Effects of alimentary lipemia and inflammation on platelet CD40-ligand

INTRODUCTION: In patients with chronic hypercholesterolemia, the CD40-CD40L dyad is upregulated, contributing to the initiation and progression of atherosclerosis. Our aim was to describe the role of postprandial lipemia and inflammatory stimulation on platelet and monocyte activation and CD40-ligand (CD40L) levels. METHODS AND RESULTS: Before and 2 h after consumption of a defined fatty meal, whole blood samples of 31 healthy subjects were incubated with endotoxin (LPS). CD40-ligand and CD62P expression on platelets, tissue-factor expression on monocytes and platelet-monocyte aggregates were measured with flow cytometry. Soluble CD40-ligand plasma levels were measured with an ELISA. After the meal, serum triglyceride levels increased from 137.6+/-60.5 mg/dl to 201.5+/-75.0 mg/dl. Expression of CD40L and CD62P on platelets and plasma levels of soluble CD40L were significantly decreased. No significant changes after the meal were observed concerning tissue factor expression on monocytes and platelet-monocyte aggregates. Addition of LPS showed no significant effect concerning CD40L or CD62P expression on platelets, whereas the amount of platelet-monocyte aggregates significantly increased under LPS stimulation after the fatty meal. CONCLUSIONS: Acute alimenatry lipemia leads to a decreased expression of CD40L on platelets and a reduced plasma level of sCD40L, suggesting an increased turnover in the CD40L system. CONDENSED ABSTRACT: Before and after a fatty meal, blood samples of 31 healthy subjects were incubated with LPS. After the meal, expression of CD40L and CD62P on platelets and plasma levels of soluble CD40L were significantly decreased. Addition of LPS showed no effect concerning CD40L or CD62P expression, whereas the amount of platelet-monocyte aggregates significantly increased under LPS stimulation after the fatty meal.     

Differences of soluble CD40L in sera and plasma: implications on CD40L assay as a marker of thrombotic risk

INTRODUCTION: Soluble CD40L (sCD40L) ELISA has emerged as a promising predictor of poor outcomes in acute coronary syndrome. Yet many blood processing techniques have been used with little consideration of their effect on the results. METHODS: We measured sCD40L by ELISA in 10 patients with thrombocytopenia and 12 with normal or high platelet counts and 8 healthy controls using three sampling techniques: serum clotted on ice (serum-I) or at room temperature (serum-RT) and platelet poor plasma (PPP). RESULTS: Serum-RT samples, compared to serum-I, gave significantly higher CD40L values (p=0.003), demonstrating that ex vivo sCD40L release by activated platelets is inhibited by cold temperature. Although serum-I and PPP were comparable in patients with normal platelet counts, serum-I gave significantly higher values than PPP in the thrombocytosis group (p=0.01), suggesting that cold inhibition is insufficient in the latter group. To estimate the fraction of sCD40L that was microparticle-bound CD40L (mp-CD40L), 16 samples underwent 0.1-microm filtration. 50.6% of sCD40L was mp-CD40L in serum-RT, whereas 21.3% and 29.9% were observed in serum-I and PPP, respectively. Lastly, plasma sCD40L was assayed in 46 patients with and 35 without thrombosis. Plasma sCD40L did not correlate with platelet count in non-thrombotic, non-inflammatory patients but did (p<0.01) in those with thrombosis. CONCLUSIONS: Sample processing and temperature profoundly affect sCD40L assay. Serum-I and PPP minimize the release of sCD40L ex vivo and better represent sCD40L in vivo. However, PPP may be preferable particularly in patients with thrombocytosis. The existence of mp-CD40L highlights the importance of centrifuge conditions.     

