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CD36 as a Multiple-Ligand Signaling Receptor in Atherothrombosis
Abstract and Figures
The glycoprotein CD36, also known as glycoprotein IIIb/IV or FAT, is expressed on the surface of platelets, monocytes, microvascular endothelial cell, smooth muscle cells, cardiomyocytes and other cells of the cardiovascular system. In spite of its abundant presence, CD36 has remained for long a mysterious protein with a poorly understood role. In this paper, we review how CD36 can affect cellular responses by interaction with a variety of ligands, in particular thrombospondin-1, oxidized lipoproteins and fatty acids. Furthermore, given the structure of CD36 with two transmembrane domains and short cytoplasmic tails, we consider how this receptor can induce intracellular signaling, likely in junction with other cellular receptors or associated proteins in the membrane. Current literature points to activation of Src-family and mitogen-activated protein kinases, as well as to activation of the NFκB and Rho pathways. The new insights make CD36 attractive as a therapeutic target to suppress platelet and monocyte/macrophage function and thereby atherothrombosis.
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... Remarkably, platelets express the transmembrane CXC chemokine ligand CXCL16, also known as SR-PSOX, which possesses the capacity to bind to PS on the RBC's surface [80,167]. Furthermore, platelet-RBC interactions may be fostered via the platelet surface glycoprotein CD36, which displays a propensity for binding to PS [168]. More recently, a platelet-RBC interaction was shown to be mediated by FasL/FasR pathway, which triggers PS externalization on RBCs and thereby enhances their procoagulant activity in circulation [169]. ...
Diabetes Mellitus (DM) is a complex metabolic disorder associated with multiple microvascular complications leading to nephropathy, retinopathy, and neuropathy. Mounting evidence suggests that red blood cell (RBC) alterations are both a cause and consequence of disturbances related to DM-associated complications. Importantly, a significant proportion of DM patients develop varying degrees of anemia of confounding etiology, leading to increased morbidity. In chronic hyperglycemia, RBCs display morphological, enzymatic, and biophysical changes, which in turn prime them for swift phagocytic clearance from circulation. A multitude of endogenous factors, such as oxidative and dicarbonyl stress, uremic toxins, extracellular hypertonicity, sorbitol accumulation, and deranged nitric oxide metabolism, have been implicated in pathological RBC changes in DM. This review collates clinical laboratory findings of changes in hematology indices in DM patients and discusses recent reports on the putative mechanisms underpinning shortened RBC survival and disturbed cell membrane architecture within the diabetic milieu. Specifically, RBC cell death signaling, RBC metabolism, procoagulant RBC phenotype, RBC-triggered endothelial cell dysfunction, and changes in RBC deformability and aggregation in the context of DM are discussed. Understanding the mechanisms of RBC alterations in DM provides valuable insights into the clinical significance of the crosstalk between RBCs and microangiopathy in DM.
... erythroblasts, adipocytes, endothelial cells, skeletal myocytes, and cardiac myocytes. CD36 belongs to the class B scavenger receptor family [1], which plays an important role in platelet aggregation, atherosclerosis, cardiopathy, diabetes, and malaria [2][3][4][5][6]. ...
Background
CD36 deficiency is closely associated with fetal/neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness, and other hemorrhage disorders, particularly in Asian and African populations. There is a clinical need for rapid and high-throughput methods of platelet CD36 (pCD36) phenotyping to improve the availability of CD36 typing of donors and assist clinical blood transfusions for patients with anti-CD36 antibodies. Such methods can also support the establishment of databases of pCD36-negative phenotypes.
Study Design and Methods
A sandwich enzyme-linked immunosorbent assay (ELISA) for CD36 phenotyping of human platelets was developed using anti-CD36 monoclonal antibodies. The reliability of the assay was evaluated by calculating the intra-assay and inter-assay coefficients of variation (CV). A total of 1,691 anticoagulant whole blood samples from healthy blood donors were randomly selected. PCD36 expression was measured using a sandwich ELISA. PCD36 deficiency was confirmed by flow cytometry (FC). Mutations underlying pCD36 deficiency were identified using polymerase chain reaction sequence-based typing (PCR-SBT).
