共信号分子CD40/CD40L质粒标准品的构建

背景：目前,对CD40/D40L的研究主要集中在可溶性和蛋白方面,对其在转录水平上的研究文献不多,可能与其无商品化质粒标准品有关。 目的：构建检测CD40/CD40L mRNA表达水平差异的标准品。 设计、时间及地点：单一样本实验,于2009-04在苏州大学附属第一医院检验科实验室完成。 材料：取手术切除的子宫肌瘤组织,经患者知情同意。 方法：以手术切除的子宫肌瘤组织细胞的总RNA为模板、Random 6mers为引物反转录合成cDNA,用该cDNA为模板PCR扩增人CD40/CD40L基因相应的cDNA片断,构建PMD-18-CD40/CD40L重组质粒,鉴定测序后,用荧光定量PCR制作标准曲线。 主要观察指标：①RNA质量检测结果。②扩增的目的片段电泳结果。③CD40/CD40L基因测序结果。④标准品的扩增情况。 结果：组织提取的总RNA完整性良好,构建的CD40/CD40L质粒经PCR扩增后分别得到136 bp,200 bp的清晰条带,测序结果与目的片段完全一致,且质粒的原始浓度为2.66×1013、2.45×1013 copies/mL,倍比稀释至2.0×106 copies/mL均能得到良好的标准曲线(R2=1)。 结论：实验成功构建了CD40/CD40L基因荧光定量PCR标准质粒。     

急性脑梗死患者中性粒细胞粘附分子表达的变化及意义探讨

目的探讨急性脑梗死(cerebral infarction,CI)患者中性粒细胞(polymorphonuclear neutrophil,PMN)表面粘附分子CD62L和CD11b/CD18表达及其意义。方法选择急性血栓形成性脑梗死患者40例,年龄、性别等与之匹配的健康志愿者30例作为对照。运用流式细胞仪对所有对象检测外周血中PMN表面粘附分子CD62L和CD11b/CD18表达。结果相对于正常组粘附分子CD62L平均抗体阳性表达率(73.316±1.276)%,CI组(61.058±8.925)%显著降低(P<0.001);相对于正常组CD11b/CD18平均阳性表达率(20.031±0.540)%,CI组(55,598±0.540)%显著升高。CI组内部CD62L、CD11b/CD18相关分析,二者呈明显负相关,相关系数为r=-0.259(P<0.001)。结论在急性血栓形成性脑梗死的急性期,PMN处于活化状态,表现为粘附分子CD62L表达的下调和CD11b/CD18表达的上调。以细胞粘附为表现的PMN活化加快了血栓的进程,可能是血栓形成的重要发病原因之一。     

中国东北地区CD40及CD40L基因多态性与散发帕金森病的相关性分析

目的 探究CD40基因rs1883832位点及CD40L基因rs1126535位点单核苷酸多态性与帕金森病(Parkinson disease,PD)的相关性。方法 本研究纳入285名中国东北地区汉族健康人和396例PD患者。根据其发病年龄将PD组患者再分为早发PD组(发病年龄≤50岁)和晚发PD组(发病年龄 50岁),收集一般临床资料,提取外周血基因组DNA,利用MALDI-TOF-PEX技术检测rs1883832位点及rs1126535位点多态性分布情况,分析其与帕金森病的相关性。结果 EOPD组与对照组rs1883832位点基因型分布有统计学差异(P 0. 05),等位基因频率在两组间没有统计学差异。PD组和LOPD组与对照组在rs1883832位点及rs1126535位点上,基因型及等位基因型频率无统计学差异。结论 CD40基因rs1883832位点单核苷酸多态性与中国东北地区汉族早发帕金森病相关,T等位基因可能是EOPD的危险因素。     

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.     

