| BackgroundNonvalvular atrial fibrillation (NVAF) is a major cause of stroke and thromboembolism. Although anticoagulant therapy is recommended for patients with NVAF at high risk for stroke,33%-38%of patients receiving anticoagulant therapy still experience stroke. The mechanisms underlying thromboembolism in NVAF are still incompletely understood. The activation of platelets may initiate thrombosis. Abundant thrombin is generated as a result of a cascade of coagulation factors activated on the phospholipid surface of activated platelets, thereby leading to thrombosis.However, antiplatelet therapy in NVAF patients has been challenged. Compared with placebo, with aspirin treatment, the risk of thromboembolism in patients with AF decreased by only21%, which was less effective than warfarin. Insufficiency of the present antiplatelet therapy may be associated with the diversity of platelet activation pathways. The molecular mechanisms that underlie platelet activation in NVAF are poorly defined but may involve the etiology of NVAF.The potential effects of inflammation, oxidative stress and metabolic disorders in the NVAF-related prothrombotic state have aroused much attention. However, these risk factors have not been considered together in targeting prevention of NVAF-related platelet activation. To determine a vector exhibiting all the mechanisms in NVAF, we can study the potential mechanisms of platelet activation and explore new, effective targets for preventing thrombosis in NVAF. Recently, studies of microvesicles (MVs) have enlightened us.MVs are submicrometer vesicles formed by activated cells. MVs released into the circulation can act as messengers delivering a variety of cargos, including cell surface receptors, proinflammatory cytokines, signature proteomes, and even mRNA, to target cells. Thus, MVs may be the vectors combining multiple pathogenic mechanisms of platelet activation. Platelet-derived microvesicles (PMVs), with surface exposure of phosphatidylserine (PS, with strong affinity for Annexin V), are the main culprit in the development of thrombosis. CD36, a class B scavenger receptor expressed on platelets, monocytes and several other cells. Its role in platelet function has remained obscure. Platelet CD36could recognize and capture PS on the surface of MVs, thus generating an MV-CD36complex. Accordingly, we speculated that the PMVs, generated in response to pathogenesis or accompanying disorders of NVAF, could be endogenous CD36ligands that transmit an activating signal to platelets to induce thrombosis. We investigated healthy controls and the NVAF patients with "low to moderate risk" or "high risk" for stroke to determine the triggers for the release of platelet-derived microvesicles and Annexin V-positive microvesicles and to explore the potential relationship among microvesicles, platelet CD36and platelet activation.Objectives1. To screen for the risk factors which trigger the release of the microvesicles and platelet-derived microvesicles in patients with NVAF.2. To determine the plasma levels of platelet-derived microvesicles and Annexin V-positive microvesicles in NVAF patients, as well as their relationship to stroke risk.3. To determine the expression of platelet CD36and its relationship to stroke risk in NVAF patients.4. To explore the relationship among platelet-derived microvesicles, Annexin V-positive microvesicles, platelet CD36and platelet activation indexes in patients with NVAF.Subjects and methodsWe included210patients with persistent or paroxysmal NVAF (mean age62.23±11.47years;127men) and35healthy controls (mean age55.71±7.43;17men). All subjects completed a questionnaire on age, gender, present condition, family and medical histories of cardiovascular risk factors and complications. Height, weight, body mass index (BMI), waist circumference, hip circumference and blood pressure (BP) were measured. Most subjects underwent2D transthoracic echocardiography and carotid ultrasonography evaluation. Furthermore, fasting blood sample was collected from each subject after12-14hours fast to determine the fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), creatinine (Cr) and uric acid (UA); Measurement of8-iso-prostaglandinF2α, interleukin6(IL-6), advanced glycosylation end products (AGEs) and soluble P-selectin involved ELISA commercially; The quantification of platelet-derived microvesicles and Annexin V-positive microvesicles in platelet free plasma (PFP) involved use of a homemade ELISA; The