Analysis of serum soluble CD40 ligand in patients with influenza virus-associated encephalopathy

CD40 ligand (CD40L) is mainly expressed on activated platelets and CD4+T cells, and it can be cleaved from the cell surface, releasing a soluble CD40L (sCD40L). Most sCD40L is derived from activated platelets. A previous paper revealed that the platelet number of patients with influenza virus-associated encephalopathy (IE) was correlated with the outcome. We determined the utility of sCD40L as a predictor for the prognosis of IE. We measured the serum concentration of sCD40L and the platelet number on the day of hospitalization in 34 patients with IE, 16 with influenza virus-associated febrile seizures (IFS), 19 with influenza virus infection without complications (Flu), and 7 with Epstein-Barr virus (EBV) infection. The serum sCD40L concentrations in IE and IFS were significantly lower than those in controls, Flu, and EBV infections. Serum sCD40L concentrations in the IE group were 0.70+/-0.43 ng/ml for deceased patients, 1.73+/-1.36 ng/ml for those with sequelae, and 3.85+/-2.91 ng/ml for those without sequelae. There was no significant difference in platelet number between IE patients with and without sequelae, while the platelet number of deceased patients with IE was significantly lower than in controls, Flu, and IFS. Serum sCD40L concentration on the day of hospitalization was more correlated with the outcome of IE than platelet number. Our findings suggest that the serum sCD40L concentration during acute IE is important for predicting the prognosis at an early stage.     

Regulation of CD40L (CD154) and CD62P (p-selectin) Surface Expression upon GPIIb-IIIa Blockade of Platelets from Stable Coronary Artery Disease Patients

Introduction

The aim of this study was to further characterize the effect of the antiplatelet agents, aspirin and eptifibatide, on the surface expression of CD40L and CD62P on platelets from patients with stable coronary artery disease.

Materials and methods

Platelet function was evaluated using standard light transmission aggregometry. Measurements of CD62P and CD40L were carried out by flow cytometry and ELISA assays.

Results

All patients had the expected level of platelet aggregation inhibition in response to 20 μM ADP in the presence of increasing eptifibatide concentrations. Platelet activation by adenosine diphosphate (ADP) or thrombin agonist peptide (TRAP) increased CD62P and CD40L surface density in the presence of aspirin by 1.9 - 2.8 -fold. Aspirin treatment did not prevent either CD62P or CD40L expression. Eptifibatide pretreatment at pharmacologically relevant concentrations blocked agonist-induced increases in CD62P platelet surface density. A marked percentage of platelets still expressed low levels of surface CD62P suggesting slight platelet activation even with potent platelet inhibition. Eptifibatide also blocked agonist-induced increases in CD40L surface expression and decreased the percent of platelets positive for surface CD40L. Decreased expression of CD40L was due to an inhibition of CD40L translocation and not caused by enhanced shedding from the surface, as soluble CD40L (sCD40L). Eptifibatide concentrations that effectively blocked platelet aggregation correlated with total inhibition of increased CD62P and CD40L surface density.

Conclusion

Blockade of the GPIIb-IIIa receptor on platelets from coronary artery disease patients may have significant bearing on reducing proinflammatory and procoagulant events mediated by CD62P and sCD40L.     

Release of soluble CD40 ligand after platelet activation: studies on the solubilization phase

sCD40L is released from platelets as a soluble, proteolyzed form of CD40 ligand (CD40L; CD154) which is exposed on the surface after platelet activation. Ethylenediaminetetraacetate (EDTA), the CD40-blocking antibody G28-5, and GPIIb-IIIa antagonists are known to inhibit the solubilization when added prior to activation. It is assumed that the surface expression of CD40L is a result of a separate fast process and that the solubilization is secondary to this. The release of sCD40L in this solubilization phase has been studied; that is, inhibitory substances were added to platelet-rich plasma (PRP) 10 min after addition of the activation agonist (100 microM SFLLRN), at which time the secretion phase was over as tested with beta-thromboglobulin (beta-TG). G28-5 (10 microg/ml) and EDTA (5 mM) inhibited the solubilization phase which did not require the presence of an activation agonist. Prostaglandin E1 (PGE1; 20 microM) and cytochalasin D (C8273; 60 and 100 microM), which exert their effects intracellularly, inhibited the solubilization even in the presence of abciximab (ReoPro; 40 microg/ml). The intracellular effect was not related to CD40L-containing microparticles as demonstrated by ultracentrifugation. Intracellular alkalinization by preincubation of PRP with 20 mM NH4Cl for 60 min resulted in a small but reproducible reduction in the amount of extracellular sCD40L. SFLLRN induced solubilization of CD40L also from the platelets of a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa, albeit at a lower rate than from normal platelets, and fibrinogen enhanced the solubilization from washed normal platelets. The data show that the solubilization of CD40L not only depends on reactions on the platelet surface but also that intracellular structures are engaged even during the solubilization phase.     

