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. 2014 Nov;12(11):1906-17.
doi: 10.1111/jth.12712. Epub 2014 Oct 13.

Exosome Poly-Ubiquitin Inhibits Platelet Activation, Downregulates CD36 and Inhibits Pro-Atherothombotic Cellular Functions

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Exosome Poly-Ubiquitin Inhibits Platelet Activation, Downregulates CD36 and Inhibits Pro-Atherothombotic Cellular Functions

S Srikanthan et al. J Thromb Haemost. .
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Abstract

Introduction: Activated platelets shed microparticles from plasma membranes, but also release smaller exosomes from internal compartments. While microparticles participate in athero-thrombosis, little is known of exosomes in this process.

Materials & methods: Ex vivo biochemical experiments with human platelets and exosomes, and FeCl3 -induced murine carotid artery thrombosis.

Results: Both microparticles and exosomes were abundant in human plasma. Platelet-derived exosomes suppressed ex vivo platelet aggregation and reduced adhesion to collagen-coated microfluidic channels at high shear. Injected exosomes inhibited occlusive thrombosis in FeCl3 -damaged murine carotid arteries. Control platelets infused into irradiated, thrombocytopenic mice reconstituted thrombosis in damaged carotid arteries, but failed to do so after prior ex vivo incubation with exosomes.CD36 promotes platelet activation, and exosomes dramatically reduced platelet CD36.CD36 is also expressed by macrophages, where it binds and internalizes oxidized LDL and microparticles, supplying lipid to promote foam cell formation. Platelet exosomes inhibited oxidized-LDL binding and cholesterol loading into macrophages. Exosomes were not competitive CD36 ligands, but instead sharply reduced total macrophage CD36 content. Exosomal proteins, in contrast to microparticle or cellular proteins, were highly adducted by ubiquitin. Exosomes enhanced ubiquitination of cellular proteins, including CD36, and blockade of proteosome proteolysis with MG-132 rescued CD36 expression. Recombinant unanchored K48 poly-ubiquitin behaved similarly to exosomes, inhibiting platelet function, macrophage CD36 expression and macrophage particle uptake.

Conclusions: Platelet-derived exosomes inhibit athero-thrombotic processes by reducing CD36-dependent lipid loading of macrophages and by suppressing platelet thrombosis. Exosomes increase protein ubiquitination and enhance proteasome degradation of CD36.

Keywords: CD36 protein; cell-derived microparticles; exosomes; platelets; thrombosis.