急性缺血性卒中患者外周血白细胞表面黏附分子β2整合素CD11c、CD18表达的研究

目的探讨急性缺血性卒中(AIS)患者外周血中性粒细胞(PMN)和单核细胞(MNL)表面黏附分子β2整合素CD11c、CD18表达的变化及意义.方法采用流式细胞术,单克隆抗体标记测定28例AIS患者(AIS组)发病72 h内及7 d时外周血PMN、MNL表面CD11c、CD18的表达量,并与 28名健康对照者(健康对照组)比较.结果 AIS组患者发病72 h内PMN、MNL表面CD11c、CD18表达的平均荧光强度分别为20.82±5.88、218.25±89.00、34.78±14.56、和286.75±95.50,7 d时分别为18.60±5.52、185.52±68.44、31.97±14.47和247.00±88.06;显著高于健康对照组(15.63 ±3.01、150.76±41.20、 20.20±8.50和186.38±52.97)(均P<0.01);发病7 d时CD11c、CD18的表达虽有所下降, 但与72 h检测结果相比差异无显著性.结论 AIS时激活的 PMN、MNL表面β2整合素CD11c、CD18表达上调, CD11c、CD18可能参与了AIS的炎性反应.     

重症肌无力患者外周血CD4+T细胞OX40的表达及作用机制研究

目的 分析重症肌无力(MG)患者外周血CD4+T细胞协同刺激分子OX40表达及其对FoxP3+CD4+CD25+调节性T细胞(Treg)的调控作用,初步探讨OX40在MG免疫学发病中的作用机制.方法 以流式细胞技术检测42例MG患者及38名健康对照的外周血OX40+CD4+T细胞、FoxP3+CD4+CD25+Treg表达水平,比较OX40表达在MG患者不同临床疾病状态、Osserman分型、临床绝对评分、胸腺病理类型等情况下的差异,并分析OX40对FoxP3+CD4+CD25+Treg细胞的影响.结果 (1) MG患者外周血OX40+CD4+T细胞占淋巴细胞百分比高于健康对照组(P<0.01).(2)MG患者OX40+CD4+T细胞百分比在发作或加重期高于缓解期(P＜0.05);在临床绝对评分呈中、重度患者OX40+CD4+T细胞百分比高于轻度患者(均P＜0.05);Osserman Ⅱ、Ⅳ型患者OX40+CD4+T细胞百分比高于Ⅰ型患者(均P＜0.05);胸腺增生及胸腺瘤患者OX40+CD4+T细胞百分比高于胸腺正常患者(P＜0.05,P＜0.01).(3)MG患者外周血OX40+CD4+T细胞百分比与FoxP3+CD4+CD25+Treg细胞百分比呈负相关(r=-0.843,P=0.01).结论 协同刺激分子OX40参与MG发病,可能通过抑制FoxP3+CD4+CD25+Treg细胞生成发挥作用.     

急性缺血性脑卒中患者外周血白细胞CD11c/CD18表达的变化

目的探讨外周血白细胞CD11c/CD18表达与急性缺血性脑卒中(acute ischemic stroke,AIS)发病的关系。方法采用流式细胞术和单克隆抗体标记测定28例AIS患者(AIS组)发病72h内及病程第7d2次外周血中性粒细胞(PMN)和单核细胞(MNL)表面CD11c、CD18的表达量,以平均荧光强度(MFI)表示其相对含量,并与28名健康对照者(对照组)比较。结果AIS组发病72h内外周血PMN表面CD11c、CD18MFI分别为20.82±5.88、218.25±89.00;病程7d时为18.60±5.52、185.52±68.44;均显著高于对照组CD11c、CD18MFI(15.63±3.01、150.76±41.20);发病72h内外周血MNL表面CD11c、CD18MFI分别为34.78±14.56、286.75±95.50,病程7d时为31.97±14.47、247.00±88.06,均显著高于对照组CD11c、CD18MFI(20.20±8.50和186.38±52.97)(P<0.05~0.01);发病7d时CD11c、CD18MFI虽有所下降,但与发病72h内比较差异无显著性(P>0.05)。结论AIS时激活的PMN、MNL表面β2整合素CD11c、CD18表达上调,CD11c、CD18可能参与了炎症反应。     