A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation

Introduction

Prasugrel, a P2Y₁₂ adenosine diphosphate (ADP) receptor antagonist effectively inhibits ADP-mediated platelet activation and aggregation, and may be useful in reducing vaso-occlusive crises in sickle cell disease (SCD). In this study, we assess the effect of prasugrel on biomarkers of platelet activation and coagulation in patients with SCD.

Materials and Methods

Twelve adult patients with SCD and 13 healthy subjects were examined before and after 12 ± 2 days of 5.0 or 7.5 mg/day oral prasugrel. Assessed cellular biomarkers included monocyte- and neutrophil-platelet aggregates, activated glycoprotein IIb-IIIa (GPIIbIIIa), P-selectin, CD40 ligand (CD40L), tissue factor (TF) expression on circulating platelets and on monocyte-platelet aggregates, and platelet-erythrocyte aggregates. Soluble biomarkers included CD40L, prothrombin fragment 1.2 (F1.2), thromboxane B₂ (TXB₂), P-selectin, and TF.

Results

Patients with SCD had increased platelet baseline activation compared to healthy subjects, as measured by percentages of monocyte-platelet aggregates, neutrophil-platelet aggregates, and platelets expressing CD40L. Likewise, baseline levels of soluble F1.2 and TXB₂ were elevated in patients with SCD compared to healthy subjects. After 12 days of prasugrel, patients with SCD had a significant reduction in platelet-monocyte aggregates that was not observed in healthy subjects. Following prasugrel administration, those with SCD maintained higher levels of monocyte-platelet aggregates and soluble F1.2, but had lower levels of platelet-erythrocyte aggregates and soluble TF compared to healthy subjects.

Conclusions

These results provide evidence for chronic platelet activation in the SCD steady state, activation that was in part attenuated by prasugrel, thereby suggesting that ADP may mediate platelet activation in SCD.     

重症肌无力患者血清中可溶性CD40配体表达及临床意义

目的:研究重症肌无力(MG)患者血清中可溶性CD40配体(sCD40L)及抗乙酰胆碱受体抗体(AchRab)的水平,探讨sCD40L在MG发病中的作用。方法:研究对象为25例MG患者,分为急性期组13例,非急性期组12例,设正常对照组15例。取静脉血,分离血清,采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)测定血清中可溶性CD40配体及抗乙酰胆碱受体抗体(AchRab)的含量。结果:MG急性期组中sCD40L及AchRab的含量比非急性期组及正常对照组显著升高(P<0.01),非急性期组sCD40L及AchRab的含量比正常对照组显著升高(P<0.05)。各组sCD40L与AchRab均呈正相关。结论:sCD40L含量增高可能与MG发生和加重有关。     

Mechanism of collagen-induced release of 5-HT, PDGF-AB and sCD40L from human platelets: Role of HSP27 phosphorylation via p44/p42 MAPK

Collagen plays a crucial role in hemostasis and thrombosis by activating platelets and reportedly induces the phosphorylation of heat shock protein (HSP) 27 in human platelets. However, the exact role of HSP27 phosphorylation in human platelets has not yet been clarified. In the present study, we investigated the mechanism of collagen-induced HSP27 phosphorylation and the role in human platelets. The collagen-effect on the phospholylation of HSP27 was dose-dependent in the range between 0.03 and 1.0 μg/ml. The phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) was also stimulated by collagen. PD98059, a specific inhibitor of MAPK kinase (MEK1/2), reduced collagen-induced HSP27 phosphorylation as well as p44/p42 MAPK phosphorylation. PD98059 significantly suppressed collagen-induced releases of serotonin (5-HT), platelet-derived growth factor (PDGF)-AB and soluble CD40 ligand (sCD40L) while it had little effect on the platelet aggregation. These results strongly suggest that the collagen-induced phosphorylation of HSP27 via p44/p42 MAPK is sufficient for releases of 5-HT, PDGF-AB and sCD40L from human platelets.     