Results
The sandwich ELISA for pCD36 phenotyping had high reliability (intra-assay CV, 2.1–4.8%; inter-assay CV, 2.3–5.2%). The sandwich ELISA was used to screen for CD36 expression on platelets isolated from 1,691 healthy blood donors. Of these, 36 samples were pCD36-negative. FC demonstrated absence of CD36 expression on monocytes in three of the 36 cases. In the present study population, the frequency of CD36 deficiency was 2.13% (36/1,691), of which 0.18% (3/1,691) was type I deficiency and 1.95% (33/1,691) was type II deficiency. In addition, we used PCR-SBT to characterize the gene mutations in exons 3–14 of the CD36 gene in 27 cases of CD36 deficiency and discovered 10 types of mutations in 13 pCD36-negative samples.
Conclusion
The present study describes the development and characterization of a highly reliable sandwich ELISA for high-throughput screening for pCD36 expression. This novel method is feasible for clinical applications and provides a useful tool for the establishment of databases of pCD36-negative phenotype donors.
... Determination of binding regions of NEGR1 and CD36 proteins NEGR1 has a relatively simple structure containing three consecutive C2-type Ig-like domains. In contrast, the extracellular region of CD36 contains only the CLESH (CD36, lysosomal integral membrane protein-2 [LIMP-2], Emp sequence homology; residues 93-155) and proline-rich (243-375) domains (27) (Fig. 3A, B). It was predicted that CD36 contains a lipid binding pocket at residues 127-279 (28), including a hydrophobic patch that might loop down into the plasma membrane (29). ...
Neuronal growth regulator 1 (NEGR1) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein associated with several human pathologies, including obesity, depression, and autism. Recently, significantly enlarged white adipose tissue (WAT), hepatic lipid accumulation, and decreased muscle capacity were reported in Negr1-deficient mice. However, the mechanism behind these phenotypes was not clear. In the present study, we found NEGR1 to interact with cluster of differentiation 36 (CD36), the major fatty acid translocase in the plasma membrane. Binding assays with a soluble form of NEGR1 and in-situ proximal ligation assays indicated that NEGR1–CD36 interaction occurs at the outer leaflet of the cell membrane. Furthermore, we show that NEGR1 overexpression induced CD36 protein destabilization in vitro. Both mRNA and protein levels of CD36 were significantly elevated in the WAT and liver tissues of Negr1-/- mice. Accordingly, fatty acid uptake rate increased in NEGR1-deficient primary adipocytes. Finally, we demonstrated that Negr1-/- mouse embryonic fibroblasts (MEFs) showed elevated reactive oxygen species levels and decreased adenosine monophosphate-activated protein kinase activation compared with control MEFs. Based on these results, we propose that NEGR1 regulates cellular fat content by controlling the expression of CD36.
... However, non esterified fatty acids cannot circulate freely in plasma, they are bound to albumin [130], which has seven fatty acid binding sites [131]. Interaction with CD36 is associated with activation of Src family kinases and mitogen activated protein kinases, as well as with the participation of Rho GTPases and tran scription factor NF κB [132]. Still to be found out does albumin binding and transcytosis require the involvement of these or other signaling pathways. ...
Being one of the main proteins in the human body and many animal species, albumin plays a crucial role in the transport of various ions, electrically neutral molecules and in maintaining the colloidal osmotic pressure of the blood. Albumin is able to bind almost all known drugs, many nutraceuticals and toxic substances, determining their pharmaco- and toxicokinetics. However, albumin is not only the passive but also the active participant of the pharmacokinetic and toxicokinetic processes possessing a number of enzymatic activities. Due to the thiol group of Cys34, albumin can serve as a trap for reactive oxygen and nitrogen species, thus participating in redox processes. The interaction of the protein with blood cells, blood vessels, and also with tissue cells outside the vascular bed is of great importance. The interaction of albumin with endothelial glycocalyx and vascular endothelial cells largely determines its integrative role. This review provides information of a historical nature, information on evolutionary changes, inflammatory and antioxidant properties of albumin, on its structural and functional modifications and their significance in the pathogenesis of some diseases.
