脑出血后白细胞浸润与血小板CD62p、CD42b表达关系的临床研究

目的 研究脑出血患者血肿周围脑组织的白细胞浸润及其与血小板CD62p、CD42b表达的关系。方法 37例行微创手术的脑出血患者，手术前用流式细胞术(FCM)检测血小板CD62p、CD42b的表达，手术过程中取引流出的血肿周围脑组织进行HE染色，观察白细胞浸润，并对两者进行相关性分析。结果 脑出血后4h，血肿周围已出现白细胞浸润，脑出血后48—72h白细胞浸润达到高峰；白细胞浸润与血小板CD62p的表达呈正相关性(rCD62p＝0．74，P＜0．01)，与血小板CD42b的表达呈负相关性(rCD42b＝-0．51，P＜0．01)。结论 脑出血后血小板活化，活化的血小板可能参与了血肿周围的白细胞浸润。     

脑出血患者血小板CD42b、CD62p的表达及临床意义

目的研究脑出血急性期血小板表面糖蛋白CD42b、CD62p的表达及其与血肿周围脑水肿形成的关系。方法20例中等量脑出血患者,利用流式细胞仪(FCM)测定其血小板CD42b和CD62p的表达,并根据头颅CT测量血肿周围脑水肿带的大小,对两者进行相关性分析。结果与正常健康人比较,脑出血急性期血小板CD42b的表达减少,CD62p的表达增加;血小板CD42b、CD62p表达量与血肿周围脑水肿带的大小有相关性(rCD42b=-0.489,P<0.05;rCD62p=0.646,P<0.01)。结论脑出血急性期血小板活化,活化的血小板可能参与了血肿周围脑水肿形成这一病理损伤过程。     

急性脑梗死患者CD62P与红细胞内、外钙离子浓度变化的研究

目的观察急性脑梗死患者的血小板CD62P,红细胞内、外钙离子(Ca2+)浓度变化,旨在探讨血小板活化因子(PAF)、Ca2+稳态失调对大脑的损害.方法采用流式细胞免疫技术,以抗血小板单克隆抗体及钙离子荧光剂为分子探针,检测了40例急性脑梗死患者,36例健康查体者的血小板CD62P、红细胞内钙(IECa2+)及血清钙(SCa2+)浓度.结果脑梗死患者CD62P、IECa2+明显高于对照组,SCa2+则明显低于对照组,两组比较差异非常显著(p＜0.01).IECa2+随着CD62P的增高而增高,两者呈正相关(p＜0.05);而血清Ca2+则随着IECa2+的增高而降低,两者呈负相关(p＜0.05).CD62P、IECa2+及SCa2+与病情程度、梗塞面积大小相关,病情重、面积大者CD62P、IECa2+增高显著,SCa2+降低亦显著.结论PAF在Ca2+稳态失调中起着重要作用;PAF及Ca2+稳态失调与脑梗死的发生发展密切相关.     

血小板活化指标与短暂性脑缺血发作的转归

目的探讨短暂性脑缺血发作(TIA)患者疾病转归与血小板活化指标α颗粒膜糖蛋白(CD62p)、溶酶体颗粒膜糖蛋白(CD63)、血小板膜表面糖蛋白(CD41)的关系.方法57例TIA患者随访2个月,分别在服药后1 d、1周、1个月和2个月时采用流式细胞仪检测血小板CD62p、CD63和CD41表达的百分率,同时监测病情发展趋向.结果TIA缓解组、TIA反复发作组与TIA进展为脑梗死组的血小板CD62p、CD63、CD41阳性百分率逐级升高(P＜0.05～＜0.001),尤其以进展为脑梗死组指标升高最为明显(P＜0.01～0.001).结论血小板活化CD62p,CD63及CD41可作为TIA发展演变的估测指标.     

血小板活化特异性标志物PAC-1和CD62p与急性脑梗死病情严重程度的相关性

目的 探讨急性脑梗死(acute cerebral irlflarction,ACI)患者血小板膜糖蛋白(glycoprotein Ⅱb/Ⅲa,GPⅡb/Ⅲa)纤维蛋白原受体PAC-1和P选择素(P-Selectin,CD62p)与ACI患者病情严重程度的相关性.方法 采用流式细胞仪技术分别检测58例ACI患者急性期(<7d)外周血PAC-1和CD62p含量,同时采用Barthel指数和NIHSS量表评分法对ACI患者进行神经功能学评分,并选取20名健康人作为对照组进行参考值测定.结果 与对照组(0.22±0.13;4.21±1.11)相比,ACI患者PAC-1含量(0.92±0.40)和CD62p含量(7.07±3.07)在急性期(<7 d)显著升高(P<0.05;P<0.05),且与ACI患者病情严重程度成正相关(P<0.05;P<0.01).结论PAC-1和CD62p与ACI患者病情严重程度呈正相关.早期检测PAC-1和CD62p水平对于预防ACI发生、评估病情轻重以及判断预后有着重要的意义.     

