Human milk oligosaccharides reduce platelet-neutrophil complex formation leading to a decrease in neutrophil beta 2 integrin expression

Human milk is thought by many authorities to be preferable to formula as a source of nutrients for infants. Some of the benefits may stem from its high concentration of unbound oligosaccharides (5-10 g/L). These sugars have structural similarities to selectin ligands, known to mediate important cell-cell interactions in the immune system. Platelet-neutrophil complexes (PNC) exist in healthy individuals but have been implicated in disease states. Formation of these complexes requires selectins and as such, could be influenced by human milk oligosaccharides (HMO). Here, we investigate this possibility by examining the effect of HMO on the formation of PNC and activation of associated neutrophils. We collected blood from 10 healthy volunteers, activated platelets with adenosine 5'-diphosphate, and added HMO, oligosaccharide standards, or phosphate-buffered saline as a control. We determined the influence of HMO on PNC formation and adjacent neutrophil activation with fluorescein-activated cell sorter analysis after labeling with antibodies for the platelet marker CD42a and the neutrophil activation marker CD11b. Within physiologically achievable concentrations (6.25-125 microg/mL), an acidic HMO fraction reduced PNC formation up to 20%, which was similar to the effect seen with high concentrations of sialyl-Lewis x. Associated neutrophils showed a dose-dependent decrease in beta 2 integrin expression, up to 30%, at high but physiological concentrations. The neutral HMO fraction had no effect. These results support the hypothesis that acidic HMO serve as anti-inflammatory components of human milk and thus, contribute to the lower incidence of inflammatory diseases such as necrotizing enterocolitis in breast-fed versus formula-fed infants.     

Effects of hemoglobin vesicles on resting and agonist-stimulated human platelets in vitro

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alphaIIbbeta3 on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.     

The effects of live Neisseria meningitidis and tumour necrosis factor-alpha on neutrophil oxidative burst and beta2-integrin expression

The effects of human recombinant tumour necrosis factor-alpha (TNF-alpha) on neutrophil (PMNL) oxidative burst and on CD11b/CD18 and CD14 expression after stimulation with pathogenic or nonpathogenic Neisseria meningitidis were studied using chemiluminescence and flow cytometry. PMNL oxidative burst increased more when stimulated with the apathogenic 29E strain than with the pathogenic B strain both when studied by chemiluminescence and by flow cytometry. When TNF-alpha was added to whole blood or PMNL together with bacteria a significant increase in the oxidative burst was seen for the B strain only. When whole blood was preincubated for 30 min with TNF-alpha the increase in oxidative burst was significant for both meningococcal strains. TNF-alpha caused a significant increase in PMNL CD 11b/CD18 expression after 30 min of incubation at 37 degrees C. TNF-alpha added simultaneously with the bacteria induced a significant increase in PMNL CD11b/CD18 in both strains. Incubation with the B strain alone caused a low but significant increase in CD11b/CD18 expression, but the addition of TNF-alpha increased this expression to the same high level as incubation with TNF-alpha alone or the 29E strain alone. Only TNF-alpha and the 29E strain caused significant increases in CD14 expression. In conclusion, human PMNLs react differentially when stimulated with pathogenic and nonpathogenic N. meningitidis and the activating effect of TNF-alpha is variable depending on the bacteria involved.     

Increased CXCR4-dependent HIV-1 fusion in activated T cells: role of CD4/CXCR4 association

Activation of peripheral CD4+ T cells resulted in augmented fusion with X4 human immunodeficiency virus type 1 (HIV-1) envelope-expressing cells without parallel increases in the surface expression of CD4 or CXC chemokine receptor 4 (CXCR4). Our study used biochemical methods and biological assays to correlate the increased fusion potential of activated T cells with changes in CXCR4 isoforms and CD4-CXCR4 association. Western blot analyses of CXCR4, precipitated from resting T cells, identified several CXCR4 species with molecular weights of 47, 50, 62, and 98 kDa. After 24 h stimulation with phytohemagglutinin/interleukin-2, a marked reduction was seen in the 47-kDa, with a concomitant increase in the amounts of 50 and 62-64 kDa CXCR4. T cell activation also induced an increase in the coprecipitation of CXCR4 with CD4. The 62-kDa CXCR4 predominantly coprecipitated with CD4 and was shown to be ubiquitinated. Stripping of CD4 from the cell surface with pronase treatment prior to cell lysis only partially reduced coprecipitation of CD4 with the 62-kDa CXCR4, revealing a pool of intracellular CD4-CXCR4 complexes. Coprecipitation of CXCR4 with CD4 was reduced in activated cells treated with Brefeldin A and Monensin, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Confocal microscopy confirmed the colocalization of CD4 and CXCR4 within CD63+ endocytic compartments. These findings demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and accumulation of ubiquitinated 62-64 kDa CXCR4 species, which preferentially associated with CD4. The CD4-CXCR4 complexes may shuttle between late endosomes and the cell surface.     