... However, non-esterified FAs cannot circulate freely in plasma as they are bound to albumin [205], which has seven FA binding sites [206]. Interactions with CD36 are associated with the activation of Src-family kinases and mitogen-activated protein kinases, as well as with the participation of Rho-GTPases and NFκB transcription factors [207]. Whether binding and transcytosis of albumin requires these or other signaling pathways remains to be investigated. ...
Being one of the main proteins in the human body and many animal species, albumin plays a decisive role in the transport of various ions—electrically neutral and charged molecules—and in maintaining the colloidal osmotic pressure of the blood. Albumin is able to bind to almost all known drugs, as well as many nutraceuticals and toxic substances, largely determining their pharmaco- and toxicokinetics. Albumin of humans and respective representatives in cattle and rodents have their own structural features that determine species differences in functional properties. However, albumin is not only passive, but also an active participant of pharmacokinetic and toxicokinetic processes, possessing a number of enzymatic activities. Numerous experiments have shown esterase or pseudoesterase activity of albumin towards a number of endogeneous and exogeneous esters. Due to the free thiol group of Cys34, albumin can serve as a trap for reactive oxygen and nitrogen species, thus participating in redox processes. Glycated albumin makes a significant contribution to the pathogenesis of diabetes and other diseases. The interaction of albumin with blood cells, blood vessels and tissue cells outside the vascular bed is of great importance. Interactions with endothelial glycocalyx and vascular endothelial cells largely determine the integrative role of albumin. This review considers the esterase, antioxidant, transporting and signaling properties of albumin, as well as its structural and functional modifications and their significance in the pathogenesis of certain diseases.
... Activated platelets may further activate other platelets (94), recruit immune cells, damage glomerular cell, and cause fibrosis in DN. Once platelets are activated, platelets will express the CD36 molecule (CD36), protein kinase C eta (PRKCH), and coagulation factor II thrombin receptor like 2 (F2RL2) on the surface of platelets, which will cause more platelets to be activated (116)(117)(118)(119)(120)(121)(122)(123). Platelet hyper function is observed in DM (92,93) and DN (93) patients, indicating that platelets may play an important role in the development of DN. ...
Diabetic nephropathy (DN) is the main cause of end stage renal disease (ESRD). Glomerulus damage is one of the primary pathological changes in DN. To reveal the gene expression alteration in the glomerulus involved in DN development, we screened the Gene Expression Omnibus (GEO) database up to December 2020. Eleven gene expression datasets about gene expression of the human DN glomerulus and its control were downloaded for further bioinformatics analysis. By using R language, all expression data were extracted and were further cross-platform normalized by Shambhala. Differentially expressed genes (DEGs) were identified by Student's t-test coupled with false discovery rate (FDR) (P < 0.05) and fold change (FC) ≥1.5. DEGs were further analyzed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to enrich the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. We further constructed a protein-protein interaction (PPI) network of DEGs to identify the core genes. We used digital cytometry software CIBERSORTx to analyze the infiltration of immune cells in DN. A total of 578 genes were identified as DEGs in this study. Thirteen were identified as core genes, in which LYZ, LUM, and THBS2 were seldom linked with DN. Based on the result of GO, KEGG enrichment, and CIBERSORTx immune cells infiltration analysis, we hypothesize that positive feedback may form among the glomerulus, platelets, and immune cells. This vicious cycle may damage the glomerulus persistently even after the initial high glucose damage was removed. Studying the genes and pathway reported in this study may shed light on new knowledge of DN pathogenesis.
... A possible explanation might reside in the ligand-bound interaction of CD36 with a range of associated proteins in the membrane to transmit further signals, including clathrinbinding proteins. However, the mechanism is not completely clear [43]. In contrast to these results, the investigation of lipid raft-mediated endocytosis showed that in both cell types, MβCD did not influence the effect of the drugs on cell growth (Supplementary Figure S1B), suggesting that C18GEM and GEM are not dependent on lipid rafts for entering into the cells. ...
Background:
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance.
Methods:
We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs.