赛莱乐对多梗死性痴呆患者CD62P、CD63P、P10、DI的变化影响

目的观察赛莱乐治疗对多梗死性痴呆(MID)患者CD62P、CD63、P10、DI变化的影响.方法将禁用阿司匹林及尼莫地平的78例MID患者分2组,治疗组48例,应用赛莱乐150mg静滴,1次/日×15天;对照组30例,复方丹参20ml静滴,1次/日×15天,观测治疗前、后CD62P、CD63、P10及DI变化.结果治疗后治疗组CD62P、CD63、P10表达明显降低,DI明显增强(P＜0.01)与对照组比差异显著(P＜0.05).CD62、P10与DI呈负相关(P＜0.05),CD63与DI无相关.结论赛莱乐可降低CD62P、CD63、P10表达,抑制血小板活化,增强红细胞变形能力,对MID恢复有益.     

奥扎格雷对急性脑梗死患者血小板CD62p和CD63表达的影响及其疗效

目的观察奥扎格雷对急性脑梗死(ACI)患者血小板CD62p、CD63表达的影响及其疗效。方法将64例ACI患者随机分为奥扎格雷治疗组和血塞通治疗组(对照组),采用流式细胞术检测ACI患者治疗前后及正常人(正常组)血小板CD62p、CD63的表达;观察奥扎格雷治疗组和对照组的临床疗效并进行比较。结果ACI患者血小板CD62p、CD63表达水平明显高于正常组(均P<0.01);奥扎格雷治疗组与对照组治疗后血小板CD62p、CD63表达水平较治疗前均有明显下降(P<0.05～0.01),奥扎格雷治疗组又明显低于对照组,差异有显著性(均P<0.05)。奥扎格雷治疗组的基本痊愈率、显著进步率、总有效率明显高于对照组(均P<0.05)。结论ACI发病后血小板CD62p、CD63表达水平显著增高;奥扎格雷有明显抑制血小板表达CD62p、CD63的作用,对ACI的治疗效果显著。     

脑出血患者急性期血小板活化与血肿内炎性反应关系的研究

目的研究脑出血患者血肿内白细胞浸润及其与血小板P选择素(CD62p)、血小板膜凝血酶敏感蛋白(TSP)表达的关系。方法33例行微创手术的脑出血患者,手术时留取血肿液及静脉血,用流式细胞术(FCM)检测其血小板CD62p、TSP的表达及白细胞计数,并对血肿内白细胞浸润与血小板CD62p及TSP之间进行相关性分析。结果脑出血组血肿液中血小板CD62p、TSP表达(20.25±1.82,37.04±3.63)显著高于静脉血(7.72±2.19,12.32±2.81)(均P<0.01);且均显著高于正常对照组(3.81±1.78,8.67±2.86)(均P<0.01)。脑出血后血肿内白细胞浸润与血肿液中血小板CD62p、TSP的表达呈显著正相关(r=0.5374,P=0.0013;r=0.5468,P=0.0010),与静脉血中血小板CD62p、TSP的表达亦呈显著正相关(r=0.4401,P=0.0104;r=0.4288,P=0.0128),前者比后者相关性更为显著。结论脑出血急性期血肿内出现以白细胞浸润为特征的炎性反应,并出现血小板活化,活化的血小板可能参与了血肿内的炎性反应。     

缺血性脑血管病中血小板聚集功能的研究

目的:探讨血小板聚集功能在缺血性脑血管病(ICVD)发病中作用。方法:对发病72h内的ICVD患者72例以及40名健康对照者进行血小板最大聚集率(MAR)、血浆血栓素B2(TXB2)、6-酮-前列腺素F1α(6-keto-PGF1α)、P-选择素水平和血清血小板活化因子(PAF)水平以及血小板膜糖蛋白(GP)Ⅲa的基因PLA多态性检测。结果:ICVD患者血小板MAR以及血TXB2、TXB2/6-keto-PGF1α比值、P-选择素和PAF水平均显著高于对照者(P<0.05),所有受试者GPⅢa基因型均为PLA1/A1纯合基因型,未发现该位点碱基变异。结论:ICVD患者急性期血小板活化功能增强、聚集性增加,但此与血小板GPⅢ基因PLA多态性无关。     

