共价交联的肝素/海藻酸盐水凝胶表面修饰膨体聚四氟乙烯人工血管**☆

背景：虽然膨体聚四氟乙烯人工血管植入体具有易于缝合、质地柔软和抗压迫等诸多优良性能，但由于血栓形成等原因，使这些材料的应用受限。为了解决前述问题，目前的工作主要集中在对现有人工血管材料表面修饰与改性上，最终使其达到血管植入的要求。 目的：用共价交联的肝素-海藻酸钠水凝胶对小口径膨体聚四氟乙烯人工血管进行表面修饰和改性，考察其血液相容性和组织相容性。 设计：观察性实验。 单位：哈尔滨工业大学生物医学工程中心，纳米医药与生物传感器实验室。 材料：实验所用直径4 mm的膨体聚四氟乙烯人工血管为W.L Gore & Associates, Inc.产品，海藻酸钠和1-乙基-3-3-二甲基氨丙基碳化二亚胺购自美国Sigma公司，肝素购于Calbiochem公司，全氟磺酸和壳聚糖购于美国Aldrich公司。人α-凝血酶和抗凝血酶III购于Haematologic Technologies，S-2238购于Chromogenix。 方法：实验于2006-05/2007-06在哈尔滨工业大学生物医学工程中心的纳米医药与生物传感器实验室完成。首先用全氟磺酸修饰膨体聚四氟乙烯表面，然后用肝素-海藻酸钠凝胶进行灌注修饰，以乙二胺为交联剂，1-乙基-3-3-二甲基氨丙基碳化二亚胺为引发剂，将多糖分子进行共价交联。用接触角表征了涂层前后人工血管表面亲水性能的变化，扫描电镜表征了材料表面形貌及血小板黏附，衰减全反射-傅立叶变换红外光谱表征了材料表面的化学结构，然后用活化部分凝血激酶时间、凝血酶原时间、溶血试验以及凝血酶失活试验表征了涂层后人工血管表面的血液相容性。 主要观察指标：①接触角。②用扫描电镜表征材料表面形貌及血小板黏附情况。③衰减全反射-傅立叶变换红外光谱。④活化部分凝血激酶时间、凝血酶原时间。⑤溶血度。⑥凝血酶失活试验。 结果：① 修饰后的人工血管，衰减全反射-傅立叶变换红外光谱结果显示在1 626 cm-1处出现了-CO-NH-基团的峰位。②修饰后人工血管的接触角由（125±1）°降低为（84±2）°。③修饰后的人工血管，具有较长的活化部分凝血激酶时间和凝血酶原时间、较低的溶血度0.065%、较少数量的血小板黏附。④凝血酶失活实验结果显示，凝胶灌注修饰后的人工血管，对凝血酶的活性有较强的抑制作用，因此具有血栓形成的性能且稳定性好。 结论：肝素-海藻酸钠凝胶修饰的膨体聚四氟乙烯具有良好血液相容性及组织相容性，可应用于小口径人工血管。     

碳纤维增强聚醚醚酮与钛合金血液相容性的比较

背景：碳纤维增强聚醚醚酮是聚醚醚酮的改进复合材料,改善了纯聚醚醚酮强度不足的缺点,扩大了其在各方面的使用范围。但若要成为应用于人体内的医用材料,必须具有良好的血液相容性。 目的：比较碳纤维增强聚醚醚酮和钛合金对人血细胞的影响,判断碳纤维增强聚醚醚酮是否具有良好的血液相容性。 设计、时间及地点：对比观察实验,于2008-06/08在吉林大学第三临床医学院中心实验室完成。 材料：取实心钛合金股骨干髓内针1根及含20%碳纤维的碳纤维增强聚醚醚酮样条若干,机加工成棒形试样（长5 mm,直径1 mm）各20个。 方法：在相同条件下,将钛合金和碳纤维增强聚醚醚酮2种材质的试件分别放置于新鲜人血中,通过观察溶血率,血小板激活程度以及白细胞激活程度判断材料的血液相容性。 主要观察指标：应用游离血红蛋白直接测定法测量材料溶血率;流式细胞术测量血小板CD62p、CD63含量和白细胞表面黏附分子CD11b/CD18表达。 结果：2种材料溶血率,血小板CD62p含量、CD63含量、白细胞表面黏附分子CD11b/CD18表达水平差异无显著性意义（P > 0.05）。 结论：碳纤维增强聚醚醚酮和钛合金对人血红细胞、白细胞和血小板无显著影响,均具有良好的血液相容性。     