Circulating CD40 ligand is elevated only in patients with more advanced symptomatic peripheral arterial diseases

INTRODUCTION: CD40 and its ligand participate in atherosclerosis formation, progression and destabilization. Increased soluble CD40 ligand (sCD40L) was observed in hypercholesterolemia, unstable angina and conditions with platelet activation. To date, there is no report on the association of sCD40L with angiographic peripheral artery disease (PAD) disease severity. MATERIALS AND METHODS: From March 1999 to April 2004, consecutive patients having angiographically documented PAD and given consents for pre-procedural serum sample use were recruited into this study. The key PAD lesions should be > or = 70% diameter stenotic at the lower limbs and patients were dichotomized into two groups depending on total PAD lengths. Peer angiographic control subjects were those free of coronary disease, PAD and major medical diseases. The serum samples were thawed and analyzed for sCD40L, monocyte chemotactic protein 1 (MCP-1) and hs-CRP in a single batch. RESULTS: A total of 63 well-defined lower-limb PAD patients and 30 control subjects were studied. Patients with PAD lengths >5 cm (N=38) presented higher sCD40L than those with lesions < or = 5 cm (N=25) (5430+/-459 vs. 3889+/-507 pg/ml, p=0.037) and control subjects (5430+/-459 vs. 3973+/-551 pg/ml, p=0.037). However, there was no significant difference in circulating MCP-1 (375+/-49 vs. 310+/-49 pg/ml and 297+/-24 pg/ml, respectively, p=0.371) or hs-CRP (0.64+/-0.16 vs. 0.51+/-0.15 mg/ml and 0.46+/-0.15 mg/dl, respectively, p=0.682) across three groups. PAD patients with associated coronary lesions did not differ in circulating CD40L, MCP-1 or hs-CRP from without and control subjects. CONCLUSIONS: Soluble CD40L was significantly elevated in patients with more advanced symptomatic PAD and might be an indicator for disease extent stratification. The distribution of sCD40L in PAD was not affected by coronary involvement or not.     

Shear-induced von Willebrand factor-mediated platelet surface translocation of the CD40 ligand

Background: Platelets, which can adhere to damaged vascular surfaces and release bioactive substances upon activation, may play important roles in regulating local inflammatory responses. We focused on the surface translocation of CD40 ligand (CD40L) molecules when the platelets are exposed to a high shear stress. Method: Blood specimens were obtained from eight apparently healthy adult donors. The number of CD40L molecules appearing on the surface of platelets after exposure of platelet-rich plasma to a shear rate of 10,800 s⁻¹ was determined by quantitative flow cytometry. Results: The number of anti-CD40L IgG molecules bound per platelet increased from 15±80/platelet before to 355±122/platelet after exposure of the platelets to a shear rate of 10,800 s⁻¹ (p<0.01), but not after their exposure to the relatively low shear rate of 1200 s⁻¹. This shear-induced platelet surface translocation of CD40L, mediated by the von Willebrand factor (VWF)–GP Ib interaction, was enhanced in the presence of a low concentration of epinephrine (100 nM), which by itself, however, could not cause platelet activation. Our results demonstrate that fluid force induces the appearance of CD40L on the surface of platelets, and also that this phenomenon is enhanced in the presence of a low concentration of epinephrine, corresponding to that released by sympathetic stimulation.     

Is soluble CD40 ligand a mediator of angiogenesis in patients with coronary artery disease?

