression level on platelets is elevated in patients with acute myocardial infarction [96]. Flotillins are raft-associated integral membrane proteins and belong to the SPFH superfamily [97]. Flotillins bind the inner leaflet of a plasma membrane raft and serve as scaffolds facilitating the assembly of multiprotein complexes. Flotillin-1 and flotillin-2 have the same domain architecture, comprising two domains: the N-terminal SPFH domain and the C-terminal flotillin domain [98]. The SPFH and flotillin domains mediate inner membrane binding and oligomerization of flotillins, respectively. The membrane association of flotillins is determined by the acyl chain(s) attached and the interaction of protein hydrophobic regions with the cytosolic leaflet of membranes. Flotillin-1 is S-palmitoylated on Cys34 located within the first hydrophobic stretch (amino acids 10-36). Flotillin-2 is N-myristoylated on Gly2 and S-palmitoylated on three cysteine residues; Cys4, Cys19, and Cys20, which are located in the first hydrophobic region (amino acids . The second hydrophobic region locates in the middle part of the SPFH domain (amino acids 134-150/151). The binding of cholesterol by flotillins is mediated by the cholesterol recognition/interaction amino acid (CRAC) motif(s) located within the SPFH domain (amino acids 117-124 in flotillin-1; 120-127 and 157-169 in flotillin-2).
Platelets store sphingosine-1-phosphate (S1P) abundantly and release this bioactive lipid extracellularly upon stimulation [99,100]. S1P induces platelet shape change and aggregation reactions and stimulates vascular endothelial cell spreading and migration [101]. Platelet-derived S1P plays an important role in vascular biology. S1P is synthetized from sphingosine by sphingosine kinases. Recently, flotillin-1 and flotillin-2 have been shown to recruit sphingosine to lipid rafts and maintain cellular S1P levels [102]. Sphingosine binding is mediated by the SPFH domain of flotillins, but the exact identities of the hydrophobic sequences of the flotillins involved are not known. Flotillins also interact with numerous signaling proteins such as receptors, protein kinases, G proteins, and adaptors [98]. Therefore, flotillin-based microdomains can serve as platforms mediating the formation of multiprotein complexes and transmembrane signal transduction at the plasma membrane.

Stomatin
Stomatin is a raft-associated integral membrane protein and belongs to the SPFH superfamily [103]. Stomatin is composed of the N-terminal 24-residue basic domain, hydrophobic intramembrane domain (residues 26-54), cholesterol recognition/interaction amino acid consensus (CRAC, residues 55-68), SPFH domain (residues 57-256), coiled-coil domain, oligomerization and lipid-raft-association domain (ORA, residues 263-273), and C-terminal domain. Stomatin is S-palmitoylated on Cys30 and Cys87. The α-helical segments of stomatin flexibly move along with the membrane surface, with such movement potentially leading to membrane bending via lipid raft clustering through the formation of homo-oligomeric complexes of SPFH-domain proteins [97]. Stomatin is localized at the platelet α-granular membrane. The lipid-raft marker proteins flotillin-1 and flotillin-2 are present in the plasma membrane but excluded from α-granules. The activation of platelets by thrombin leads to translocation of stomatin to the plasma membrane [59]. Lipid raft-associated stomatin enhances cell fusion. With its unique molecular topology, stomatin forms molecular assemblies within lipid rafts, and promotes membrane fusion by modulating fusogenic protein engagement [104]. During platelet activation, the α-granular membrane undergoes fusion with the platelet plasma membrane and granular secretion. Stomatin may have a role in the α-granular membrane fusion.

Prohibitin
Prohibitin is also a raft-associated integral membrane protein and belongs to the SPFH superfamily [105]. Prohibitins, comprising the two homologous members PHB1 and PHB2, are ubiquitously expressed and highly conserved. Prohibitin is composed of the N-terminal hydrophobic stretch, SPFH domain, and coiled-coil domain. Prohibitin is S-palmitoylated on Cys69 [106]. Prohibitins are distributed in lipid rafts, as determined by sucrose density centrifugation. In addition, prohibitins are associated with protease-activated receptor 1 (PAR1). Platelet aggregation, integrin αIIbβ3 activation, granular secretion, and calcium mobilization stimulated by low-concentration thrombin are reduced by the blockade of prohibitins with anti-prohibitin antibody [72]. Prohibitins are involved in PAR1-mediated platelet aggregation.

