单采血小板不同保存条件膜糖蛋白的变化

目的　探讨单采血小板经不同方法保存后膜糖蛋白 (glycoproteins ,GP)的变化。 方法　采用流式细胞术 (flowcytometry ,FCM)对 17例机采血小板经 2种方法保存后测定血小板膜GPCD4 1a、CD4 1b、CD4 2a、CD6 2p和纤维蛋白原受体 (fibrinogenreceptor,PAC 1)。 结果　 2 2°C保存后CD6 2 p的表达率和平均荧光强度 (meanflu orescenceintension ,MFI)均随时间的延长而逐渐增高 ,由采集时的 10 .96 %± 5 .5 4 %和 31.98± 16 .33至保存后的第 6天增高到 91.31%± 7.79%和 79.80± 19.6 8;与当日比较P <0 .0 0 1。 - 80℃保存 3个月后的CD6 2p的表达率除与 2 2℃保存 1d时相同外 (P >0 .0 5 ) ,均低于 2 2℃保存 2～ 6d(P <0 .0 0 1) ;PAC 1的表达率于保存后1d开始增高 ,到保存第 3天又逐渐下降。CD4 1a无论是表达率还是MFI保存后均高于采集当天 (P <0 .0 0 1)。CD4 1b和CD4 2a保存后变化范围不大。结论　 (1)血小板膜GP的表达率和MFI随保存时间的延长而增高 ,PAC 1保存到第 3天后又逐渐下降 ,以CD6 2 p、PAC 1及CD4 1a变化为大 ;(2 )血小板加保护剂于 - 80℃保存稳定性较好     

全血流式细胞术检测血小板膜糖蛋白Ib

目的 建立全血流式细胞术检测血小板膜糖蛋白Ｉｂ（ＧＰＩｂ）的实验方法,并探讨了体外循环（ＣＰＢ）期间血小板ＧＰＩｂ的变化及意义。方法 抽取献血员及二尖瓣置换病人的血液,经免疫荧光染色,通过流式式细胞仪检测并分析结果。结果 经多聚甲醛（ＰＦＡ）固定的血标本可减少非特异染色。ＣＰＢ期间血小板ＧＰＩｂ呈降低趋势,ＣＰＢ结束２小时达最低点（ｔ＝３．７１４,Ｐ〈０．０１）,术后一天回复术前水平（ｔ＝１．９２     

全血流式细胞术检测血小板膜糖蛋白Ib

建立全血流式细胞术检测血小板膜糖蛋白Ｉｂ（ＧＰＩｂ）的实验方法，并探讨了体外循环期间血小板ＧＰＩｂ的变化及意义。抽取献血员及二尖瓣转换病人的血液，经免疫荧光染色，通过流式细胞仪检测并分析结果。经多聚甲醛固定的血标本可减少非特异染色。富含血小板血浆与相应的全血相比，其ＧＰＩｂ相对荧光强度值减小。体外循环期间血小板ＧＰＩｂ呈降低趋势，体外循环结束２小时达最低点（ｔ＝３．７１４，Ｐ〈０．０１），术后一天     

血小板减少性紫癜患者血小板膜糖蛋白测定及临床意义

血小板减少性紫癜患者（ＩＴＰ）是一组由自身血小板抗体引起的综合度，这些抗体作用的血小板膜靶抗原可多种多样，其中血小板膜糖蛋白（ＧＰ）Ⅱｂ／Ⅲｓ是一种主要的靶抗原，少数针对ＧＰＩｂ／ＩＸ复合物，为探讨ＩＴＰ与血小板膜表面糖蛋白关系，采用流式细胞仪分析方法，测定了２８例ＩＴＰ患者血小板的ＧＰⅡａ，ＧＰⅡｂ、ＧＰⅢａ、ＧＰＩｂ、ＧＰＩＸ１７例正常工作对照组，ＩＴＰ患者的ＧＰ均明显低于正常对照组，患者的Ｇ     

流式细胞术分析急性脑缺血患者血小板膜糖蛋白改变

应用流式细胞术（ＦＣＭ）检测急性脑缺血患者血小板膜糖蛋白Ⅱｂ／Ⅲａ复合物（ＧＰⅡｂ／Ⅲａ），ＧＰ１ｂ，α－颗粒膜蛋白（ＧＭＰ－１４０），假血管性血友病因子（ｖＷＦ）等的改变，结果显示病例组ＧＰ阳性细胞百分率及平均荧光强度均较对照线明显升高（Ｐ〈０．００１～０．０５），表明急性离缺血患者血小板处于激活状态，并探讨了ＧＰ在脑缺血发病中的作用ＦＣＭ为研究脑缺血患者血小板活化程度和抗血小板药物疗效提供了一     