Results:
PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters' expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions.
Conclusion:
We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment.
... Studies have highlighted the pro-tumorigenic and prometastatic actions of CD36 in various cancer types, including oral squamous cell carcinoma, breast cancer, and melanoma [12]. However, due to its multifunctional role, the potential metastasis-promoting activity of CD36 in GC and other cancers remains to be fully characterized [13]. ...
Evidence indicates that the lipid scavenger receptor CD36 has pro-metastatic functions in several cancers. Although CD36 expression correlates with an unfavorable prognosis in gastric cancer (GC), its specific contribution to disease onset, progression, and/or metastasis remains unclear. Using bioinformatics analyses, we ascertained that CD36 expression was increased in metastatic GC specimens in The Cancer Genome Atlas and Gene Expression Omnibus databases and correlated with poor prognosis. In addition, higher CD36 expression was associated with lymph node metastasis (p < 0.05) and poor prognosis (p = 0.030) in 79 Chinese GC patients. Basal CD36 expression levels correlated positively with migration, invasion, and expression of epithelial-to-mesenchymal transition (EMT) markers in GC cell lines, a relationship confirmed by knockdown and overexpression experiments. Importantly, analysis of gene expression changes in CD36-knockdown GC cells led us to identify the chromatin-associated protein DEK as a c-Myc target that mediates activation of the GSK-3β/β-catenin signaling pathway to trigger EMT. These findings further our understanding of the mechanisms governing metastatic dissemination of GC cells and suggest the therapeutic potential of strategies targeting CD36.
Chronic stress leads to post-traumatic stress disorder (PTSD) and metabolic disorders including fatty liver. We hypothesized that stress-induced molecular mechanisms alter energy metabolism, thereby promoting hepatic lipid accumulation even after a stress-free recovery period. In this context, we investigated fibroblast growth factor-21 (FGF21) as protective for energy and glucose homeostasis. FGF21 knockout mice (B6.129S6(SJL)-Fgf21tm1.2Djm; FGF21KO) and control mice (C57BL6; WT) were subjected to chronic variable stress. Mice were examined directly after acute intervention (Cvs) and long-term after 3 months of recovery (3mCvs). In WT, Cvs reduced insulin sensitivity and hepatic lipid accumulation, whilst fatty acid uptake increased. FGF21KO mice responded to Cvs with improved glucose tolerance, insulin resistance but liver triglycerides and plasma lipids were unaltered. Hepatic gene expression was specifically altered by genotype and stress e.g. by PPARa and SREBP-1 regulated genes. The stress-induced alteration of hepatic metabolism persisted after stress recovery. In hepatocytes at 3mCvs, differential gene regulation and secreted proteins indicated a genotype specific progression of liver dysfunction. Overall, at 3mCvs FGF21 was involved in maintaining mitochondrial activity, attenuating de novo lipogenesis, increased fatty acid uptake and histone acetyltransferase activity. Glucocorticoid release and binding to the FGF21 promoter may contribute to prolonged FGF21 release and protection against hepatic lipid accumulation. In conclusion, we showed that stress favors fatty liver disease and FGF21 protected against hepatic lipid accumulation after previous chronic stress loading by i) restored physiological function, ii) modulated gene expression via DNA-modifying enzymes, and iii) maintained energy metabolism.