expression of platelet GPⅡ b/Ⅲa and CD36were detected by flow cytometry.Results1. Characteristics in NVAF patients:relationship to stroke riskPatients were classified as being at "low to moderate risk" or "high risk" for stroke according to the CHADS2stroke risk scheme:1point each for presence of congestive heart failure, hypertension, age older than75or diabetes mellitus; and2points for history of stroke or transient ischemic attack. Patients with CHADS2score0or1were considered at "low to moderate risk" and those with score≥2at "high risk". Of the210NVAF patients,113(53.81%) were at high risk of stroke (mean age:66.68±10.46years,62males and51females),97(46.19%) were at low to moderate risk of stroke (mean age:57.05±10.41years,65males and32females).2. Clinical characteristics of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke(1) There were no significant differences in sex, body mass index (BMI), diastolic blood pressure (DBP), cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c) and platelet counts among healthy controls, NVAF patients at "low to moderate risk" and NVAF patients at "high risk" for stroke (P>0.05).(2) There were no significant differences in age, sex, BMI, DBP, fasting blood glucose, cholesterol, cholesterol, TG, LDL-c, HDL-c, UA, creatinine and platelet counts between healthy controls and NVAF patients at low to moderate risk for stroke (P>0.05). Systolic blood pressure (SBP) and waist-to-hip ratio (WHR) were significantly higher in the patients at low to moderate risk compared with healthy controls (P<0.01).(3) By definition, the high-risk NVAF patients were older and more likely to have congestive heart failure, hypertension, diabetes mellitus, or history of stroke than those at low to moderate risk (P<0.001for all); Furthermore, SBP, fasting blood glucose, creatinine, white blood cell counts were significantly higher in the patients at low to moderate risk compared with healthy controls and the low to moderate-risk NVAF patients (P<0.01); There was no significant differences in WHR between the high-risk NVAF patients and the low to moderate-risk NVAF patients (P>0.05). WHR was higher in the high-risk NVAF patients compared with healthy controls (P <0.001).3. Clinical medication of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for strokeCompared with the NVAF patients at low to moderate risk for stroke, the application proportion of warfarin, calcium channel blockers, beta-receptor blockers and statins had no significant differences in the high-risk NVAF patients (P>0.05); However, the patients in the high-risk group received more anti-platelet therapy and ACEI/ARB compared with those with low to moderate risk (P<0.01); The healthy controls were free of any medication.4. Comparison of ultrasonic parameters among the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke(1) The transthoracic echocardiography results:There were no significant differences in thickness of inter-ventricular septum (IVST), thickness of posterior wall of left ventricle (LVPWT) and left ventricular ejection fraction (LVEF) between NVAF patients at low to moderate risk for stroke and healthy controls (P>0.05), but the diameter of left atrium (LAD), the diameter of left atrium (RAD), ratio of early transmitral flow velocity to early mitral annular diastolic velocity (E/E’) and the pulmonary capillary wedge pressure (PCWP) were higher in patients at low to moderate risk (P<0.01); The high-risk NVAF patients showed significantly decreases in LVEF (P<0.01), with significantly increases in LAD, RAD, IVST, LVPWT, E/E’and PCWP (P<0.01) compared with healthy controls; Compared with the NVAF patients at low to moderate risk for stroke, the high-risk patients showed significantly decreases in LVEF (P<0.05), with increases in E/E’(P<0.05) and no differences in PCWP (P>0.05).(2) The carotid ultrasonography results:The mean intima-media thickness (IMT) and plaque score increased significantly in the NVAF patients at low to moderate risk for stroke compared with healthy controls (P<0.01); Furthermore, the high-risk NVAF patients showed significantly increases in the mean IMT and plaque score than healthy controls (P<0.001); Compared with patients at low to moderate risk, the high-risk NVAF patients showed significantly increases in plaque score (P<0.05), with no differences in IMT (P>0.05).5. Plasma markers of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke(1) Compared with healthy controls, oxLDL and8-iso-PGF2a (indexes of oxidative stress) were significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.01); Meanwhile, oxLDL and8-iso-PGF2a were significantly higher in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.05).