BACKGROUND: In cardiovascular disease, soluble CD40 ligand (sCD40L) has been associated with an adverse prognosis. Angiogenesis has been implicated in the progression of coronary artery disease (CAD) and sCD40L has pro-angiogenic effects in vitro. Angiogenesis itself is regulated by many mediators, such as vascular endothelial growth factor (VEGF) and the angiopoietins (Ang). Ang-1 promotes vascular maturation whilst Ang-2 destabilises the blood vessel and permits vascular growth with VEGF. Hence, selective elevation of VEGF and Ang-2 suggest a state of vascular plasticity and increased angiogenesis. We hypothesised raised plasma levels of VEGF and Ang-2, but not Ang-1, and correlations with raised sCD40L levels and CAD severity/collateralisation in patients with CAD. METHODS: We recruited 153 patients attending diagnostic angiography for CAD and 47 healthy controls. Patients with previous revascularisation or unequivocally normal angiograms were excluded. The coronary atheroma score (CAS) and coronary stenosis score (CSS), and the presence of collaterals, were assessed by 2 blinded observers. Plasma sCD40L, VEGF, Ang-1 and -2 levels were measured by ELISA. RESULTS: Plasma levels of sCD40L, VEGF and Ang-2, but not Ang-1, were higher in CAD patients compared to controls. Both plasma VEGF (r=0.526, p<0.001) and Ang-2 (r=0.429, p<0.001) were correlated with sCD40L, but not with CAS, CSS or collateralisation. On stepwise multivariate regression analysis, plasma sCD40L was an independent predictor of plasma VEGF (p=0.002), and Ang-2 (p<0.001) levels. CONCLUSION: These data suggest abnormal indices of angiogenesis in CAD, which may be associated with increased CD40-CD40L interactions in patients with CAD. Plasma sCD40L, VEGF and Ang-2 levels were not correlated to angiographic CAD/collateralisation.     

Platelet CD40L at the interface of adaptive immunity

Initiated by the finding that platelets express functional CD40 ligand (CD40L, CD154), many new roles for platelets have been discovered in unanticipated areas, including the immune response. When current literature is considered as a whole, the picture that is emerging begins to show that platelets are able to significantly affect, for better or worse, the overall health and condition of the mammalian host. Animal models have made significant contributions to our expanding knowledge of platelet function, much of which is anticipated to be clinically relevant. While still mostly circumstantial, the evidence supports a critical role for CD40L in many normal and disease processes.     

Ginkgolide B inhibits platelet release by blocking Syk and p38 MAPK phosphorylation in thrombin-stimulated platelets

Introduction

Atherosclerosis is a chronic vascular inflammatory disease. Platelets play a critic role in the initiation of vascular inflammation in atherosclerosis. In the present study, we investigated the effects of ginkgolide B on the inhibition of platelet release and the potential mechanisms.

Methods

Experiments were performed in freshly human platelets. Platelet aggregation and ATP release were measured with a Lumi-aggregometer. Thrombin (0.5 U/ml) was used to induce platelet activation. Protein expression and phosphorylation was examined by Western blotting.

Results

The results showed that ginkgolide B significantly suppressed ATP release by 50.8% in thrombin-activated platelets. Ginkgolide B completely abolished the expression of platelet factor 4 (PF4) and CD40 Ligand (CD40L). Moreover, ginkgolide B fully attenuated the phosphorylation of Syk and p38MAPK. Similarly, R788 (a syk inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited the expression PF4 and CD40L, respectively. Furthermore, the combination of low concentrations of ginkgolide B and R788 or SB203580 has synergistic inhibition on the expression of PF4 and CD40L. Ginkgolide B partially reduced calcium efflux by 52.7% in thrombin-stimulated platelets.

Conclusion

Ginkgolide B potently inhibited the expression of PF4 and CD40L in thrombin-activated platelets. Ginkgolide B partially decreased ATP release and Ca^2 + efflux. The mechanism might be associated with the inhibition of Syk and p38 MAPK phosphorylation. These results demonstrated that ginkgolide B might be a promising drug on inhibiting platelet function and reducing inflammation in atherosclerosis.     