GPR40 is highly expressed in pancreatic islets and its activation increases glucose-stimulated insulin secretion from pancreas. Therefore, GPR40 is considered as a target for type 2 diabetes mellitus (T2DM). Since nonalcoholic fatty liver disease (NAFLD) is associated with T2DM and GPR40 is also expressed by hepatocytes and macrophages, it is important to understand the role of GPR40 in NAFLD. However, the role of GPR40 in NAFLD in animal models has not been well defined. In this study, we fed wild-type or GPR40 knockout C57BL/6 mice high-fat diet (HFD) for 20 weeks and then assessed the effect of GPR40-deficiency on HFD-induced NAFLD. Assays on metabolic parameters showed that HFD increased bodyweight, glucose, insulin, insulin resistance, cholesterol and alanine aminotransferase (ALT), and GPR40-deficiency did not mitigate the HFD-induced metabolic abnormalities. In contrast, we found that GPR40-deficiency was associated with increased bodyweight, insulin, insulin resistance, cholesterol and ALT in control mice fed low fat diet (LFD). Surprisingly, histology and Oil Red O staining showed that GPR40-deficiency in LFD-fed mice was associated with steatosis. Immunohistochemical analysis showed that GPR40-deficiency also increased F4/80, a macrophage biomarker, in LFD-fed mice. Furthermore, results showed that GPR40-deficiency led to a robust upregulation of hepatic fatty acid translocase (FAT)/CD36 expression. Finally, our in vitro studies showed that GPR40 knockdown by siRNA or GPR40 antagonist increased palmitic acid-induced FAT/CD36 mRNA in hepatocytes. Taken together, this study indicates that GPR40 plays an important role in homeostasis of hepatic metabolism and inflammation and inhibits nonalcoholic steatohepatitis by possible modulation of FAT/CD36 expression.
Deficiency of the membrane protein FAT/CD36 causes a marked defect in fatty acid uptake by various tissues and is genetically linked to insulin resistance in rats and humans. Here, we examined insulin responsiveness of CD36(-/-) mice. When fed a diet high in complex carbohydrates and low (5%) in fat, these animals cleared glucose faster than the wild-type. In vivo, uptake of 2-fluorodeoxyglucose by muscle was increased severalfold, and in vitro, insulin responsiveness of glycogenesis by the soleus was enhanced. Null mice had lower glycogen levels in muscle and liver, lower muscle triglyceride levels, and increased liver triglyceride content-all findings consistent with increased insulin-sensitivity. However, when the chow diet was switched to one high in fructose, CD36(-/-) mice but not wild-type mice developed marked glucose intolerance, hyperinsulinemia, and decreased muscle glucose uptake. High-fat diets impaired glucose tolerance equally in both groups, although CD36 deficiency helped moderate insulin-responsive muscle glucose oxidation. In conclusion, CD36 deficiency enhances insulin responsiveness on a high-starch, low-fat diet. It predisposes to insulin resistance induced by high fructose and partially protects from that induced by high-fat diets. In humans, CD36 deficiency may be an important factor in the metabolic adaptation to diet and in susceptibility to some forms of diet-induced pathology.
A peptide from the C-terminal domain of thrombospondin-1 (Arg-Phe-Tyr-Val-Val-Met-Trp-Lys; known as 4N1-1) has been reported to induce platelet aggregation and to bind to the integrin-associated protein (IAP), which is also known as CD47. In this study, it was discovered that 4N1-1 or its derivative peptide, 4N1K, induces rapid phosphorylation of the Fc receptor (FcR) γ chain, Syk, SLP-76, and phospholipase C γ2 in human platelets. A specific inhibitor of Src family kinases, 4-amino-4-(4-methylphenyl)-7-(t-butyl) pyrazola[3,4-d]pyrimidine, prevented phosphorylation of these proteins, abolished platelet secretion, and reduced aggregation by approximately 50%. A similar inhibition of aggregation to 4N1-1 was obtained in the presence of Arg-Gly-Asp-Ser in mouse platelets deficient in FcR γ chain or SLP-76 and in patients with type I Glanzmann thrombasthenia. These results show that 4N1-1 signals through a pathway similar to that used by the collagen receptor glycoprotein (GP) VI. The αIIbβ3-independent aggregation induced by 4N1-1 was also observed in fixed platelets and platelets from patients with Bernard-Soulier syndrome, which are deficient in GPIbα. Surprisingly, the ability of 4N1-1 to stimulate aggregation and tyrosine phosphorylation was not altered in platelets pretreated with anti-IAP antibodies and in IAP-deficient mice. These results show that the C-terminal peptide of thrombospondin induces platelet aggregation through the FcR γ-chain signaling pathway and through agglutination. The latter pathway is independent of signaling events and does not use GPIbα or αIIbβ3. Neither of these pathways is mediated by IAP.