(2) Compared with healthy controls, IL-6was significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.05); Furthermore, IL-6was significantly higher in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.001).(3) Compared with healthy controls, the levels of AGEs (markers of glucose metabolic disorders) was significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.001); Furthermore, AGEs was significantly higher in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.01).6. Microvesicles of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for strokeCompared with healthy controls, PMVs and Annexin V-positive PMVs were significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.01); PMVs and Annexin V-positive PMVs were significantly higher in the high-risk NVAF patients compared with healthy controls (P<0.001); Furthermore, PMVs and Annexin V-positive PMVs were higher in the high-risk NVAF patients compared with those at low to moderate risk (P<0.01).7. Platelet markers of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke(1) The expression of platelet CD36was significantly increased in the NVAF patients at low to moderate risk for stroke compared with healthy controls (P<0.001); Furthermore, mean fluorescent intensity (MFI) of platelet CD36was significantly increased in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.01).(2) Platelet activation in patients was assessed by surface detection of GPⅡ b/Ⅲa (PAC-1binding) and degranulation of P-selectin, both of which enhanced significantly in the high-risk group compared with those in the low to moderate-risk group (P<0.05) and the healthy controls (P<0.001) although the patients in the high-risk group received more anti-platelet therapy. Furthermore, platelet GP Ⅱb/Ⅲa and plasma soluble P-selectin enhanced significantly in the NVAF patients at low to moderate risk compared with the healthy controls (P<0.001).8. Correlations of the CHADS2scores and the platelet activation markersThe CHADS2scores was correlated with platelet GP Ⅱb/Ⅲa significantly (r=0.264, P<0.001); Meanwhile, the CHADS2scores was correlated with plasma soluble P-selectin significantly (r=0.448, P<0.001).9. Correlations of the CHADS2scores and plasma levels of PMVsThe CHADS2scores was correlated with plasma levels of PMVs (r=0.213, P<0.001); Meanwhile, the CHADS2scores was correlated with plasma levels of Annexin V-positive PMVs (r=0.449, P<0.001).10. Correlations of the CHADS2scores and plasma markersThe CHADS2scores was correlated with plasma levels of8-iso-PGF2a significantly (r=0.353, P<0.001); the CHADS2scores was correlated with plasma levels of ox-LDL significantly (r=0.338, P<0.001); the CHADS2scores was correlated with IL-6significantly (r=0.410, P<0.001); the CHADS2scores was correlated with AGEs significantly (r=0.511, P<0.001).11. Correlations of PMVs and the plasma markersPMVs was associated with8-iso-PGF2a significantly (r=0.320, P<0.001); PMVs was associated with IL-6significantly (r=0.150, P=0.027); PMVs was associated with AGEs significantly (r=0.226, P=0.001).12. Correlations of Annexin V-positive PMVs and the plasma markersAnnexin V-positive PMVs was associated with8-iso-PGF2a significantly (r=0.228, P=0.004); Annexin V-positive PMVs was associated with oxLDL significantly (r=0.321, P<0.001); Annexin V-positive PMVs was associated with IL-6significantly (r=0.176, P=0.008); Annexin V-positive PMVs was associated with AGEs significantly (r=0.228, P=0.001).13. Correlations of plasma soluble P-selectin and microvesiclesPMVs was correlated with plasma soluble P-selectin (index of platelet activation) significantly (r=0.184, P=0.007); Annexin V-positive PMVs was also correlated with plasma soluble P-selectin significantly (r=0.173, P=0.009). Those results indicated that PMVs and Annexin V-positive PMVs may be involved in the process of platelet activation in NVAF patients.14. Associations of platelet CD36and platelet activation markersMFI of platelet CD36was correlated with platelet GPⅡb/Ⅲa significantly (r=0.296, P<0.001); Meanwhile, platelet CD36was correlated with plasma soluble P-selectin significantly (r=0.248> P<0.001). Moreover, multivariate linear regression showed that the CD36contributed to the activation of platelet activation (β=0.314, P=0.011for GP Ⅱb/Ⅲa and β=0.114, P=0.045for