Detection of circulating platelet-monocyte complexes in persons infected with human immunodeficiency virus type-1

Activated platelets form transient aggregates with monocytes in circulation and have a half-life of approximately 30-60 min. These complexes are increased in various inflammatory conditions and are an early marker of myocardial infarction. HIV-1 infection is associated with chronic inflammation, and increased CD16? inflammatory monocytes have been observed in these individuals, probably as a result of increased interaction with platelets. However, narrow detection period and platelet activation during sample processing pose significant problems in detecting platelet-monocyte complexes (PMCs). A method was standardized addressing these difficulties, to enumerate PMCs involving CD16? or CD16? monocytes in whole blood using flow cytometry. Blood collected from healthy individuals was treated with either collagen (for platelet activation) or LPS (for monocyte activation) and subsequently used to study effect of these treatments on PMC formation. This method was also validated for the ex vivo quantitation of PMCs in blood obtained from persons infected with HIV. The in vitro results demonstrated that platelet activation, but not monocyte activation, resulted in significant increase in PMC formation. There was a significant increase in CD16? PMCs and platelet activation, in samples obtained from persons infected with HIV as compared to those without HIV infection. Furthermore, PMC percentages correlated positively with platelet activation. These findings improve the ability to detect PMCs and shed light on HIV pathogenesis.     

类风湿关节炎患者CD62P、CD41/CD61的表达及意义

目的:通过检测类风湿关节炎(RA)患者血小板表面活化标志物CD41/CD61、CD62P的表达,研究血小板活化与RA及RA活动性的关系。方法:采用流式细胞技术检测RA患者及健康对照组血小板CD41/CD61、CD62P的表达阳性率。结果:血小板CD62P阳性率在RA活动组及RA组患者分别高于缓解组和健康对照组,CD41、CD61阳性率差别在各组间无统计学意义。结论:(1)RA患者血小板CD62P表达增高与疾病活动有关;(2)血小板CD62P高表达可能是RA患者好发心血管疾病的原因之一;(3)CD41/CD61可能与RA炎症及其好发心血管病无关。     

The role of thiols and disulfides in platelet function

Disulfide bonds formed in newly synthesized proteins in the endoplasmic reticulum of cells are important for protein structure and stability. Recent research, however, emphasizes a role for thiol-disulfide reactions with disulfide bond rearrangement as a dynamic process in cell and protein function, and in platelet function in particular. Protein disulfide isomerase was found on the platelet surface where it appears to play an important role in the platelet responses of aggregation and secretion, as well as activation of the platelet fibrinogen receptor, the alphaIIbbeta3 integrin. Additionally, sulfhydryl groups in alphaIIbbeta3 have been implicated in the activation of this integrin. Physiologic concentrations of reduced glutathione generate sulfhydryls in alphaIIbbeta3 and potentiate sulfhydryl-dependent reactions in alphaIIbbeta3. Sulfhydryl labeling in alphaIIbbeta3 is inhibited by phenylarsine oxide, a reagent that binds to vicinal thiols. As vicinal thiols are in equilibrium with disulfide bonds, they provide redox-sensitive sites in alphaIIbbeta3 able to respond to external or cytoplasmic reducing equivalents. Furthermore, protein disulfide isomerase and sulfhydryls are now implicated in platelet adhesion by a second platelet integrin, the alpha2beta1 collagen receptor. Most recently, extracellular sulfhydryls in the P2Y12 ADP receptor were found to be required for platelet activation by this receptor. We here provide an overview of this field with a focus on recent developments, and conclude with a working model.     

The beta 1 integrin, very late activation antigen-4 on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers.