多系统萎缩患者血清YKL-40和CD40水平变化及相关性因素研究

目的探讨多系统萎缩(MSA)患者血清几丁质酶3样蛋白1(YKL-40)、CD40水平的变化,以及其与发病年龄、病程、疾病严重程度的相关性。方法病例组为2013-03-01—2014-11-30期间大连医科大学附属第一医院神经内科的住院MSA患者30例,其中很可能MSA24例,可能MSA6例,均符合2008年修订Gilman诊断标准;对照组为同期年龄和性别相匹配的30例健康体检者。利用酶联免疫吸附试验(ELISA)检测血清YKL-40与CD40水平,并分析两者与MSA患者性别、年龄、发病年龄、病程和疾病严重程度〔以统一多系统萎缩评估量表(UMSARS)Ⅱ评分进行评估〕的相关性。结果 MSA患者血清YKL-40水平〔38.32(11.74)ng/mL〕较对照组〔47.2(13.88ng/mL〕降低(t=238.00,P=0.002);对照组不同性别间〔男(36.23±9.74)ng/mL,女(36.50±10.86)ng/mL,t=0.519,P=0.477)〕及病例组不同分型间〔MSA-C型:40.67(13.09)ng/mL,MSA-P型:38.24(9.90)ng/mL,U=104.00,P=0.739〕比较血清YKL-40水平差异均无统计学意义。MSA患者血清中CD40水平〔120.39(39.47)pg/mL〕较对照组〔116.12(35.85)pg/mL〕差异无统计学意义(t=439.00,P=0.871)。对照组血清YKL-40水平与年龄无明显相关性(P0.05),病例组血清YKL-40水平与患者发病年龄、病程和UMSARSⅡ评分均无相关性(均P0.05);ROC曲线分析显示血清YKL-40水平对MSA无明确诊断价值(AUC=0.264,P=0.002)。结论 MSA患者血清YKL-40水平较健康人显著降低。MSA患者YKL-40水平与发病年龄、病程和UMSARSⅡ评分无相关性。     

重症肌无力患者CD40配体高表达及其临床意义

目的:研究重症肌无力(MG)患者CD40配体(CD40L)的表达,探讨CD40L在MG中的作用。方法: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。分离血中单个核细胞,分别培养并用不同刺激剂(PHA、PMA和A23187)刺激。培养前后用流式细胞仪检测CD40L阳性细胞率。结果:①MG急性期组CD40L阳性细胞率比正常对照组及非急性期组显著增高(均为P<0.01);②经PHA或PMA和A23187刺激后,非急性期组CD40L阳性细胞率也比正常对照组显著增高(P<0.01)。结论:CD40L的高表达与MG发生和加重有关。     

糖皮质激素对多发性硬化患者外周血淋巴细胞CD80和CD4+CD25+T细胞表达的影响

目的探讨糖皮质激素(GC)对多发性硬化(MS)患者外周血淋巴细胞CD80和CD4+CD25+T细胞表达的影响。方法利用流式细胞仪检测21例MS急性期患者GC治疗前后外周血淋巴细胞CD80和CD4+CD25+T细胞阳性率,并与正常对照组比较;比较MS患者治疗前后扩展功能障碍状况量表(EDSS)评分的变化。结果MS患者急性期外周血淋巴细胞CD80的阳性率[(5.031±1.782)%]较正常对照组[(6.436±2.035)%]明显下降(P<0.05),经GC治疗后CD80的阳性率[(6.467±1.882)%]明显增高(P<0.01);CD4+CD25+T细胞阳性率治疗前后与正常对照组间差异均无统计学意义;治疗后EDSS评分[(3.64±1.79)分]较治疗前[(4.26±1.68)分]明显下降(P<0.01)。结论GC可上调MS患者淋巴细胞CD80的表达,抑制细胞免疫,促进MS病情缓解。     

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.     

Diurnal variation of soluble CD40 ligand in patients with acute coronary syndrome. Soluble CD40 ligand and diurnal variation

Introduction

We sought to investigate whether day-night variations occur in the concentration of circulating soluble CD40 ligand in patients with acute coronary syndrome, as this may have practical implications.

Materials and Methods

We assessed 70 consecutive ST-segment elevation myocardial infarction patients admitted into the Coronary Care Unit and 50 control subjects. Each subject was studied under strictly controlled light/dark conditions. Blood samples were drawn at 09:00 h (light phase) and 02:00 h (dark phase). Nocturnal blood samples were drawn by a trained nurse, with the help of a minute torch with a dim red light in order to avoid any direct lighting on the patient during sleep. The soluble CD40 ligand was measured using a commercially available ELISA.