急性脑梗死患者血小板表达血小板内皮细胞黏附分子-1、P选择素的改变及其意义

目的观察急性脑梗死(AC I)患者血小板表达血小板内皮细胞黏附分子-1(CD31)、P选择素(CD62p)的改变及其意义。方法采用全血流式细胞术测定53例AC I患者发病48 h内血小板CD31、CD62p的表达水平,并与有脑梗死易患因素组及健康对照组比较。结果AC I组血小板表达CD31、CD62p[(90.91±15.39)%,(7.00±2.96)%]明显高于易患因素组和健康对照组(均P<0.001);AC I组中合并高血压或糖尿病患者血小板CD62p表达高于无高血压和糖尿病的患者(均P<0.01);血小板CD31、CD62p的表达与脑梗死体积正相关(r=0.39,P<0.05;r=0.63,P<0.01)。结论AC I发病后血小板表达CD31、CD62p显著增高,其表达程度与脑梗死体积以及是否合并高血压或糖尿病有关。     

脑出血患者急性期血小板活化的临床意义

目的探讨脑出血(ICH)急性期血小板活化的临床意义。方法33例行微创手术的ICH患者,术前经头颅CT测量血肿周围脑水肿带的大小,术中留取血肿液及静脉血,用流式细胞术(FCM)检测其血小板表面糖蛋白P-选择素(CD62p)和血小板膜凝血酶敏感蛋白(TSP)的表达,并与所测水肿带大小进行相关性分析。结果CD62p、TSP在ICH组的表达显著高于健康对照组,ICH组血肿液中其表达量显著高于静脉血中的表达;血肿液中血小板CD62p、TSP的表达与血肿周围脑水肿程度呈显著正相关(r=0.4781,r=0.5183,均P<0.005);静脉血中血小板CD62p、TSP表达量与血肿周围脑水肿程度呈显著正相关(r=0.4058,r=0.4193,均P<0.05),前者比后者相关性更为显著。结论ICH急性期血小板活化,活化的血小板可能参与了血肿周围脑水肿的形成。     

血小板激活及血小板参数变化在脑梗死发病机制中的作用

目的研究血小板激活以及血小板参数变化在脑梗死发病机制中的作用。方法采用流式细胞术测定急性脑梗死患者168例和健康对照者40名外周血P选择素(CD62p)、溶酶体蛋白(CD63)的阳性表达率,同时测定血小板计数(PLT)、血小板平均体积(MPV)和血小板最大聚集率(MAR)。并进行比较及相关因素分析。结果(1)脑梗死患者CD62p、CD63及MPV、MAR明显高于健康对照组,并且上述指标急性期均高于恢复期(均P<0·01);(2)全前循环梗死(TACI)亚型的脑梗死患者CD62p、CD63及MPV、MAR显著高于部分前循环梗死(PACI)、后循环梗死(POCI)及腔隙性梗死(LACI)亚型,在PACI及POCI亚型中上述各测定值较LACI亚型显著增高,差异均具有显著性(均P<0·01);而在PACI及POCI亚型之间差异并无显著性(P>0·05);(3)PLT在脑梗死患者急性期、恢复期与健康对照组之间以及牛津郡社区卒中项目(OCSP)各亚型之间差异无显著性(均P>0·05)。(4)CD62p、CD63呈显著正相关(r=0·826,P<0·01),且与MPV及MAR亦呈明显正相关(r=0·703、0·698,均P<0·01);但与PLT之间无相关性(均P>0·05)。结论脑梗死患者血小板的大量激活及其体积和最大聚集率的升高参与了脑梗死的病理过程,监测MPV和MAR较PLT更能反映脑梗死的病情程度,为应用抗血小板聚集药物提供依据。     

Platelet activation patterns are different in mouse models of diabetes and chronic inhibition of nitric oxide synthesis