Polymorphonuclear leucocyte (PMNL) accumulation in extravascular tissues and inflammatory exudates is dependent on their migration through blood vessel endothelium and then through connective tissue. Previously we utilized a barrier of human synovial and dermal fibroblasts (HSF or HDF) grown on microporous filters, as a model of PMNL migration through connective tissue. Those studies showed that beta 2 (CD18) and the beta 1 integrins, very late activation antigen-5 (VLA-5) and VLA-6, in part mediate this PMNL migration. Here we report that VLA-4, which can also be expressed at low levels on activated PMNL, is also involved in PMNL migration induced by C5a through fibroblast (HSF and HDF) barriers, because monoclonal antibody (mAb) to VLA-4 significantly inhibited (by 20-30%) PMNL migration. Blocking the function of CD18, VLA-5 or VLA-6 was not required for detection of the VLA-4-mediated migration. Combination treatment with mAb to VLA-4 and with mAb to VLA-5 or to VLA-6 further inhibited PMNL migration, irrespective of whether CD11/CD18 mechanisms were blocked with anti-CD18 mAb or not. Treatment of PMNL with a peptide based on the VLA-4-binding domain in the CS-1 fragment of fibronectin, but not a control peptide, inhibited PMNL migration to a comparable extent to treatment with mAb to VLA-4. A low level of VLA-4 was expressed on C5a-activated PMNL, detected by immunofluorescence flow cytometry. These results suggest that VLA-4 can be mobilized by human peripheral blood PMNL and can, in addition to VLA-5, VLA-6 and CD11/CD18 integrins, mediate PMNL migration through connective tissue. This is in marked contrast to PMNL transendothelial migration, where beta 1 integrins appear to play no significant role.     

血小板生成素及血小板生成素Ⅱ对人血小板活化的影响

目的:检测血小板生成素(TPO)及TPOⅡ在体外对人血小板活化的影响。方法:取正常健康成人富血小板血浆,分别与血小板生成素(rhTPO)、TPOⅡ及磷酸盐溶液孵化,通过荧光单克隆抗体标记血小板表面CD62P及CD41分子,用流式细胞术测定CD62P/CD41比率,观察各组血小板活化率。结果:rhTPO、TPOⅡ组血小板活化率与PBS组无明显差异,P＞0.05。结论:血小板生成素不会导致血小板异常活化,可应用于血小板生成减少的各种情况。     

益气活血解毒法对不稳定性心绞痛患者血管内皮保护与抑制血小板活化作用

观察益气活血解毒法治疗不稳定性心绞痛患者 ,血管内皮保护与抑制血小板活化作用。将 43例不稳定性心绞痛患者 ,随机分为 2个治疗组 :单用西药治疗组 (对照组 )与益气活血解毒法治疗组 (中药组 )。 2组均给予同样的常规西药方案治疗 ,中药组加静脉滴注黄芪注射液 (每日 2 0ml,含黄芪 40 g) ,静脉滴注络泰注射液 (三七总皂甙粉针剂 ,每日 40 0mg) ,口服解毒护心胶囊 (每粒含黄连超细粉碎物 0 .4g、茶多酚 0 .0 8g ,一次 3粒 ,一日 3次 ) ,疗程 2周。 2 0例健康人为正常组。流式细胞仪检测血小板P -选择素 (CD 6 2 p) ,酶联免疫检测血浆假血友病因子 (vWF)。作治疗前后及组间统计学比较。结果 :两治疗组治疗前CD 6 2p、vWF均未见显著差异 ,均显著高于健康组 (P <0 .0 1)。两治疗组治疗后CD 6 2 p、vWF均显著下降 (P <0 .0 5或P <0 .0 1) ,且中药组非常显著地低于对照组 (P <0 .0 1)。结论 :黄芪、三七总皂甙与黄连等配伍体现的益气活血解毒法 ,对不稳定性心绞痛患者 ,有显著的保护血管内皮功能、抑制血小板活化作用。     

Determination of activated platelets: evaluation of methodology and application for patients with idiopathic thrombocytopenic purpura]

Platelet activation causes a change in surface expression of several endogenous proteins, such as CD62P, CD63 and CD40L. Therefore, it is possible to analyze the functional in vivo status of the circulating platelet population directly by flow cytometry. In this study we developed the method to be suitable for use in clinical studies. We used EDTA-2K as anticoagulant since the sample anticoagulated with EDTA-2K, sodium citrate or ACD-A showed no difference in the data of activated platelets. We determined whether fixation of sample is necessary. The samples stained before or without fixation showed abnormally high level of activated platelets, indicating that fixation is necessary before staining. It is controversial whether activated platelets circulate in patients with idiopathic thrombocytopenic purpura(ITP). We measured activated platelets in patients with ITP using our optimised method. The percentages of CD62P, CD63 and CD40L positive platelets were significantly high in patients with ITP and 24%, 55% and 36% (respectively) of ITP patients showed elevated level of activated platelets. These data indicate that activated platelets circulate in ITP patients.     