Thrombospondin (TSP) 2, and its close rela- tive TSP1, are extracellular proteins whose functions are complex, poorly understood, and controversial. In an attempt to determine the function of TSP2, we dis- rupted the Thbs2 gene by homologous recombination in embryonic stem cells, and generated TSP2-null mice by blastocyst injection and appropriate breeding of mu- tant animals. Thbs2 2 / 2 mice were produced with the expected Mendelian frequency, appeared overtly nor- mal, and were fertile. However, on closer examination, these mice displayed a wide variety of abnormalities. Collagen fiber patterns in skin were disordered, and ab- normally large fibrils with irregular contours were ob- served by electron microscopy in both skin and tendon. As a functional correlate of these findings, the skin was fragile and had reduced tensile strength, and the tail was unusually flexible. Mutant skin fibroblasts were de- fective in attachment to a substratum. An increase in total density and in cortical thickness of long bones was documented by histology and quantitative computer to- mography. Mutant mice also manifested an abnormal bleeding time, and histologic surveys of mouse tissues, stained with an antibody to von Willebrand factor, showed a significant increase in blood vessels. The basis for the unusual phenotype of the TSP2-null mouse could derive from the structural role that TSP2 might play in collagen fibrillogenesis in skin and tendon. However, it seems likely that some of the diverse mani- festations of this genetic disorder result from the ability of TSP2 to modulate the cell surface properties of mes- enchymal cells, and thus, to affect cell functions such as adhesion and migration.
A null mutation in the scavenger receptor gene CD36 was created in mice by targeted homologous recombination. These mice produced
no detectable CD36 protein, were viable, and bred normally. A significant decrease in binding and uptake of oxidized low density
lipoprotein was observed in peritoneal macrophages of null mice as compared with those from control mice. CD36 null animals
had a significant increase in fasting levels of cholesterol, nonesterified free fatty acids, and triacylglycerol. The increase
in cholesterol was mainly within the high density lipoprotein fraction, while the increase in triacylglycerol was within the
very low density lipoprotein fraction. Null animals had lower fasting serum glucose levels when compared with wild type controls.
Uptake of3H-labeled oleate was significantly reduced in adipocytes from null mice. However, the decrease was limited to the low ratios
of fatty acid:bovine serum albumin, suggesting that CD36 was necessary for the high affinity component of the uptake process.
The data provide evidence for a functional role for CD36 in lipoprotein/fatty acid metabolism that was previously underappreciated.
The transmembrane glycoprotein CD36 has been identified in isolated cell studies as a putative transporter of long-chain fatty
acids. To examine the physiological role of CD36, we studied FA uptake and metabolism by tissues of CD36 null mice after injection
with two fatty acid analogs. Compared to controls, uptake was substantially reduced (50–80%) in heart, skeletal muscle, and
adipose tissues of null mice. The reduction in uptake was associated with a large decrease in fatty acid incorporation into
triglycerides, which could be accounted for by an accumulation of diacylglycerides. Thus CD36 facilitates a major fraction
of fatty acid uptake by myocardial, skeletal muscle, and adipose tissues, where it is highly expressed. Its role in other
tissues where its expression is low and cell-specific could not be determined in these studies.
Introduction: Platelets bind plasma-derived thrombospondin-1 (TSP1) in part via CD36. Whether and how TSP1 activates platelets is still debated.
Methods: Activation properties of suspended platelets were measured by flow cytometry. TSP1 was immobilized on coverslips in the presence or absence of collagen, and (Fluo-4-loaded) platelets were allowed to adhere under stasis or high shear flow. Thrombus formation was assessed by (confocal) fluorescence microscopy.
Results: TSP1 failed to increase activation or aggregation of platelets in suspension. However, immobilized TSP1 supported platelet adhesion, spreading, strong [Ca 2+]i flux and procoagulant activity, even in the absence of autocrine and integrin αIIbβ3 stimulation. These responses were significantly inhibited by a panel of CD36 blockers (50-55%, p<0.05). Similar results were obtained with platelets spreading on the supposed CD36 ligand, ox-LDL. Co-coating of TSP1 and collagen enhanced shear-dependent platelet activation and thrombus formation, again in part via CD36. Deficiency in murine TSP1 led to a marked reduction in thrombus formation.