用流式细胞术分析人血小板膜糖蛋白Ⅱb/Ⅲa

用流式细胞术分析人血小板膜糖蛋白Ⅱｂ／Ⅲａ王美健林其燧潘家绮赖悦云血小板膜糖蛋白（ＧＰ）Ⅱｂ／Ⅲａ属整合素家族成员，是纤维蛋白原受体，介导血小板聚集。在先天性血小板无力症时，血小板ＧＰⅡｂ／Ⅲａ较少、缺失或异常。单克隆抗体作为分子探针对于研究血小板膜...     

脑出血患者血小板膜表面糖蛋白分子的表达及其临床意义

脑出血已成为致死、致残的一种常见疾病,其病理过程较复杂.近几年来,对于血小板活化与脑血管病发病关系受到重视.研究证实,CD42b和CD62p两者都是活化血小板特异性的糖蛋白分子标志物,是体内血小板活化的敏感性指标[1-2],本研究利用流式细胞仪技术测定脑出血患者入院时、发病后第3天、第7天、第21天的血小板CD42b和CD62p的表达,探讨它们在脑出血发生发展过程中的变化和意义.     

血小板膜糖蛋白Ⅰbα表达水平与急性脑梗死相关性研究

目的探讨血小板膜糖蛋白Ⅰbα(GPⅠbα)表达水平与急性脑梗死(ACI)的相关性。方法采用流式细胞术(FCM)分别检测119例ACI患者与117例健康对照者GPⅠbα的表达水平并进行比较分析。结果 ACI组的GPⅠbα表达水平低于对照组,其差异有统计学意义(P<0.05)。非条件Logistic回归分析显示GPⅠbα表达降低是ACI发生的独立危险因素。结论 GPⅠbα的表达水平与ACI的发生有关。     

流式细胞术分析急性脑缺血患者血小板膜糖蛋白改变

应用流式细胞术（FCM）检测急性脑缺血患者血小板膜糖蛋白Ⅱb／Ⅲa复合物（GPⅡb／Ⅲa）、GPⅠb、α-颗粒膜蛋白（GMP－140）、假血管性血友病因子（vWF）等的改变，结果显示病例组GP阳性细胞百分率及平均荧光强度均较对照组明显升高（P＜0．001～0．05），表明急性脑缺血患者血小板处于激活状态，并探讨了GP在脑缺血发病中的作用，FCM为研究脑缺血患者血小板活化程度和抗血小板药物疗效提供了一种新方法。     

血细胞分离机对血小板膜糖蛋白影响的观察

目的　了解血小板经血细胞分离机单采后膜糖蛋白的变化。方法　利用流式细胞仪及CD41a、CD41b、CD42a、CD61、CD62 p、PAC 1单克隆抗体对 2 1份机采血小板前后的膜糖蛋白表达率和平均荧光强度 (meanfluores cenceintension ,MFI)进行检测。结果　血小板经血细胞分离机单采后 ,CD41a、CD41b、CD42a的阳性表达率和MFI、CD61的阳性表达率和CD62 p、PAC 1的MFI与采集前比较无明显变化 (均为P >0 .0 5 ) ;CD6 2 p、PAC 1的阳性表达率增高 (均为P <0 .0 5 ) ,CD61的MFI降低 (P <0 .0 1)。结论　血小板经血细胞分离机单采后仍保存了膜糖蛋白的完整性 ,虽有一定比率的血小板活化 ,但MFI无明显改变     

血小板膜糖蛋白及其多态性的研究

血小板是动脉血栓形成的主要参与者，其功能是通过其表面的受体糖蛋白来实现的，血小板膜糖蛋白的基因多态性能够改变其抗原性，从而影响血小板的粘附、聚集和活化反应。本文将对目前进行深入研究的几种血小板膜糖蛋白的结构、功能及其多态性进行论述。     

Biochemistry of platelet membrane glycoproteins

Platelet membranes are rich in glycoproteins which mediate the key functions of platelets, adhesion and aggregation, by their binding of specific adhesive proteins. The membrane glycoproteins were initially identified by surface labeling with 125I or 3H followed by SDS-polyacrylamide gel electrophoresis. Many of these glycoproteins are now cloned and sequenced and have been found to be members of major gene families, such as integrin, leucine rich glycoprotein (LRG), selectin, etc. In this chapter, the structure and the functions of membrane glycoproteins are briefly reviewed.     