Differential expression of platelet activation markers in aspirin-sensitive asthmatics and normal subjects

Background Activation of platelets and expression of adhesion molecules (e.g. CD62P and CD63) which mediate interactions between platelets and other cells may be important in the pathogenesis of aspirin-sensitive asthma. Objective To determine the expression of CD62P and CD63 on platelets from aspirinsensitive asthmatic (ASA +), aspirin-tolerant asthmatic (ASA-) and normal subjects and to assess the modulatory effect of aspirin on platelet CD62P and CD63 expression following stimulation with either platelet-activating factor (PAF), arachidonic acid (AA) or collagen (COL). Methods Platelet-rich plasma was obtained from 10 ASA +, 10 ASA—and 10 normal control subjects, and expression of CD62P and CD63 was measured by flow cytometry. Platelets were stimulated with PAF (10, 80 nM), AA (0.1, 1 mM) or COL (80, 800 μg/mL) with or without aspirin (concentration range 0.4–4 mg/mL). Results In the absence of aspirin, CD62P expression induced by AA and COL was greater in ASA+ patients compared with control subjects (P<0.001) while CD62P expression with PAF, AA and COL was reduced in ASA—when compared wilh ASA+ and control subjects (P < 0.001). CD63 expression with PAF and AA was reduced in both ASA+ and ASA- patients compared with control subjects (P<0.001). Aspirin inhibited the expression of both CD62P and CD63 after agonist stimulatitin. Greater inhibition of CD62P expression was observed in ASA+ compared with ASA- patients (P<0.001) and normal subjects (P<0.05) while greater inhibition of CD63 expression was observed in normal subjects compared with both ASA+ and ASA- patients (P<0.05). In ASA+ patients and normal subjects, stimulation with PAF and COL resulted in only one platelet population while in contrast with 1 mM AA two populations were observed. Conclusions Fnhanced AA- and collagen-induced platelet CD62P expression in ASA+ patients compared with normal subjects and greater inhibition by aspirin of CD62P expression in ASA+ may be relevant to the pathogenesis of this syndrome. Reduced expression of CD62P and CD63 in platelets of ASA- patients following stimulation with PAF and AA may also have implications for the role of platelets and these mediators in the pathogenesis of other forms of asthma.     

Increased expression of surface activation markers on neutrophils following migration into the nasal lumen

BACKGROUND : The sequence of events following the recruitment of a free-flowing neutrophil in the peripheral circulation, via adhesion, migration and release of mediators, to a neutrophil on the surface of the nasal epithelium is a co-ordinated process. Little is known about the state of neutrophil activation following this course of events. OBJECTIVES : To investigate the expression of surface activation markers on neutrophils, reflecting activation during their recruitment to the nose, and to see whether the inflammatory process during allergic rhinitis influences this process. METHOD : Nine healthy controls and 12 patients with grass pollen-induced intermittent allergic rhinitis were investigated during the peak of the pollen season. The expression of CD11b, CD66b and CD63 on the neutrophil cell surface, as a reflection of activation, was analysed using flow cytometry. Neutrophils were derived from peripheral blood and nasal lavage fluid. In addition, eosinophil cationic protein (ECP) and myeloperoxidase (MPO) as well as L-, P- and E-selectins in the nasal lavage fluid were analysed using RIA and ELISA, respectively. RESULTS : A marked increase in the expression of all three CD markers on the neutrophil cell surface was noticed following migration from the bloodstream to the surface of the nasal mucosa. At the peak of the grass pollen season, the MPO levels increased, reflecting an increase in the total number of nasal fluid neutrophils. In parallel, the expression of CD11b was further augmented. The expression of the CDb11b was reduced on neutrophils remaining in the circulation. In addition, the level of L-selectin was reduced on neutrophils derived from the blood during allergic inflammation. CONCLUSION : Neutrophils might become activated during their transfer from the blood to the surface of the nasal mucosa, but these changes may also be due to depletion of activated neutrophils in the blood via activated endothelial/epithelial adhesion and chemoattractant measures. The increased expression of surface activation markers during allergic rhinitis suggests roles for neutrophils in the inflammatory process.     