急性进展性脑梗死患者外周血清可溶性CD40配体、外周血单个核细胞胞核核因子κB表达的变化及灯盏生脉胶囊的干预作用

目的 探讨急性进展性脑梗死（acute progressive cerebral infarction,APCI）患者各期病程中外周血清可溶性CD40配体（serum soluble CD40 ligand,sCD40L）水平及外周血单个核细胞（peripheral blood mononuclear cells,PBMC）胞核核因子κB（nuclear factor-κB,NF-κB）p65表达率的变化及灯盏生脉胶囊的干预作用。 方法 将起病24&#8197;h以内的100例APCI患者随机分为常规组和试验组,每组各50例患者,选择同期住院的急性脑梗死患者50例作对照组,所有病例均采用常规治疗,试验组除常规治疗外还给予灯盏生脉胶囊治疗。治疗前后检测常规组和试验组患者神经功能缺损评分（China Stroke Scale,CSS）,常规组、试验组和对照组梗死灶体积、外周血清sCD40L水平及PBMC胞核NF-κB p65表达率的变化。 结果 治疗后试验组患者的CSS和梗死灶体积的进展程度明显低于常规组（P＜0.05）,常规组和试验组患者各期病程中外周血清sCD40L水平及PBMC胞核NF-κB p65表达率均明显高于对照组（P＜0.05）,试验组患者各期病程中外周血清sCD40L水平及PBMC胞核NF-κB p65表达率明显低于常规组（P＜0.05）,治疗期间不良反应轻微。 结论 CD40-CD40L信号通路及PBMC胞核NF-κB表达的过度上调而介导的炎症、凋亡机制可能是APCI的分子生物学因素,灯盏生脉胶囊能通过下调此分子生物学因素而改善其预后。     

阿尔茨海默病与轻度认知障碍患者血浆sCD40及sCD40L浓度研究

目的 探索阿尔茨海默病(Alzheimer’s disease,AD)及遗忘型轻度认知障碍(amenstic mild cognitive impairment,a MCI)患者血浆可溶性CD40(soluble CD40,s CD40)和可溶性CD40配体(soluble CD40 ligand,s CD40L)浓度变化特点及其临床意义。方法 采用酶联免疫吸附法检测20例AD患者、35例a MCI患者和32名正常对照者血浆s CD40和s CD40L浓度水平,简易精神状态检查表(mini-mental state examination,MMSE)评估患者认知功能。结果 AD组、a MCI组和对照组血浆s CD40浓度中位数(上下四分位数)分别为[123.3(97.4,149.5)pg/m L]、[102.9(63.6,124.0)pg/m L]和[70.66(51.0,90.8)pg/m L],3组间s CD40浓度差异均有统计学意义(P0.05)。AD组、a MCI组和对照组血浆s CD40L浓度中位数(上下四分位数)分别为[537.0(316.0,1134.0)pg/m L]、[316.0(190.0,546.0)pg/m L]和[167.0(107.5,478.0)pg/m L],3组间s CD40L浓度差异均有统计学意义(P0.05)。a MCI组血浆s CD40L浓度与MMSE得分呈负相关(r=-0.74,P0.01)。结论 s CD40和s CD40L浓度水平在AD和a MCI患者血浆中异常增高,因此s CD40和s CD40L可作为AD的早期检测指标,并提示CD40-CD40L信号可能参与AD的发生发展。     

Platelet soluble CD40L and matrix metalloproteinase 9 activity are proinflammatory mediators in Behçet disease patients

Platelets are the major source of plasma-soluble CD40L (sCD40L), an important inflammatory mediator. This study explored the impact of platelet-derived sCD40L on Beh?et disease (BD), an autoinflammatory vasculitis. We also searched for influences by platelet matrix metalloproteinases (MMP) -2 and MMP-9, implicated in several inflammatory diseases, on CD40L shedding from platelet membrane. Platelet activation were studied by flow cytometry and aggregometry, surface expression of CD40L and platelet-leukocyte aggregates by flow cytometry, sCD40L by ELISA, cellular CD40L and CD40 levels by Western blot and MMPs activity by gelatin zymography. The effect of sCD40L on MMP9 expression was studied in cultured MEG-01 cells. Plasma and platelet-released sCD40L levels were higher in BD patients. No differences in platelet activation and in platelet-leukocyte aggregates formation were observed between BD patients and controls. Plasma and platelet MMP-9 levels were increased in BD patients, whereas there was no difference in platelet MMP-2 activity. Since a correlation between plasma sCD40L and platelet MMP-9 activity was observed, we studied the influence of sCD40L on MMP-9 levels in the megakaryoblastic cell line MEG-01. Treatment of MEG-01 cells with recombinant sCD40L increased MMP-9 but did not change MMP-2 levels. In conclusion, sCD40L release from platelets was mediated by MMP-9, and MMP-9 expression was in turn upregulated by sCD40L in the MEG-01 cell line. We conclude that platelets and megakaryocytes might participate in a positive feedback loop occurring between sCD40L and MMP-9 which would contribute to the proinflammatory state observed in BD.     