soluble P-selectin).Conclusions(1) Compared with controls and NVAF patients at low to moderate risk for stroke, the high-risk NVAF patients had higher levels of oxidative stress, inflammation and AGEs;(2) Compared with controls and NVAF patients at low to moderate risk for stroke, the plasma levels of PMVs and Annexin V-positive PMVs were significantly enhanced in the high-risk NVAF patients;(3) Compared with controls and NVAF patients at low to moderate risk for stroke, the high-risk NVAF patients had higher levels of platelet CD36and platelet activation;(4) PMVs and Annexin V-positive PMVs were associated with markers of oxidative stress, inflammation and AGEs significantly, indicating that oxidative stress, inflammation and AGEs might be the important stimuli of production of PMVs in NVAF;(5) PMVs and Annexin V-positive PMVs were associated with markers of platelet activation significantly, indicating that PMVs might be the important mediators of platelet activation mechanisms in NVAF.(6) Platelet CD36was correlated with markers of platelet activation significantly, indicating that CD36might involved in the signal pathway of platelet activation in NVAF. BackgroundNonvalvular atrial fibrillation (NVAF) is a. major cause of stroke and thromboembolism. It is still controversial whether the prothrombotic state and thrombosis in NVAF due to the atrial arrhythmia alone or the coexisting pathological factors. The results of AFFIRM and RACE trials did not demonstrate any superiority of a rhythm control versus a rate control strategy on occurrence of ischemic stroke. So, the thrombosis is independent of AF itself. The underlying pathological factors, such as oxidative stress and inflammation are speculated be the principal culprit of platelet activation in NVAF.The upstream diseases of NVAF, including coronary heart diseases, hypertension, diabetes mellitus, could enhance the oxidative stress and inflammatory state. The process of non-enzymatic glycation of proteins and lipids is markedly accelerated in the setting of inflammation and oxidative stress. The resulting new products are defined as advanced glycation endproducts (AGEs), which mediate the atrial fibrosis and contribute to the pathogenesis of NVAF. We have demonstrated that, the plasma levels of oxidative stress, inflammation and AGEs enhanced significantly in NVAF patients at high risk for stroke compared with those at low to moderate risk. So, oxidative stress, inflammation and accumulated AGEs might not only result in occurrence of NVAF, but also be linked to thrombogenesis. Platelets contribute to thrombosis by assembling into aggregates and by stimulating blood coagulation. Activated platelets play a well established role in thrombosis of NVAF. How the fundamental disorders of NVAF, such as oxidative stress, inflammation and accumulated AGEs, drive the prothrombotic state in atrial fibrillation? Recently, studies of microvesicles (MVs) have enlightened us.MVs are membranous submicrometer vesicles formed by activated or apoptotic cells. They carry abundant signaling proteins and participate in multiple physiopathologic processes. The most MVs in circulation are platelet-derived microvesicles (PMVs), which are the main culprit in the development of thrombosis. The plasma levels of PMVs and Annexin V-positive PMVs enhanced significantly in NVAF patients at high risk for stroke compared with those at low to moderate risk. Thus, MVs may be the vectors bearing multiple pathogenic mechanisms of platelet activation. To find the source of platelet activation in NVAF, we can study the potential mechanism of the prothrombotic state and explore new, effective targets for preventing thromboembolism.CD36, a multifunctional membrane receptor expressed on platelets, monocytes and several other cells, takes part in multiple physiopathological processes by engaging with multiple ligands. Platelet CD36could recognize and capture PS on the surface of PMVs, thus generating a PMV-CD36complex. The PMV-CD36complex was speculated be the key part of platelet activation. CD36can be a signaling molecule, but the detailed signal pathway of the PMV-CD36complex is unknown. Chen et al. showed that platelet CD36mediates mitogen-activated protein kinase (MAPK) activation induced by oxLDL. The modified lipids on oxLDL mediate its combination with CD36, and the surface of microvesicles contains multiple modified lipids (such as PS). Therefore, we speculated that the PMV-CD36complex could activate