TSLPR 抗体抑制ApoE 敲除小鼠动脉粥样硬化血栓形成

目的:探讨TSLPR 抗体在抑制动脉粥样硬化血栓形成中的作用。方法:8 周龄载脂蛋白E-/ - 小鼠,给予高脂饲养12 周,期间分别周期性尾静脉注射TSLPR 抗体和同型无关IgG 抗体,检测各组小鼠动脉粥样硬化血栓形成时间,血小板聚集和释放功能,血小板膜糖蛋白表达。结果:与对照组相比,TSLPR 抗体处理组血栓形成时间明显长,血小板聚集功能和释放功能明显低,血小板活化指标CD62p、CD63、PAC-1 表达明显低,静息指标CD42b 表达明显高。结论:静脉注射TSLPR 抗体可以明显抑制血小板聚集功能和释放功能,降低血小板活化程度,从而抑制动脉粥样硬化血栓形成。     

Leukocytes-depleting filters preferentially remove activated leukocytes and reduce the expression of surface adhesion molecules during the simulated extracorporeal circulation of human blood

The effect of leukocyte-depleting filters on the total and activated leukocyte counts and the expression of surface adhesion molecules CD11b, CD18, and CD62L during the in vitro extracorporeal circulation of human blood was studied. A 200 ml blood sample was taken from 10 patients undergoing CABG surgery. The blood was circulated for 60 minutes within an experimental extracorporeal circuit. A leukocyte-depleting filter was attached in five circuits (filtered group). In five other circuits, no filter was used (controls). Total leukocyte counts were determined manually. Activated leukocytes were identified using nitroblue tetrazolium staining. The expression of CD11b, CD18, and CD62L was measured with flow cytometry. At 60 minutes, total leukocyte counts were reduced by 49% from the baseline values in the filtered group and 10% in the control group (p < 0.0001). Activated leukocyte counts decreased by 86% in the filtered group and increased by 116% in the control group (p < 0.0001). In the filtered group, the expression of CD11b, CD18, and CD612L decreased by 60%, 21%, and 79%, respectively, and in the control group it increased by 24%, 6%, and 28% (p < 0.0001). Leukocyte-depleting filters preferentially remove activated leukocytes and reduce the expression of CD11b, CD18, and CD62L during the in vitro extracorporeal circulation of human blood.     

急性脑梗死患者外周血淋巴细胞的早期活化与其黏附分子表达的关系研究

目的：探讨脑梗死患者外周血T淋巴细胞早期活化与黏附分子CD54和血小板P选择素（CD62P）、溶酶体颗粒糖蛋白63（CD63）的动态变化及临床意义。方法：用流式细胞仪检测32例脑梗死患者发病3d、7d外周血T淋巴细胞CD69和CD54及血小板CD62P、CD63的表达水平,并与35例健康者进行比较。结果：急性脑梗死（MS）患者发病72h内、7d外周血CD3^＋CD69^＋与CD3^＋CD54^＋及血小板CD62P与CD63表达阳性率均显著高于对照组（P〈0．01）,7d时各值虽较72h略降低,但差别无统计学意义（P〉0．05）;急性脑梗死患者发病72h内CD3^＋CD69^＋与CD3^＋CD54^＋、CD62P和CD63的表达无明显相关性（r值分别为-0．218、-0．117、-0．224,P〉0．05）;而CD54与CD62P、CD63的表达呈明显正相关（r=0．468,P〈0．01;r=0．397,P〈0．05）。结论：急性脑梗死后T淋巴细胞活化程度增强,其细胞表面CD54分子表达增高,同时血小板表面CD62P和CD63分子表达增加,活化的T淋巴细胞和血小板介导了白细胞与血小板及内皮细胞的黏附,加速了脑梗死的发生和发展,进一步加重了脑组织的损伤。     

老年慢性肺心病患者血小板活性与高敏C反应蛋白的关系

目的探讨老年慢性肺心病患者血小板活化标志物PAC-1、CD62p的变化与高敏C反应蛋白（hs-CRP）的关系。方法用三色全血流式细胞术测定36例老年慢性肺心病患者外周血中血小板PAC-1、CD62p的表达水平,采用免疫比浊法测定hs-CRP,并与28例老年健康对照者及28例非老年健康对照者比较。结果老年慢性肺心病组PAC-1、CD62p、hs-CRP均明显高于老年健康对照组及非老年健康对照组（均P〈0.001）。老年健康对照组PAC-1、CD62p阳性表达率亦高于非老年健康对照组（P〈0.05）。老年慢性肺心病患者PAC-1、CD62的变化与hs-CRP呈正相关。结论老年慢性肺心病患者血小板明显活化,其活化程度与hs-CRP关系密切。     