Currently, there are several animal models of diabetes mellitus and hypertension, but relatively little is known about blood platelet function in these models. The aim of this work was to characterise and compare platelet reactivity and activation in db/db mice (mouse model of diabetes) and mice receiving L-NAME (model of chronic inhibition of NO synthesis), using various platelet function assays. We found higher platelet activation (circulating resting platelets) in db/db mice than in db/+ heterozygotes, as evidenced by elevated expressions of CD62P and CD40L and a lower expression of CD42b. The expression of COX-1 was significantly increased, and the phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser¹⁵⁷ significantly reduced in platelets from db/db mice. Similarly, we observed platelet hyperreactivity in db/db mice following the in vitro responses to 20 μg/ml collagen (reflected by increased expressions of CD62P and CD40L, and reduced CD42b), 20 μM ADP (reduced CD42b) and lower concentrations of thrombin (0.025 U/ml) (increased CD62P, JON/A, bound vWF, and bound fibrinogen). Otherwise, platelet hyporeactivity was revealed for higher thrombin (0.25 U/ml) (reduced CD62P and bound vWF), while hyperreactivity occurred for CD40L and bound Fg in db/db mice compared to non-diabetic control, db/+. Plasma levels of sCD40L, but not of sCD62P, were increased in db/db mice; also plasma TXB₂ concentrations were over 3.5-fold higher in this group than in the heterozygous db/+ mice (P < 0.01). In contrast, in the mice administered with L-NAME, no statistical differences in expressions of platelet activation markers were found between mice supplemented with L-NAME and controls. Likewise, the TXB₂ level did not differ between L-NAME mice and controls, but L-NAME mice had significantly higher plasma levels of sCD62P and sCD40L than controls. In conclusion, these two studied models differ in the overall picture of blood platelet activation and reactivity, as they demonstrated opposite time sequence patterns of platelet activation in circulating blood. More generally, our study provides another argument for the opinion that multiparametric analysis of platelet function offers a much better tool for investigation and minimizes the likelihood of artefacts.     

动脉瘤性蛛网膜下腔出血后脑血管痉挛与血浆花生四烯酸代谢产物的关系

目的探讨动脉瘤性蛛网膜下腔出血(aSAH)病人血浆花生四烯酸代谢产物的含量与脑血管痉挛(CVS)发生、发展之间的关系。方法选取34例aSAH病人作为研究组,在aSAH后第1、3、7、14天抽取外周静脉血测定血栓素B2(TXB2)和6-酮-前列腺素F1α(6-Keto-PGF1α)的含量。另选取同时期健康成人6例作为对照组,进行对比研究。结果研究组发生CVS 23例,其中表现为迟发性缺血性神经功能障碍(DIND)的症状性CVS 10例,无症状性CVS(无DIND)13例。与对照组相比,研究组各时间点血TXB2含量均明显升高,而血6-Keto-PGF1α含量变化无显著性差异;与无CVS病人相比,CVS病人血TXB2含量在各时间点均明显升高,而血6-Keto-PGF1α含量仅在第7天时明显降低;与无DIND病人相比,DIND病人血6-Keto-PGF1α含量在第1、3、14天明显降低,而血TXB2含量仅在第3天时明显升高。结论 aSAH后CVS、DIND的发生、发展与血TXB2、6-Keto-PGF1α含量变化可能存在相关性。     

脑梗死患者血浆ICAM-1、CD62p、CD63的动态变化及其临床意义

目的 探讨脑梗死患者血浆细胞间黏附分子（ICAM-1）、血小板表面P选择素（CD62p）、溶酶体颗粒糖蛋白53（CD63）的动态变化及其临床意义。方法 用流式细胞仪观察60例脑梗死患者发病3d、7d、14d外周血中ICAM-1、CD621，CD63的变化，并与20名健康者进行比较。结果 脑梗死3d、7d上述3项指标明显高于14d及正常对照组（均P〈0．05），而3d与7d间则差异不显著（P〉0．05）；ICAM-1与CD62p、CD63间无相关关系（r=0．1385、0．1632，均P〉0.05）；CD62p与CD63呈显著正相关（r=0.746，P〈0．05）；3项指标与临床神经功能缺损程度评分无相关性（r=0.1462、0.2145、0．368，均P〉0．05）。结论 脑梗死后ICAM-1表达增强，介导了粒细胞与脑血管内皮发细胞间的黏附，同时血小板表面CD62p、CD63表达增强，反映了血小板的活化程度与功能状态，评介导了血小板与中性粒细胞及内皮细胞间的黏附，促进了脑梗死的发生和发展，加重了脑组织的损伤。     

Soluble CD40L in peripheral artery disease. Relationship with disease severity, platelet markers and the effects of angioplasty

