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。

Comparison of the changes in platelet membrane glycoproteins between patients with hemorrhagic thrombopathy and healthy people

BACKGROUND: It has been confirmed that platelet aggregation defect presents in patients with hemorrhagic thrombopathy, and platelet membrane glycoproteins play a significant role in the process of platelet aggregation.OBJECTIVE: To observe the expression changes of platelet membrane glycoproteins between patients with hemorrhagic thrombopathy and healthy people.DESIGN: Case-control analysis.SETTING: Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College,Huazhong University of Science and Technology.PARTICIPANTS :① Seventy-nine patients with hemorrhagic thrombopathy who received treatment from January 2001 to March 2003 in the Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were enrolled in this study. The patients, including 31 male and48 femlae, averaged (35.76±14.14)years old. They all agreed to participate in this study and met with the diagnosis of hemorrhagic thrombopathy revised in 1996. Informed consents were obtained from all the patients and their relatives.The course of multiple bleeding symptoms was 1 to 14 years. And the bleeding sites were totally 167: extremity petechia was found in 64 cases, rhinorrhagia in 33 cases, hypermenorrhea (more than 150 mL in each cycle) in 29 cases, gingival bleeding in 28 cases, ocular fundus bleeding in 8 cases and conjunctival hemorrhage in 5 cases.②lnclusive criteria: All the cases presented clinical manifestation of multiple focal bleeding; The laboratory tests including platelet counts,bleeding time and coagulation profiles were examined with no marked abnormal changes; According to hemorrhage sites, the patients were examined by specialists of internal medicine,otorhinolaryngology, gynecology, stomatology and ophthalmology, and no specific diagnostic focuses of infection were found; They had normal blood pressure and heart rate, and those with thrombocytasthenia had been ruled out through ristocetin test. ③Another 34 healthy volunteer donors, 15 males and 19 females with an average age (30.12±7.14) years,were selected as normal control group.METHODS: ① Detection of platelet aggregation ratio: Adenosine diphosphate (ADP, final concentration 1.90 μmol/L),arachidonic acid(AA, final concentration 0.37 μmol/L),and platelet activating factor(PAF, final concentration 150 nmol/L)were used as aggregation inductors, and Chronolog 430 type aggregometry was used to detect platelet maximum agglutination ratio. When maximum agglutination ratio was within 21％ and 40％, it was considered as aggregation defect, and when below 20％, it was considered as aggregation absence.② Test of platelet membrane glycoprotein: 3.6 mL cubital venous blood was drawn from the patients and mixed with 20 g/L disodium ethylenediamine tetraacetic acid(EDTA-NA2) for the purpose of anticoagulation at 1:9, and the same procedure was performed on the blood samples from the normal control group. The plasma was isolated and collected, after being centrifuged, the supernatant liquid was discarded, and paraform was added to fix the rest of the platelet solution. The platelet concentration was adjusted to 3×107 L-1. 100 μL regulated platelet solution was extracted, and fluorescein isothiocyanate (FITC)-marked CD42b (anti-GP Ⅰ b),CD41 (anti-GP Ⅱ b), CD61 (anti-GP Ⅲ a), CD42b/CD42a (anti-GP Ⅰ b/Ⅸ ), CD41/ CD61 (anti-GP Ⅱ b/Ⅲ a) and CD62p(anti-P-selectin)clonal antibodies 200 μL were added respectively and well mixed, and then cultured for 30 minutes at room temperature away from light. Finally phosphate buffer solution was added to the solution till the volume reached 1 mL,and FACS420 flow cytometer was used to analyze the samples, one sample for 10000 platelets, and the results were demonstrated by positive fluorescence percentage.MAIN OUTCOME MEASURES: ① Platelet aggregation ratio. ② Expression of platelet membrane glycoproteins.RESULTS: All the patients and the healthy subjects were involved in the analysis of results. ①All the cases were found to have platelet aggregation defect or absence which could be induced by single or more platelet aggregating agents. No abnormal platelet aggregations were found in the control group. ② The fluorescence intensities of GPIb/Ⅸ, GP Ⅱ b/Ⅲa complexes, GPIb, GPⅢa and P-selectin in patients with hemorrhagic thrombopathy were lower than those in the healthy subjects (t =2.50-5.57,all P ＜ 0.05). The fluorescence intensity of GP Ⅱb in patients with hemorrhagic thrombopathy was slightly higher than that in the healthy subjects, but no significant difference was found. (t =0.86, P ＞0.05).CONCLUSION: Abnormal expressions of platelet membrane glycoproteins may be partial pathogenesis of hemorrhagic thrombopathy.