Results

Soluble CD40 ligand levels showed no diurnal variations in control subjects. In the ST-segment elevation myocardial infarction group, however, soluble CD40 ligand concentration (pg/mL) in the light phase was significantly higher than that in the dark phase (167.3 ± 63.2 vs 118.9 ± 48.3 pg/mL, p < 0.001).

Conclusions

The study shows for the first time the existence of diurnal variations in soluble CD40 ligand levels in ST-segment elevation myocardial infarction patients, which indicates the need for standardizing the time of blood sampling for the assessment of this molecule, at least in studies involving ST-segment elevation myocardial infarction patients.     

Expression of CD40 induces neural apoptosis

The tumor necrosis factor receptor superfamily includes 12 members, some of which (e.g., tumor necrosis factor receptor I and FAS) induce cell death triggered by ligand binding. Another member of the superfamily, the neurotrophin receptor p75^NTR, induces neural apoptosis, with apoptosis being inhibited by binding of ligand to the receptor. As such, it is a candidate for the mediation of neurotrophin dependence. Here, we show that CD40, a superfamily member that is closely related to p75^NTR, also induces neural apoptosis, but apoptosis is inhibited by binding of the G28-5 monoclonal antibody to CD40. These results provide further support for a model in which some members of the tumor necrosis factor receptor superfamily induce apoptosis triggered by ligand binding, whereas other members may, at least under certain conditions, induce apoptosis in the absence of ligand binding, with apoptosis being inhibited by binding of ligand or monoclonal antibody. J. Neurosci. Res. 50:383–390, 1997. © 1997 Wiley-Liss, Inc.     

重症肌无力患者CD40配体表达的信号传导途径初步探讨

目的研究重症肌无力(MG)患者CD40配体(CD40L)的表达,探讨MG患者CD40L表达的信号传导途径.方法研究对象为急性期MG患者13例.分离血中单个核细胞,分组用刺激剂刺激进行单个核细胞培养(1)纯培养组不加任何刺激剂进行细胞培养;(2)PMA组用PMA和A23187刺激;(3)PHA组用PHA刺激;(4)BLM组加PKC抑制剂BLM后再加PHA刺激培养.培养后用流式细胞仪检测CD40L阳性细胞率及RT-PCR法检测单个核细胞CD40L mRNA表达.结果MG急性期BLM组CD40L阳性细胞率和CD40L mRNA与纯培养组差异无显著性(P＞0.05),而显著低于PMA组和PHA组(P＜0.01).结论在MG中,CD40L的表达可能依赖于PKC活性介导的T细胞活化途径.     

Neutrophil CD40 enhances platelet-mediated inflammation

INTRODUCTION: CD40 is a transmembrane protein expressed on monocytes, macrophages, endothelial cells, and platelets. Platelets are the richest source of soluble CD40 ligand (sCD40L) and interact with monocytes and endothelial cells via CD40. While CD40 was recently reported to be present on neutrophils, the detailed mechanism of its interaction with platelets via CD40-CD40L has not been examined. MATERIALS AND METHODS: The existence of neutrophil CD40 was verified by real-time PCR and western blot. Platelet sCD40L release was measured by ELISA. Neutrophil superoxide generation was measured by chemiluminescence and confocal microscopy. The neutrophil-platelet conjugates were measured by flow cytometry. RESULTS AND CONCLUSION: The presence of neutrophils enhances stimulation-induced platelet release of sCD40L. The addition of platelets leads to an enhancement of neutrophil superoxide and reactive oxygen species (ROS) generation. The specificity of the CD40-CD40L pathway in the neutrophil-platelet interaction was confirmed by using recombinant soluble CD40L (rsCD40L) and an anti-CD40L antibody. The involvement of the PI3 kinase/Akt pathway in neutrophil superoxide production was revealed by using LY294002 in isolated neutrophils/platelets experiments, as well as during whole blood aggregation-mediated neutrophil-platelet conjugation. N-acetylcysteine, a scavenger of ROS, eliminates both neutrophil superoxide generation and sCD40L release from activated platelets. These data suggest that activated neutrophils release ROS in a PI3 kinase-dependent manner, contributing to platelet activation and further sCD40L release in a redox-controlled positive feed-back loop. In conclusion, our results define a new pathway by which platelets and neutrophils interact and modulate each other's function, and may be relevant in understanding acute thrombo-inflammatory processes.     

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。     

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.     

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.