Although soluble CD40L (sCD40L, possibly derived from platelets and pro-inflammatory in vitro) may be implicated in thrombosis and haemostasis, there are little data in peripheral artery disease (PAD). We hypothesised the following: (a) that sCD40L relates to the clinical severity of PAD; and (b) that peripheral artery angioplasty acutely raises sCD40L levels. sCD40L was compared to established platelet markers soluble P selectin, platelet microparticles and platelet surface expression of CD62 and CD63. We recruited 36 healthy controls, 33 patients with intermittent claudication (IC), and 33 with symptomatically more severe critical limb ischaemia (CLI), measuring plasma markers by ELISA and membrane markers by flow cytometry. Eleven patients with CLI subsequently underwent peripheral artery angioplasty: blood was taken before and 10 minutes after the intervention. Results show that sCD40L was raised in IC at median 68 (IQR 28-333) pg/ml and in CLI at 64 (34-282) pg/mL compared to 35 (IQR 28-55) pg/ml in the healthy controls (p=0.009). Levels were no different between IC and CLI. The same distribution pattern was present for soluble P selectin, %platelets CD62+ve and CD63+ve. sCD40L failed to correlate significantly with ABPI (p=0.264), unlike %platelets CD62+ve (p=0.0032) and CD63+ve (p=0.009). Pre-angioplasty sCD40L level of 72 (35-610) ng/ml rose to 100 ng/ml (IQR=60-237)(p=0.018) post-angioplasty. Plasma sCD40L, in addition to other platelet indices, is raised in peripheral atherosclerosis and is increased by peripheral artery angioplasty, although levels seem unrelated to clinical severity. Failure to correlate with other markers suggest the platelet may not be the sole source of sCD40L, and that other cells may contribute to plasma levels.     

急性缺血性脑卒中患者血小板膜糖蛋白的表达水平与临床伤残严重程度的相关性及其临床意义

目的 分析急性缺血性脑卒中患者血小板膜糖蛋白的表达水平与临床伤残严重程度的相关性及其临床意义。方法 选取本院神经内科2018年1月-2019年3月收治的120例急性缺血性脑卒中患者为研究对象,将其设定为观察组。另选取60例健康者为对照组,通过流式细胞术检测方法来检测2组研究对象的血小板膜糖蛋白CD31、CD62p、CD63以及PAC-1的表达水平,分析其与临床伤残严重程度的相关性。结果 观察组患者的血小板膜糖蛋白CD31、CD62p、CD63、PAC-1表达水平均高于对照组(P<0.05); 观察组患者血小板膜糖蛋白CD62p与PAC-1表达水平和临床伤残严重程度评分呈正相关(Pearson相关系数分别为0.178和0.241,P<0.05); CD31、CD62p和CD63的表达水平与不同神经功能缺损程度有关,其中中度和重度急性缺血性脑卒中患者的CD31、CD62p与CD63表达水平高于轻型患者(P<0.05)。结论 在急性缺血性脑卒中患者体内血小板的活化程度较健康者来说明显升高,血小板膜糖蛋白CD62p与PAC-1的表达水平对临床伤残程度有显著影响,可作为反映急性缺血性脑卒中患者病情变化和预测康复效果的指标。     

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.     

Influence of stent length and heparin coating on platelet activation: a flow cytometric analysis in a pulsed floating model

Platelets are involved in acute and subacute thrombotic occlusions of coronary stents and also may play a role in the pathophysiology of in-stent restenosis. This study sought to investigate the expression of activation dependent glycoproteins on platelets by flow cytometry and time until stent thrombosis in an in vitro model of stent thrombosis. Coronary stents were placed in parallel silicon tubings with circulating citrated platelet rich plasma to measure 1) influence of stent length on platelet antigens; 2) influence of heparin coating on platelet antigens; and 3) time until stent thrombosis. After recalcification aliquots of platelet-rich plasma were taken over 10 minutes in 2-minute intervals and immediately fixed and stabilized. For flow cytometric analysis monoclonal antibodies to CD41a (glycoprotein IIb/ IIIa), CD42b (glycoprotein Ib-V-IX), CD62p (P-selectin), and CD63 (glycoprotein 53) were used. Within 2 minutes after start of circulation, the expression of CD62p and CD63 increased. Longer stents resulted in more platelet activation than shorter stents (25 mm vs. 15 mm; p<0.001. Time until stent thrombosis was reduced (25 mm vs. 15 mm; p<0.05). Heparin coating did not significantly influence flow cytometry detectable platelet activation but prolonged time until stent thrombosis (coated vs. uncoated; p<0.005). In control tubing systems without stents platelet activation was less pronounced (p<0.0001). Antibodies to CD41a and CD42b did not show significant changes. In this model platelet activation detected by flow cytometry and time until stent thrombosis were dependent on stent length and coating. In vitro testing could be useful to optimize stent design and material.