Platelets activation in patients undergoing PTCA and their responsiveness after in vitro stimulation

We evaluated expression of platelet activation markers in blood samples of 15 patients who underwent percutaneous transluminal coronary angioplasty (PTCA) by flow cytometry. Analysis was performed before the beginning of PTCA, during initial coronary angiography and after the end of PTCA or after a stent placement, respectively. We evaluated platelet-derived microparticles, platelet-leukocyte aggregates, platelet aggregates and a membrane expression of CD62P and CD63 molecules. Responsiveness of platelets to the activation in vitro with thrombin-receptor activating protein-6 (TRAP-6) was tested simultaneously. Statistically significant differences between patient samples were found only in the expression of the activation markers CD62P (before PTCA 0.22%, during 0.39%, after 0.67%), CD63 (0.26%/0.45%/0.85%) and platelet-leukocyte aggregates (13.57%/18.39%/23.63%). In the same group the expression of all constitutive membrane markers was statistically significantly decreased: in patients undergoing PTCA was the expression of CD9: 87.98% (in comparison with control group 94.98%), CD31: 87.10% (92.78%), CD36: 87.37% (90.98%), CD41: 88.09% (95.62%), CD42a: 88.54% (94.98%), CD42a: 88.31% (94.13%).     

High levels of LDL-C combined with low levels of HDL-C further increase platelet activation in hypercholesterolemic patients

High levels of low-density lipoprotein cholesterol (LDL-C) enhance platelet activation, whereas high levels of high-density lipoprotein cholesterol (HDL-C) exert a cardioprotective effect. However, the effects on platelet activation of high levels of LDL-C combined with low levels of HDL-C (HLC) have not yet been reported. We aimed to evaluate the platelet activation marker of HLC patients and investigate the antiplatelet effect of atorvastatin on this population. Forty-eight patients with high levels of LDL-C were enrolled. Among these, 23 had HLC and the other 25 had high levels of LDL-C combined with normal levels of HDL-C (HNC). A total of 35 normocholesterolemic (NOMC) volunteers were included as controls. Whole blood flow cytometry and platelet aggregation measurements were performed on all participants to detect the following platelet activation markers: CD62p (P-selectin), PAC-1 (GPIIb/IIIa), and maximal platelet aggregation (MPAG). A daily dose of 20 mg atorvastatin was administered to patients with high levels of LDL-C, and the above assessments were obtained at baseline and after 1 and 2 months of treatment. The expression of platelets CD62p and PAC-1 was increased in HNC patients compared to NOMC volunteers (P<0.01 and P<0.05). Furthermore, the surface expression of platelets CD62p and PAC-1 was greater among HLC patients than among HNC patients (P<0.01 and P<0.05). Although the expression of CD62p and PAC-1 decreased significantly after atorvastatin treatment, it remained higher in the HLC group than in the HNC group (P<0.05 and P=0.116). The reduction of HDL-C further increased platelet activation in patients with high levels of LDL-C. Platelet activation remained higher among HLC patients regardless of atorvastatin treatment.     

Internalization of IgG-coated targets results in activation and secretion of soluble CD40 ligand and RANTES by human platelets

Platelets are crucial elements for maintenance of hemostasis. Other functions attributable to platelets are now being appreciated, such as their role in inflammatory reactions and host defense. Platelets have been reported to bind immunological stimuli like IgG complexes, and for nearly 50 years it has been speculated that platelets may participate in immunological reactions. Platelets have been reported to bind and internalize various substances, similar to other leukocytes, such as macrophages and dendritic cells. In the present study, we tested the hypothesis that human platelets can bind and internalize IgG-coated particles, similar to leukocytes. To this end, we observed that interaction with IgG-coated beads resulted in platelet activation (as measured by CD62P expression), internalization of targets, and significant soluble CD40 ligand (sCD40L) and RANTES (regulated upon activation, normal T cell expresses and secreted) secretion. Blocking FcγRIIA with monoclonal antibody (MAb) IV.3 or inhibiting actin remodeling with cytochalasin D inhibited platelet activation, internalization, and cytokine production. These data suggest that platelets are capable of mediating internalization of IgG-coated particles, resulting in platelet activation and release of both sCD40L and RANTES.