Figures

Figure1
Figure1. Platelet exosomes are anti-thrombotic and inhibit platelet adhesion
A) Transmission electron microscopy of platelet-derived exosomes and microparticles. Platelets were stimulated with A23187 and removed by low speed centrifugation before microparticles and exosomes were isolated by differential centrifugation as stated in “Methods.” Photomicrographs of exosomes and microparticles are magnified 98,000× and 68,000×, respectively, with 50 and 200 nmeter scale bars. B) Exosomes prolong arterial occlusion time after FeCl3 induced injury. Exosomes (50 μg) were injected into wild type mice through the jugular vein along with Rhodamine 6G to fluorescently label platelets. Thrombosis was induced 10 min later by topical application of filter paper saturated with 7.5% FeCl3 to murine carotid arteries for 1 min. Time to complete cessation of blood flow was determined by intravital microscopy (n=3 experimental, 4 control; *p≤ 0.025). C) Exosome-treated platelets fail to effectively reconstitute occlusive thrombosis in FeCl3 damaged carotid arteries. Murine platelets were incubated with PBS or 50 μg/ml platelet-derived exosomes for 2 h and labeled with Rhodamine 6G. Sufficient treated platelets (109) were injected through the jugular vein into mice previously rendered thrombocytopenic by γ-irradiation to reconstitute platelet numbers [49]. Thrombosis was induced 10 min later by FeCl3 as in the preceding panel before the time to complete cessation of blood flow was determined by intravital microscopy (n=4 experimental, 4 control; *p≤ 0.03). D) Exosomes inhibit platelet ex vivo adhesion to collagen at high shear. Calcein-labeled whole human blood with was treated with buffer, exosomes (50 μg/ml), or microparticles (50 μg/ml), and then flowed through collagen-coated microchannels using the Vena8 Fluoro system (Cellix) at 67.5 dynes cm−2. Representative images of immobilized fluorescent platelets in the distal image frame (top) and fluorescent intensities quantified by ImageJ (bottom). PEX, platelet-derived exosomes; PMP, platelet-derived microparticles.
Figure 2
Figure 2. Platelet exosomes inhibit platelet function
A) Exosomes inhibit collagen-induced platelet aggregation. Washed human platelets were incubated with buffer or exosomes (50 μg/ml) derived from A23187-activated platelets before aggregation was initiated with 0.1U thrombin with stirring. Tracings are representative of 3 independent experiments. B) Exosomes from A23187-, thrombin- or LPS-stimulated platelets were incubated with human platelet rich plasma before aggregation was initiated with ADP (2 μM). Tracings are representative of 3 independent experiments. C) Exosomes from platelets activated by varied stimuli inhibit collagen-induced aggregation. Washed human platelets were incubated for 1h with buffer or 50 μg/ml exosomes isolated from platelets stimulated by the stated agonists prior to initiation of aggregation by 3 μg/ml collagen. * p<0.05 ** p< 0.01 D) Cancer cell-derived exosomes reduce platelet aggregation. Washed human platelets incubated (1 h) with exosomes spontaneously released from Lewis Lung Carcinoma cells (LLEX) were stimulated with collagen (3 μg/ml) to initiate aggregation.
Figure 3
Figure 3. Exosomal proteins are ubiquitinated, and recombinant poly-ubiquitin suppresses platelet aggregation
A) Platelet exosomal proteins are adducted by ubiquitin. Proteins from platelets, exosomes (PEX), or microparticles (PMP) were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and separately immunoblotted (IB) with anti-ubiquitin P4D1 antibody that recognizes free and polymeric ubiquitin, anti-CD63, anti-CD9, or anti-CD36 before antibody detection by peroxidase-conjugated secondary antibody. B) The proteome of exosomes derived from platelets stimulated with varied agonists are differentially conjugated with polyubiquitin. Proteins extracted from exosomes prepared from platelets stimulated with the stated agonists were resolved by SDS-PAGE and immunoblotted with P4D1 antibody to detect ubiquitin modification. C) Exosomes from human platelets and monocytes are ubiquitinated and distinct from their microparticles. Exosomes and microparticles were isolated by differential centrifugation and ubiquitin conjugation of their proteomes determined by western blotting as in the preceding panel. D) Exosomes contain surface-exposed ubiquitin. Exosomes or microparticles from activated platelets were allowed to adhere to a glass surface and stained with anti-ubiquitin antibody P4D1, or a non-immune isotype antibody, and they with Alexa594-conjugated secondary antibody to detect surface exposed ubiquitin by Total Internal Reflection Fluorescence microscopy. E) Recombinant K48 poly-ubiquitin reduces collagen-stimulated platelet aggregation. Washed platelets were incubated with 5 μg/ml recombinant poly-ubiquitin polymerized through lysine 48 prior to treatment with 3 μg/ml collagen to stimulate homotypic aggregation. The tracing is representative of 3 independent experiments and inhibition was significant. * p<0.05.
Figure 4
Figure 4. Exosomes reduce platelet CD36 expression and cargo internalization