Tetraspanin Family
Tetraspanins are a superfamily of cell-surface glycoproteins that are characterized by four transmembrane domains, intracellular N-and C-termini, and conserved sequence motifs within the larger of two extracellular regions. Tetraspanins are considered to function by self-associating to form a novel type of membrane microdomain, "tetraspanin-enriched microdomains (TEMs)". TEMs are physically and functionally distinct from lipid rafts [107]. However, gangliosides are a membrane component of TEM [108] and are involved in tetraspanin-partner interactions, as determined from the finding that the depletion of gangliosides affects the interaction between CD82 and its partners [109], suggesting that gangliosides play a critical role in the organization of TEMs. Therefore, TEMs are considered to be a subset of glycosphingolipid microdomains [110].

CD9
CD9 is found to be expressed at approximately 50,000 copies per platelet [111]. CD9 is a negative regulator on platelets, because the fibrinogen binding of integrin αIIbβ3 in response to platelet agonists is found to be mildly enhanced in CD9-deficient platelets, suggesting that CD9 limits the inside-out activation of this integrin [112]. CD9 is S-palmitoylated on six cysteine residues (Cys9, Cys78, Cys79, Cys87, Cys218, and Cys219), which are located in four internal juxta membrane regions [113].

CD151
CD151-deficient platelets exhibited impaired "outside-in" integrin αIIbβ3 signaling with defective platelet aggregation by the protease-activated receptor 4 (PAR4) agonist peptides, collagen, and ADP; impaired platelet spreading on fibrinogen; and delayed kinetics of clot retraction in vitro [114]. Furthermore, tail bleeding assay shows longer bleeding times, leading to the three-fold loss of blood and a seven-fold increase in the incidence of rebleeding [115]. CD151 is S-palmitoylated on six cysteine residues (Cys 11,15,79,80, 242, and 243). The association of a palmitoylation-deficient CD151 with CD81 and CD63 is markedly attenuated, but the interaction of the α3β1-CD151 complex with phosphatidylinositol 4-kinase was not affected [116].

CD63
In resting platelets, CD63 is localized on the membranes of α-granules and dense granules. Following platelet activation and granule exocytosis, CD63 is expressed on the plasma membrane and colocalizes with the αIIbβ3-CD9 complex. CD63-deficient platelets show slightly enhanced in vitro aggregation responses, but they do not affect thrombus formation in vivo [117]. Palmitoylation levels of CD63 and CD9 increase following thrombin activation.

Tspan32
Tspan32(TSSC6)-deficient platelets exhibit impaired clot retraction, platelet aggregation at lower doses of PAR4, and collagen and platelet spreading on fibrinogen. Tspan32-deficient mice exhibit longer bleeding times and an increase in rebleeding, as shown by tail bleeding assay [118].
A major problem in tetraspanin research is how to determine whether a particular phenotype is due to a specific effect on tetraspanin. CD151 and Tspan32 are direct binding partners of αIIbβ3 and might enhance outside-in signaling by recruiting specific signaling proteins in a subset of glycosphingolipid microdomains.

Calcium Channels (Orai 1, STIM, TRPC)
Platelet activation and aggregation depend on the increase in (Ca 2+ )i resulting from intracellular Ca 2+ release followed by store-operated Ca 2+ entry (SOCE) through Ca 2+ release-activated channels [120]. SOCE is accomplished by the pore forming unit Orai and its regulator the stromal interaction molecule (STIM). STIM1 is a transmembrane protein essential for the activation of SOCE, a major Ca 2+ influx mechanism. STIM1 is localized in the endoplasmic reticulum, communicating the Ca 2+ concentration in the stores to plasma membrane channels. Lipid rafts are required for the inactivation of SOCE by extracellular Ca 2+ mediated by the interaction between plasma-membrane-located STIM1 and Orai1 [70]. Orai1 is a novel candidate of the platelet palmitoylome [88].
Orai1 trafficking to the cell surface is impaired in Tspan18-deficient platelets, resulting in impaired Ca 2+ signaling. Tspan18 may regulate the Ca 2+ channel function of Orai1 at the cell surface by promoting its clustering [121]. A reduction in the rate of release and a maximal Ca 2+ increase are observed in Tspan18-deficient platelets. Defective aggregation of Tspan18-deficient platelets is observed in response to a collagen-related peptide at an intermediate concentration. Tspan18-deficient platelet spreading is impaired on a collagen-related peptide but normal on fibrinogen.

Acknowledgments:
We are indebted to Toshihide Kobayashi (INSERM) for providing lysenin. We are grateful to Yoshiko Ohno-Iwashita (Iwaki Meisei University) for providing BCθ. We thank Hidenori Suzuki (Keio University) for immunoelectron microscopic study.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results