MAPKs, thereby activating platelets.However, whether the PMVs mediate platelet activation induced by accompanying disorders, such as oxidative stress, inflammation and accumulated AGEs, in the context of NVAF is unclear. Thus, we speculated that the PMVs, generated in response to pathogenesis or accompanying disorders of NVAF, could be endogenous CD36ligands that transmit an activating signal to platelets to induce thrombosis.Objectives1. To determine the effects of oxidative stress, inflammation and accumulated AGEs on the production of PMVs.2. To determine the effects of PMVs bearing signals of oxidative stress, inflammation and AGEs on the platelet activation and the involved signaling transduction.3. To determine whether the signal pathway mediated by PMV-CD36complex could be a new effective target for preventing the prothrombotic state of NVAF.Methods1. Platelet isolation.All the healthy volunteers had not taken any medication for2weeks. Fasting venous blood was collected in0.109mol/L sodium citrate (ratio1:9) under minimal tourniquet pressure using a sterile22-gauge needle. Platelet-rich plasma (PRP) was prepared by centrifugation at120g for10min at room temperature. Platelets were isolated from PRP after centrifugation at800g for10min in the presence of in the presence of100nmol/L prostaglandin E1. Then platelets were washed and resuspended in modified Tyrode’s buffer (137mmol/L NaCl,2.7mmol/L KC1,12mmol/LNaHCO3,0.4mmol/L NaH2PO4,5mmol/LHEPES,0.1%glucose and0.35%bovine serum albumin, pH7.2) in the presence of100nmol/L PG-E1. Platelet concentration was adjusted to1×106/mL by use of a Z2particle counter, then used at once in all experiments.2. The effects of oxidative stress, inflammation and AGEs on production of PMVs.Resting platelets (1×106/mL) were incubated with oxidized LDL (oxLDL,50μg/mL), IL-6(1μg/mL) or AGEs (200μg/mL) for proper times at37℃respectively. The blank control (treated with buffer), isotype control and positive control (treated with10μmol/L adenosine diphosphate, ADP) were set for each group at the same time. Then the production of PMVs was detected by flow cytometry and a homemade ELISA.3. Identification of PMVs by flow cytometry.Immunofluorescent staining for PMVs:After gently blending,2.5μL platelet suspension (1×106/mL) was incubated with PEcy5-conjugated anti-CD41a antibody (5μL) in the dark for15min. Then the platelets were resuspended by1mL PBS and detected at once. Only cells and particles labeled with CD41a were gated. The PMV region was defined by FSC-SSC dot plot. The lower limit of the platelet gate was set at the left-hand border for resting platelets to distinguish between platelets and microvesicles. For each sample,10,000positive events in the platelet gate were acquired by use of FACS Calibur cytometer.4. ELISA quantification of PMVs and Annexin V-positive PMVs.After incubation with oxLDL, IL-6, AGEs or their control reagents, the platelet suspension was centrifuged at3,000g for10min. Then the supernatant was centrifuged again at13,000g for2min to avoid platelet contamination. Quantification of total PMVs and Annexin V-positive PMVs in the last supernatant was involved use of a homemade ELISA.5. Collection of PMVs derived from platelets stimulated by oxLDL, IL-6or AGEs.Platelets (1×106/mL) treated with oxLDL, IL-6or AGEs were sedimented at3000g for10min. An amount of2.0mmol/L phenylmethylsulfonyl fluoride was added to the PMV-enriched supernatants, which were then centrifuged at15,000g for1h at4℃. Then PMV pellets were washed twice (to avoid contamination of oxLDL, IL-6or AGEs) and resuspended in Modified Tyrode buffer and stored at-80℃. Freezing had no ad-verse effect on microvesicles. The protein content was measured by the Bradford Protein Assay Kit.6. Double color flow cytometry of CD36expression.For CD36quantification and CD36deficiency screening, the platelet suspension was incubated with5μL PEcy5-conjugated anti-CD41a antibody and5μL PE-conjugated anti-CD36antibody or isotype-matched control IgM in the dark for15 min. Then the platelets were resuspended by1mL PBS and detected by flow cytometry at once.7. Double color flow cytometry of platelet GPⅡb/Ⅲa.The platelet suspension (2.5μL) treated with PMVs (30μg/mL) was incubated with5μL PEcy5-conjugated anti-CD41a antibody and5μL FITC-conjugated PAC-1antibody (for activated platelet GPⅡb/Ⅲa) at room temperature in the dark for15min. Then the platelets were resuspended by1mL PBS and detected by flow cytometry at once.8. Platelet aggregation