A) Exosomes suppress platelet CD36 surface expression. Washed platelets were treated with platelet-derived exosomes (PEX, 50 μg/ml) before surface CD36 was assessed by flow cytometry relative to platelets stained with isotype control antibody or left unstained. B) NO2-LDL-enhanced platelet aggregation was suppressed by exosomes. The CD36 agonist NO2-LDL enhanced, but did not stimulate, collagen-induced platelet aggregation. Exosome pre-incubation (50 μg/ml, 1h) reduced aggregation enhanced by NO2-LDL. C. Recombinant poly-ubiquitin suppressed platelet CD36 expression. Washed platelets were treated with 1 μg/ml unconjugated poly-K48 (3–7) ubiquitin or with the non-immune control antibody from panel A before CD36 expression was assessed by flow cytometry.
Figure 5
Figure 5. Exosomes reduce macrophage CD36 expression and microparticle internalization
A) Unanchored poly-ubiquitin reduces macrophage CD36 surface expression. RAW macrophages were treated, or not, with 1 μg/ml recombinant poly-ubiquitin (poly-ub) for 1 h before surface CD36 was determined by flow cytometry relative to the fluorescence of untreated cells or those stained with control antibody. B) Temporal suppression of macrophage CD36 by exosomes. Cultured macrophages were treated with platelet-derived exosomes (PEX), microparticles (PMP), or both for the stated times before CD36 expressed on unpermeabilized cells was visualized by Alex488-labeled anti-CD36 that fluoresces green. Nuclei of immobilized cells were counterstained with DAPI fluorescing blue. The total green fluorescence of 6 images at each stated time was quantitated by ImageJ (below). C) Exosomes or recombinant polyubiquitin reduces microparticle accumulation by RAW macrophages. Platelet-derived microparticles were fluorescently labeled with calcein-AM, washed, and then incubated with RAW macrophages for 1 h in the presence or absence of 50 μg/ml exosomes (left) or 1 μg/ml poly-ubiquitin (right) prior to analysis of surface CD36 by flow cytometry. Histograms are representative of two independent blood donors.
Figure 6
Figure 6. Exosomes reduce lipid accumulation by macrophages from lipid-rich particles
A) Exosomes reduce macrophage accumulation of NO2 oxidized LDL. RAW macrophages were incubated with fluorescently DiI-labeled NO2 oxidized LDL with or without platelet-derived exosomes for 30 or 60 min before fluorescent dye accumulation was imaged and quantitated by ImageJ n=3, * p<0.05. B) Macrophage lipid loading by NO2-LDL is reduced by exosomes. The CD36-specific ligand NO2-oxidized LDL was incubated with RAW macrophages in the presence or absence of 50 μg/ml platelet exosomes overnight and washed before total cellular cholesterol content was quantified. n=3, * p<0.05
Figure 7
Figure 7. Exosomes do not activate CD36 signaling, nor are accumulated by macrophages
A) Platelet JNK phosphorylation is induced by microparticles, but not exosomes, through CD36. Cultured monocyte-derived macrophages from C57BL6 wild-type or CD36 null mice were treated with 50 μg/ml platelet-derived microparticles (left) or exosomes (right) for the stated times before cellular proteins were resolved and western blotted for phospho-JNK or total JNK. B) NO2 oxidized LDL (NO2-LDL) or microparticles, but not exosomes, increase macrophage cholesterol content. Elicited peritoneal macrophages from wild-type or CD36 null mice were incubated overnight with Ca++ ionophore-elicited platelet microparticles, oxidized LDL, or ionophore-elicited exosomes. In some incubations, the particles were s oxidized by 100 μM tert-butylhydroperoxide (TBHP) prior to incubation with macrophages. Total cellular cholesterol was determined as described in “Methods.” n=3, * p<0.05.
Figure 8
Figure 8. Exosomes increase CD36 ubiquitination and proteasome degradation
Macrophage (A) or platelet (B) surface CD36 was quantified by flow cytometry after exposure to platelet exosomes (50 μg/ml) for 1 following pretreatment, or not, with the proteasome inhibitor MG-132 (10 μM). The comparison is to isotype control antibody or unstained cells C) Macrophage CD36 ubiquitination increases after exosome or MG-132 exposure. Control or exosome- (50 μg/ml; 60 min) treated RAW macrophages were pretreated or not with 10 μM MG-132 for 1 h and then lysed. Lysates were immunoprecipitated (IP) with anti-CD36 antibody (left), resolved by SDS polyacrylamide gel electrophoresis and immunoblotted (IB) with anti-ubiquitin (UB) (top) or for total CD36 (bottom). Conversely, ubiquitin-macrophage conjugates were precipitated with anti-ubiquitin antibody (right) and then probed with anti-CD36 antibody (top) or anti-ubiquitin P4D1 antibody (bottom). D) Exosomes reduce total cellular CD36. Protein lysates from thioglycolate-elicited macrophages treated with or without exosomes derived from either F1 (F1EX) or F10 (F10EX) melanoma cells or Lewis Lung Carcinoma cells (LLEX) were resolved by SDS-PAGE and immunoblotted with anti-CD36 (top) and then re-probed with anti-β-actin. Lysates from platelets (bottom left) and the RAW264.7 macrophage cell line (bottom right) were similarly analyzed.

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