The Feverfew plant-derived compound, parthenolide enhances platelet production and attenuates platelet activation through NF-κB inhibition

Introduction

Few treatments are available that can safely and effectively stimulate new platelet production for thrombocytopenic patients. Additionally, recipients of transfused platelets may experience an inflammatory response due to stored platelets becoming unnecessarily activated, thus creating the need for suitable agents that will dampen undesirable platelet activation. We investigated the effect of the feverfew plant-derived compound, parthenolide on platelet production and platelet activation because of its well-studied ability to induce apoptosis or differentiation in some types of cancer.

Methods

Parthenolide was used to treat human megakaryoblastic cell lines, primary human and mouse megakaryocytes. Resulting platelet production and function was measured via flow cytometry. The two most common parthenolide signaling mechanisms, oxidative stress and nuclear factor-κB inhibition, were assessed within the megakaryocytes using reactive oxygen species, glutathione and luciferase reporter assays. The influence of parthenolide on ex vivo platelet activation was tested with parthenolide pretreatment followed by collagen or thrombin activation. The resulting P-selectin surface expression and released soluble CD40 ligand was measured.

Results

Parthenolide stimulates functional platelet production from human megakaryocyte cell lines, and from primary mouse and human megakaryocytes in vitro. Parthenolide enhances platelet production via inhibition of nuclear factor-κB signaling in megakaryocytes and is independent of the parthenolide-induced oxidative stress response. Additionally, parthenolide treatment of human peripheral blood platelets attenuated activation of stimulated platelets.

Conclusion

Overall, these data reveal that parthenolide has strong potential as a candidate to enhance platelet production and to dampen undesirable platelet activation.     

The CD40/CD40L system regulates rat cerebral microvasculature after focal ischemia/reperfusion via the mTOR/S6K signaling pathway

Objective: The role of CD40/CD40 ligand (CD40L) in microvascular thrombosis is now widely accepted. However, the exact mechanisms linking the CD40/CD40L system and the soluble form of CD40L (sCD40L) with microvascular thrombosis are currently a topic of intensive research. The objective of this study was to assess the potential mechanisms in CD40/CD40L system-regulated microvascular thrombosis after focal ischemia/reperfusion (I/R).

Methods: Rats were subjected to 60-min transient middle cerebral artery occlusion (MCAO). The experiments were divided into three groups: sham operation, MCAO, and MCAO + CD40 antagonist. Dynamic changes of serum-free sCD40L levels for 0, 1, 3, 5, 6, and 12 h by ELISA detecting kit after focal I/R were observed, and the CD40 expression levels in both platelet surface and vascular endothelial cell surface were measured by flow cytometry and immunofluorescence, respectively. Cerebral infarct volume was analyzed 12 h after reperfusion. mTOR/S6K signaling was determined by Western blot.

Results: A comparison of thrombus formation between MCAO and CD40 antagonist treatment rats revealed a role for CD40 and/or CD40L in the inflammation-enhanced thrombosis responses in both of the platelet and vascular endothelial cell. MCAO rats yielded an acceleration of thrombus formation that was accompanied by increased CD40 levels in serum. The brain infarction was significantly decreased in CD40 antagonist treatment group compared to MCAO model group. The mTOR/S6K signaling was activated in MACO model than that of CD40 antagonist treatment group.

Conclusions: Our findings indicate that CD40/CD40L system contributes to microvascular thrombosis and brain infarction induced by MCAO and reperfusion. The mTOR/S6K signaling pathway is involved in the regulation of cerebral microvasculature after focal I/R by CD40/CD40L.

Abbreviations:

AKT: protein kinase B; CD40L: CD40 ligand; CSF: cerebrospinal fluid; FITC: fluorescein isothiocyanate; I/R: ischemia/reperfusion; MCAO: middle cerebral artery occlusion; mTOR: mechanistic target of rapamycin; PE: P-phycoerythrin; sCD40L: soluble form of CD40L; TNF-a: tumor necrosis factor-alpha; WT: wild type.     