studies.PRP was obtained by centrifuging fasting venous blood at120g for10min at22℃, and platelet-poor plasma (PPP) was obtained by centrifuging PRP at3000g for10min. The platelet concentration of PRP was adjusted to2.5×108/mL by the addition of PPP. Aggregation was assessed by turbidimetry with a dual channel aggregometer (Chrono-log Corp., Havertown, PA, USA), with2μmol/L ADP used as an agonist. An amount of100%aggregation was defined as the light transmission of PPP, and0%was defined as the light transmission of PRP before the addition of agonists. Then the PRP treated with PMVs (30μg/mL) was stimulated with ADP (2μmol/L), and the change in light transmission was recorded.9. Immunoassay for soluble P-selectin.After the treatment described above, the platelet-PMV mixtures were centrifuged at3,000g for10min. Then the supernatant was centrifuged again at13,000g for2min to avoid platelet contamination. The last supernatant was used for detection of soluble P-selectin with use of commercially available immunoassay kits. The lower limit of sensitivity of the assay was0.5ng/mL.10. Western blot analysis.Platelets treated with PMVs or controls were lysed in2mmol/L Tris-HCl (pH7.5),150mmol/L NaCl,1mmol/L EGTA,1mmol/L EDTA,1%Triton X-100,2.5mmol/L sodium pyrophos-phate,1mmol/L Na3VO4,1mmol/L phenylmethylsulfonyl fluoride and1μg/mL leupeptin, and protein concentrations were measured with use of a Bradford protein assay kit (Beyotime). Lysate protein (40μg) was separated on10% polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. After a blocking for1h at room temperature in5%nonfat milk, the membranes were incubated with mouse anti-phospho-JNK1/2or rabbit anti-phospho-MKK4(1:1000dilution) overnight at4℃, then with HRP-conjugated secondary anti-mouse IgG or anti-rabbit IgG (1:4000dilution). The blots were developed with use of ECL detection reagent, then stripped and re-blotted with antibodies to native proteins for normalization.11. Tanshinone IIA (TS IIA) treatmentTS IIA was obtained commercially (Xi’an Honson Biotechnology, China). Platelets were incubated with various concentrations of TS IIA (5μg/mL、10μg/mL20μg/mL、50μg/mL、100μg/mL) for15min before being stimulated with various PMVs(30μg/mL). Then the expression of platelet CD36and GPⅡ b/Ⅲa were detected by flow cytometry and the phosphorylation of MKK4/JNK was detected by western blot analysis.Results1. Platelet function studies with PMVs derived from oxLDL-treated platelets (oxLDL-PMVs)(1) OxLDL treatment induced the release of PMVs and Annexin V-positive PMVsFlow cytometry detected the formation of microvesicles by labeling with PEcy5-conjugated anti-CD41a antibody (M gates). The release of microvesicles increased after stimulation with oxLDL (50μg/mL) or ADP (10μmol/L), as quantified by CD41a-positive microparticles (P<0.05). For further confirmation, the supernatant of platelets was tested by ELISA. The amount of total PMVs and Annexin V-positive PMVs increased significantly after treatment with oxLDL or ADP (P<0.05), but not native LDL.(2) OxLDLr-PMVs induced platelet activationNext, we tested whether the PMVs collected from oxLDL-treated platelets could enhance platelet activation. We incubated resting platelets (1×106/mL) with oxLDL-PMVs (30μg/mL) for30min at22℃. Platelet activation is characterized by a conformation change in glycoprotein Ⅱ b/Ⅲa (GP Ⅱ b/Ⅲa). As expected, the MFI and percentage of PAC-1(recognizing the activated platelet GP Ⅱ b/Ⅲa) were enhanced significantly compared with the control (P<0.05). OxLDL-PMVs had almost the same effect as ADP. Furthermore, oxLDL-PMVs increased platelet aggregation in response to ADP.(3) OxLDL-PMVs were ineffective in CD36-deficient plateletsWe screened2CD36-deficient male donors in our laboratory. The CD36-deficient platelets were unable to bind PE-conjugated anti-CD36antibody. We incubated the CD36-deficient platelets (1×106/mL) with oxLDL-PMVs (30μg/mL) for30min at22℃. The PMV-enhanced platelet GP Ⅱ b/Ⅲa expression and platelet aggregation were absent.(4) OxLDL-PMVs activated platelets in a CD36-and PS-dependent mannerTo define the influence of CD36and PS on PMV-platelet interaction, we determined the expression of platelet GP Ⅱ b/Ⅲa and the secretion of P-selection. oxLDL-PMVs substantially increased the expression of GP Ⅱ b/Ⅲa and secretion of P-selectin (P<0.05). In all cases, the blocking of CD36(by a CD36neutralizing antibody) or PS (by Annexin V) could diminish the enhancement by oxLDL-PMVs (P<0.05), but IgG, an isotype-matched control of the CD36neutralizing antibody, had no effect. As well, the CD36neutralizing antibody decreased the binding of PE-conjugated anti-CD36antibody to platelets, with the expression of CD36for other groups not changed significantly.