Platelet membrane glycoprotein changes during the preparation and storage of platelet concentrates

Previous studies of platelet membrane glycoproteins during blood bank storage have reported conflicting results. This study assessed two major plasma membrane glycoproteins (GP Ib and GP IIb), an alpha-granule membrane protein (GMP-140), and the concentration of platelet membrane microparticles in cell-free plasma during routine hospital blood bank platelet storage. 125I-monoclonal antibody binding was used to measure membrane glycoproteins on the surface of intact platelets and to measure the concentration of membrane microparticles in cell-free plasma. Platelet concentrates were stored at room temperature in polyolefin bags for 7 days. In this blood bank, two types of rotators are routinely used for platelet concentrate storage: a 2-rpm circular tumbler rotator and a 6-rpm elliptical rotator. Different results were obtained with the rotators. With the tumbler rotator, there was no loss of platelets and antibody binding to GP Ib remained normal. With the elliptical rotator, one third of platelets were lost into clumps during storage, and a 50 percent decrease of antibody binding to GP Ib occurred in the remaining single platelets. There was no loss of antibody binding to GP IIb with either rotator. Antibody binding to GMP-140 increased equally in both rotators indicating that the remaining single platelets had secreted about 16 percent of their alpha-granule contents. The plasma concentration of platelet membrane microparticles was greater in the bags stored in the elliptical rotator. These results indicate that it is possible to maintain the normal concentration of platelet membrane glycoproteins Ib and IIb during 7 days of room-temperature blood bank storage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of amphotericin B and fluconazole on platelet membrane glycoproteins

BACKGROUND : Fever, chills, and reduced platelet recovery may result when platelets are transfused simultaneously with amphotericin B. Amphotericin B reportedly increases the pitting of membranes in stored platelets. STUDY DESIGN AND METHODS : The effects of amphotericin B and another antifungal agent, fluconazole, on platelet membrane glycoproteins (GP) were examined by the incubation of split aliquots of fresh and stored platelet concentrates (PCs) with these drugs for 3 days in storage bags. To determine the effect of storage, PCs were stored for 5 days, and aliquots removed on Days 1 through 5 were placed in platelet storage bags with 4 micrograms per mL of amphotericin B for 2 to 6 hours. Membrane glycoprotein expression was assessed by flow cytometry with fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed against the following antigens: GPIb (CD42b), CD63 (an activation protein), P-selectin (CD62), and GPIIb/IIIa (CD41a). RESULTS : Amphotericin B produced a concentration-dependent decrease in the surface binding of CD42b MoAb with no consistent changes in the binding of CD41a, CD63, or CD62 MoAbs after a 3-day exposure. Stored but not fresh PCs showed decreased binding of MoAb CD42b after a 6-hour exposure to amphotericin B (4 micrograms/mL). Fluconazole produced no changes. When the binding of MoAb CD42b to permeabilized platelets was used to measure total platelet content, amphotericin B (4 micrograms/mL) decreased MoAb CD42b binding to a similar degree in fresh and stored platelets. Inhibition of aggregation to ADP and collagen and ADP and epinephrine was seen in stored but not fresh PCs. CONCLUSION : Therapeutic levels of amphotericin B resulted in partial loss of total platelet GPIb in fresh and stored PCs, but decreased surface expression of platelet membrane GPIb only in stored platelets. This difference between fresh and stored platelets may be related to the limited reservoir of GPIb available for redistribution to the membrane in the previously stored PCs and may account for the decreased recovery of transfused platelets observed in some patients receiving amphotericin B.     