Conclusions: TSP1 appears to be another adhesive protein that, like fibrinogen, activates platelets only when it is surface-immobilized. The resulting outside-in signaling and platelet spreading is mediated by CD36. In junction with collagen, this role of TSP1 appears to stimulate thrombus formation. This provides a novel mechanism for the role of TSP1 in primary haemostasis.
Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from alpha-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted alpha-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation. and was inhibited by cytochalasin D. an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors. as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. alphaIIbbeta3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking alphaIIbbeta3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes. as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1. in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of alphaIIbbeta3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin microfilament system which may promote platelet aggregate cohesion.
CD36 is a multifunctional cell surface glycoprotein that acts as a surface receptor for thrombospondin (TSP), and thereby
may mediate adhesive interactions between cells and substrata, platelets and other cells, and macrophages and apoptotic neutrophils.
The identity of the TSP binding site on CD36 is controversial and may involve more than one structural domain. We have constructed
a series of recombinant bacterial GST/CD36 fusion proteins that span nearly all of the CD36 molecule and have demonstrated
that fusion proteins containing the region extending from amino acid 93 to 120 formed specific, saturable, and reversible
complexes with TSP. As with intact CD36, binding was calcium-dependent, was independent of which ligand was immobilized, and
was blocked by monoclonal antibodies to both CD36 and TSP. Stoichiometry and affinity of the fusion proteins for TSP were
consistent with that of the intact protein. We also demonstrated that these fusion proteins competitively inhibited binding
of TSP to purified platelet CD36 and to cell surface CD36 on peripheral blood monocytes and CD36 cDNA-transfected melanoma
cells. These data demonstrate that the region between amino acids 93 and 120 has all of the characteristics required of the
TSP binding domain.
Cd36 is a small-molecular-weight integral membrane protein expressed in a diverse, but select, range of cell types. It has an equally diverse range of ligands and physiological functions, which has implicated Cd36 in a number of diseases including insulin resistance, diabetes, and, most notably, atherosclerosis. The protein is reported to reside in detergent-resistant microdomains within the plasma membrane and to form homo- and hetero-intermolecular interactions. These data suggest that this class B scavenger receptor may gain functionality for ligand binding, and/or ligand internalization, by formation of protein complexes at the cell surface. Here, we have overexpressed Cd36 in insect cells, purified the recombinant protein to homogeneity, and analyzed its stability and solubility in a variety of nonionic and zwitterionic detergents. Octylglucoside conferred the greatest degree of stability, and by analytical ultracentrifugation we show that the protein is monomeric. A solid-phase ligand-binding assay demonstrated that the purified monomeric protein retains high affinity for acetylated and oxidized low-density lipoproteins. Therefore, no accessory proteins are required for interaction with ligand, and binding is a property of the monomeric fold of the protein. Thus, the highly purified and functional Cd36 should be suitable for crystallization in octylglucoside, and the in vitro ligand-binding assay represents a promising screen for identification of bioactive molecules targeting atherogenesis at the level of ligand binding.
Since glycoprotein IV (GPIV) has been shown to play an important role in the interaction of platelets with collagen and thrombospondin, the aggregation and secretion of GPIV-deficient platelets were examined. Using a binding assay with monoclonal 125I-OKM5 antibody against CD36 antigen and crossed immunoelectrophoresis of the solubilized platelets against anti-GPIV antibody, the platelets from seven (4.1%) out of 170 healthy Japanese donors were found to be deficient in GPIV. The GPIV-deficient platelets showed normal aggregations in response to collagen as well as ADP, epinephrine, arachidonic acid and thrombin in comparison with GPIV-positive platelets. Polyclonal anti-GPIV antibody aggregated GPIV-positive platelets but not the GPIV-negative ones. The F(ab')2 fragments of the anti-GPIV antibody competitively inhibited the anti-GPIV-induced aggregation, but did not affect the collagen-induced aggregation of GPIV-positive platelets. These results suggest that the deficiency of GPIV does not affect platelet aggregability.