CD40-CD40 L在颈动脉粥样硬化斑块稳定性中的作用及与基质金属蛋白酶的相关性

目的 观察CD40、CD40L和基质金属蛋白酶9(MMP9)在人类颈动脉粥样硬化斑块中的表达情况,探讨CD40、CD40L在斑块稳定性中的作用及与MMP9表达的关系.方法 将因颈动脉粥样硬化狭窄(>70%)行颈动脉内膜切除术的37例手术标本分为有临床脑卒中事件组(A组,n=20)和无临床脑卒中事件组(B组,n=17),另设尸检正常颈动脉(C组,n=11)作为对照组,采用实时荧光定量聚合酶链反应法检测各组斑块中CD40、CD40L和MMP9 mRNA水平,Western blot检测各组CD40、CD40L和MMP9蛋白表达水平,免疫组织化学检测各组CD40、CD40L和MMP9表达及分布,并对CD40、CD40L与MMP9的转录及表达水平作相关性分析.结果 A、B、C 3组的CD40mRNA的相对表达量分别为:2.41±0.43、1.03±0.38和0.31±0.12;MMP9 mRNA的相对表达量分别为:6.88±1.57、1.90±0.44和0.39±0.12.A组CD40、MMP9的mRNA水平高于B组(FCD40=105.183,FMMP9=160.395,P=0.000),B组高于C组(FCD40=37.307,FMMP9=124.093,P=0.000);CD40与MMP9的mRNA水平有正的直线相关关系(r=0.929,P=0.000),但各组CD40L的mRNA水平差异无统计学意义(FA-B=0.033,P=0.857;FB-C=1.564,P=0.767;FA-C=0.219,P=0.644).A组CD40、CD40L和MMP9的蛋白表达高于B组(FCD40=104.100,P=0.000;FCD40L=129.932,P=0.000;FMMP9=13.565,P=0.021),B组高于C组(FCD40=115.848,P=0.000;FCD40L=30.482,P=0.005;FMMP9=35.557,P=0.004).免疫组织化学示CD40、CD40L和MMP9的阳性表达面积A组大于B组(FCD40=39.451,FCD40L=57.606,FMMP9=30.711,P=0.000),B组大于C组(FCD40=95.066,FCD40L=59.081,FMMP9=145.758,P=0.000);CD40、CD40L与MMP9的阳性表达面积有正相关关系(rCD40L-CD40L=0.963,rCD40-MMP9=0.959,rCD40L-MMP9=0.929,P=0.000),并且CD40、CD40L和MMP9均在斑块肩部表达明显增多.结论 CD40-CD40L在动脉粥样硬化的形成和斑块稳定性中起重要作用;其可能通过上调MMP9导致斑块不稳定;CD40L可能是通过转录后修饰影响CD40L的表达,从而产生生物学效应.     

CD40 ligand as a potential biomarker for atherosclerotic instability

Abstract

Objective: The signaling protein CD40 and its ligand, CD40L, are thought to contribute to atherosclerotic plaque formation and rupture. We sought to determine their utility as markers of cerebral atherosclerosis and neurological dysfunction.

Methods: We recruited 82 patients with acute cerebral infarction (ACI) and classified each as having large-artery atherosclerosis (LAA, 30), small-artery occlusion (36), or cardioaortic embolism (16). We also recruited 17 patients who had carotid artery stenosis (CAS) without stroke and 20 healthy individuals as controls. CD40L expression on peripheral blood monocytes (PBMCs) was detected by direct immunofluorescence with flow cytometry, and serum soluble CD40L (sCD40L) was measured by ELISA.

Results: CD40L expression on PBMCs was highest in the LAA group, followed by that in the CAS group (both P < 0·01 versus control). It was also higher in patients with atherosclerotic infarction than in those without atherosclerosis (P < 0·05). PBMC CD40L expression was sensitive and specific for detecting atherosclerosis and LAA cerebral infarction and was superior to serum C-reactive protein for predicting cerebral atherosclerosis (P < 0·01). Serum sCD40L was higher in patients with ACI than in healthy controls (P < 0·01) or patients with CAS (P < 0·01) and correlated with increased disability on three scales (all P < 0·01).

Conclusions: We conclude that patients with ACI have an upregulated CD40–CD40L system, which could be used as a clinical biomarker for assessing atherosclerotic instability and severity of neurological dysfunction.