血小板膜糖蛋白表达变化在出血性血小板病患者与健康人之间的差异

背景:已证实出血性血小板病患者血小板聚集功能存在缺陷,血小板膜糖蛋白在血小板聚集过程中具有重要作用。目的:以健康人作为正常对照,观察出血性血小板病患者血小板膜糖蛋白表达的变化。设计:病例-对照分析。单位:华中科技大学同济医学院附属协和医院中西医结合科。对象:①选取2001-01/2003-03华中科技大学同济医学院附属协和医院中西医结合科和血液专科收治的79例出血性血小板病患者,男31例,女48例,平均年龄(35.76±14.14)岁,病程1～14年,符合1996年出血性血小板病的诊断标准,患者及其家属对本实验知情同意。共计167个出血部位,其中肢体皮肤瘀斑或瘀点64例,鼻出血33例,月经过多29例,齿龈出血28例,眼底出血8例,球结膜出血5例。②纳入标准:均有多部位出血临床表现;血小板计数、出血时间、凝血象检测无明显异常改变;根据出血部位,经内科、耳鼻喉科、妇科、口腔科和眼科等科室诊视,未发现特异诊断性病灶;血压、心率正常;经瑞斯托霉素检测,排除血小板无力症。③另选取健康献血员34例作为正常对照组,男15例,女19例,平均年龄(30.12±7.14)岁。方法:①血小板聚集率测定:以二磷酸腺苷(终浓度1.90μmol/L)、花生四烯酸(终浓度0.37μmol/L),血小板活化因子(终浓度150nmol/L)作为诱聚剂,采用Chronolog430型血小板聚集仪检测正常对照与出血性血小板病患者的血小板最大聚集率。血小板最大聚集率21%～40%为反应不良,低于20%为反应缺如。②血小板膜糖蛋白检测:患者取肘静脉血3.6mL,20g/L的乙二胺四乙酸二钠1:9抗凝,正常对照组标本以同法收集。分离血浆,收集血浆层,离心去上清,加多聚甲醛室温固定,调整血小板浓度为3×107L-1。取调整好的血小板溶液100μL,分别加入异硫氰酸荧光黄标记的CD42b(抗血小板膜糖蛋白Ⅰb)、CD41(抗血小板膜糖蛋白Ⅱb)、CD61(抗血小板膜糖蛋白Ⅲa)、CD42b/CD42a(抗血小板膜糖蛋白Ⅰb/Ⅸ)、CD41/CD61(抗血小板膜糖蛋白Ⅱb/Ⅲa)、CD62p(抗P-选择素)克隆抗体200μL,混匀后室温避光孵育30min,磷酸盐缓冲液补至终体积1mL,每个样本分析10000个血小板,于FACS420型流式细胞仪上测定荧光阳性百分标记率。主要观察指标:①血小板聚集率。②血小板膜糖蛋白的表达。结果:79例出血性血小板病患者与34例健康献血员全部进入结果分析。①每例出血性血小板病患者均对一项或多项诱聚剂诱导的聚集反应不良或缺如,而正常对照组血小板聚集检测均无异常发现。②与正常对照组比较,出血性血小板病患者血小板膜糖蛋白Ⅰb/Ⅸ,Ⅱb/Ⅲa,Ⅰb,Ⅲa及P-选择素的荧光阳性标记率均明显降低(t=2.50～5.57,P均<0.05);血小板膜糖蛋白Ⅱb荧光阳性标记率略微升高,但差异无显著性意义(t=0.86,P>0.05)。结论:血小板膜糖蛋白表达异常可能是导致出血性血小板病的因素之一。     

Platelet storage lesions: analysis of platelet membrane glycoproteins and platelet-derived microparticles by fluorescence-activated flow cytometry

We have used recently developed flow cytometric techniques in combination with specific monoclonal antibodies (MoAbs): (i) to study the membrane expression of two major platelet glyco-protein (GP) complexes, GP Ib-IX and GP IIb-IIIa, and the appearance of activation-dependent membrane epitopes, and (ii) to evaluate the relative proportion of platelet-derived microparticles and their GP pattern during storage of platelet-rich plasma. Binding of fluoresceinated (FITC) LJ-P3, an anti-GP Ibalpha MoAb, to platelets continuously decreased by 50% during storage for 6 days. Binding of FITC-LJ-P4, directed to the GP IIb-IIIa complex on the platelet surface, also decreased during day 1-3, but increased again to baseline levels from day 4-6 of storage. The re-increase of GP IIb-IIIa was associated with the exposure of secretion-dependent granule membrane proteins, GMP-140 and GP-53, and the presence of thrombospondin at the platelet surface. These neoantigens are indicative of platelet activation. Moreover, the proportion of platelet-derived microparticles increased from 6 to 22% (p less than 0.001), whereby a predominant subpopulation of GP Ib-negative microparticles was identified. Thus, significant changes in platelet membrane GP occur during standard platelet preservation. These changes, resulting from time-dependent platelet activation and/or proteolysis in vitro may affect platelet GP receptor function upon transfusion.