(5) Platelet activation induced by oxLDL-PMVs was mediated by JNK signalsTo elucidate the potential effect of JNK signaling in PMV-induced platelet activation, platelets were treated with the JNK inhibitor SP600125before incubation with PMVs. Pharmacological inhibition of JNK reduced platelet activation by PMVs, including expression of GP Ⅱ b/Ⅲa and secretion of P-selectin (P<0.05). Thus, JNK signaling may contribute to PMV-induced platelet activation.(6) Phosphorylation of platelet JNK2and its upstream activator MKK4 induced by oxLDL-PMVs.To confirm the role of the JNK pathway in PMV-induced activation of platelets, we compared the phosphorylation of JNK2in CD36-deficient and-positive platelets by immunoblotting. CD36-positive but not CD36-deficient platelets exposed to oxLDL-PMVs for30min showed a significant increase in phosphorylation of JNK2and its upstream activator MKK4(P<0.05). The JNK2phosphorylation was time and dose dependent, peaking at15min to approximately1.3-fold that of baseline with30μg/mL oxLDL-PMVs and with increased concentrations of oxLDL-PMVs inducing stronger phosphorylation of JNK2.2. Platelet function studies with PMVs derived from IL-6-treated platelets (IL-6-PMVs)(1) IL-6treatment promoted the formation of PMVs and Annexin Ⅴ-positive PMVsThe formation of PMVs was identified with labeling with PEcy5-conjugated anti-CD41a antibody (M gates). We observed an increase in total PMVs and Annexin V-positive PMVs released from IL-6treated compared to untreated platelets (P<0.05)(2) IL-6-PMVs induced platelet activationThe MFI and percentage of PAC-1of the CD36-positive platelets were increased significantly stimulated by IL-6-PMVs, with similar effect as ADP (10μmol/L)(P <0.05). Furthermore, IL-6-PMVs increased platelet aggregation in response to ADP.(3) IL-6-PMVs induced platelet activation in a CD36-and PS-dependent wayThe PMV-enhanced platelet GPⅡ b/Ⅲa expression and platelet aggregation were absent when the CD36-deficient platelets (1×106/mL) were incubated with IL-6-PMVs (30μg/mL).IL-6-PMVs substantially increased the expression of GP Ⅱb/Ⅲa and secretion of P-selectin (P<0.05). In all cases, the blocking of CD36(by a CD36neutralizing antibody) or PS (by Annexin V) could diminish the enhancement by IL-6-PMVs (P <0.05).(4) Activation of platelets by IL-6-PMVs depends on MKK4/JNK pathwayInhibition of JNK blocked the effect of IL-6-PMVs on platelet activation, including the expression of integrin αHbβ3and secretion of P-selectin (P<0.05).CD36-positive but not-deficient platelets exposed to IL-6-PMVs for30min showed a significant increase in phosphorylation of JNK2and its upstream activator MKK4(P<0.05). The JNK2phosphorylation was time and dose dependent.(5) Tanshinone ⅡA (TS Ⅱ A) treatment inhibits platelet activation induced by IL-6-PMVs To test whether the signal pathway mediated by IL-6-PMV/CD36complex could be a potential target for preventing the prothrombotic state of NVAF, we treated resting platelets with doses of TS ⅡA (5μg/mL、10μg/mL.20μg/mL、50μg/mL、100μg/mL) for15min before incubation with IL-6-PMVs. We found that TSⅡA dose-dependently inhibited the activation of platelets in vitro. At the same time, we detected the platelet CD36expression and found that TS ⅡA dose-dependently decreased the expression of platelet CD36. Furthermore, TS ⅡA could downregulate the phosphorylation of MKK4/JNK2activated by IL-6-PMVs.3. Platelet function studies with PMVs derived from AGE-treated platelets (AGE-PMVs)(1) AGEs treatment promoted the formation of PMVs and Annexin V-positive PMVsWe observed an increase in total PMVs and Annexin V-positive PMVs released from AGE (200μg/mL)-treated compared to bovine serum albumin (BSA,200μg/mL)-treated platelets or untreated platelets (P<0.05).(2) Effect of AGE-PMVs on platelet activationThe MFI and percentage of platelet PAC-1were enhanced significantly after incubation with AGE-PMVs (P<0.05), which had similar effect as ADP. Furthermore, Surface exposure of CD40L (an index of platelet activation and assumed to originate from the platelet cytosol) increased after incubation with AGE-PMVs (P <0.05).(3) Activation of platelets by AGE-PMVs depends on CD36and PSExpression of GPⅡb/... |
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