膨体聚四氟乙烯人工血管；共价交联；抗凝血表面修饰；血液相容性

背景：小口径（直径小于6 mm）膨体聚四氟乙烯人工血管由于血栓形成和内膜增生等原因，在冠状和外周血液循环中的应用受到了限制，而生物活性表面肝素涂层是解决上述问题的一条有效途径。 目的：为了提高膨体聚四氟乙烯人工血管的抗凝血性能，采用新型肝素涂层——共价交联的聚乙烯醇/重氮树脂/肝素凝胶修饰小口径膨体聚四氟乙烯人工血管以提高其通畅性，并观察其血液相容性。 设计、时间及地点：观察性实验，于2006-05/2007-06在哈尔滨工业大学生物医学工程中心的纳米医药与生物传感器实验室完成。 材料：膨体聚四氟乙烯人工血管（直径4 mm），全氟磺酸和聚乙烯醇购于美国Aldrich公司，肝素购于Calbiochem公司（MW12 000~ 14 000），重氮树脂由实验室自己合成。 方法：①用全氟磺酸修饰膨体聚四氟乙烯表面。②用聚乙烯醇/重氮树脂/肝素凝胶进行灌注修饰，在紫外光照射的条件下，重氮树脂作为交联剂，将肝素和聚乙烯醇分子进行共价交联。 主要观察指标：①接触角。②衰减全反射-傅里叶变换红外光谱表征材料表面的化学结构。③活化部分凝血激酶时间、凝血酶原时间。④溶血试验。⑤血小板黏附试验。⑥凝血酶失活试验。 结果：①修饰后的人工血管的接触角降低，衰减全反射-傅立叶变换红外光谱显示在2 172 cm-1和2 224 cm-1处出现了重氮基团的特征峰位。②与未修饰的人工血管相比，修饰后的人工血管具有较长的活化部分凝血激酶时间和凝血酶原时间、较低的溶血度、很少数量的血小板黏附。③与未修饰的人工血管相比，修饰后的人工血管对凝血酶的活性有较强的抑制作用，且涂层稳定。 结论：聚乙烯醇/重氮树脂/肝素凝胶形成的肝素涂层不仅具有很好的抗血栓形成的性能，而且具有较低的溶血度，因此显著提高了人工血管的血液相容性。     

壳聚糖球形多孔微载体的血液相容性评价****☆

摘要 背景：文献报道的微载体大多为实心的、大孔的,虽然较二维微载体的比表面积有明显的增加,但距理想的三维微环境相差甚远。 目的：构建壳聚糖球形多孔微载体,通过溶血实验、凝血实验、血小板计数及聚集实验评价其血液相容性。 方法：利用液氮冷冻干燥技术成功构建浓度为1%,2%,3%的壳聚糖球形多孔微载体。选择健康成年新西兰兔为宿主,采用溶血实验、凝血实验、血小板计数及其聚集实验评价壳聚糖球形多孔微载体的血液相容性。 结果与结论：浓度为1%,2%,3%的壳聚糖球形多孔微载体的溶血率分别为1.56%,2.07%,2.31%,均小于5%,均无致溶血性;3种浓度壳聚糖球形多孔微载体样本材料对兔血时间无明显影响,三者与生理盐水阴性对照组间也无明显差别(P > 0.05);3种浓度壳聚糖球形多孔微载体样本材料对兔血小板计数无明显影响,注入浸提液前后比较和组间比较差异均无显著性意义(P > 0.05)。证实壳聚糖球形多孔微载体无致溶血性、无凝血性和无血小板聚集性,表明壳聚糖球形多孔微载体具有良好的血液相容性。 关键词：人工肝脏;壳聚糖;微载体;血液相容性;组织工程 doi:10.3969/j.issn.1673-8225.2011.03.006     

低温等离子体肝素化改性后小肠黏膜下层的血液相容性及其构建小口径血管的可行性*★

背景：栓塞是小口径血管植入后失败的主要原因，目前常常对血管支架材料进行抗凝改性，以期提高其血液相容性，从而提高血管的有效通畅性。 目的：观察低温等离子体肝素化改性后的内植物修复材料小肠黏膜下层的血液相容性，并探讨其体内构建小口径血管的可行性。 设计：单一样本实验。 单位：上海交通大学附属第六人民医院骨科、上海市四肢显微外科研究所。 材料：实验于2006-01/10在上海市四肢显微外科研究所实验室进行。小肠黏膜下层来自农场猪。 方法：①改性：将猪小肠黏膜下层用氩等离子体处理器照射处理，氩气流量20 mL/min，照射时间分别为0, 2, 4, 6, 8, 10, 12, 14 s，接着浸入肝素钠溶液24 h。②体内抗凝血实验：将20条狗分为2组，分别植入经过改性或未改性的小肠黏膜下层缝合成的3 mm口径血管支架，与股动脉直接吻合，观察6周。 主要观察指标：①血液相容性检测：通过扫描电镜观测表面形态，并通过液滴接触角、凝血时间及血小板黏附实验检测小肠黏膜下层改性前后的抗凝性。②体内抗凝血：通过彩色多普勒和组织学检测，评价血管支架直接在体内循环血流下的长期通畅性和形成血管的可行性。 结果：①改性小肠黏膜下层膜表面呈现出均匀的微结构改变，随着等离子体照射时间增加，表面液滴接触角降低；改性后凝血酶原时间、活化部分凝血活酶时间和凝血酶时间延长；血小板黏附减少。②植入体内后未改性小肠黏膜下层血管支架3 d内栓塞，改性组在6周内仍保持通畅，管腔内表面有完整内皮细胞覆盖。 结论：经低温等离子体肝素化改性后小肠黏膜下层的亲水性、抗凝性有明显提高。     

聚乳酸乙醇酸/RNAⅢ抑制肽缓释微球的血液相容性*☆

目的：研究表明RNA Ⅲ抑制肽(RNA Ⅲ inhibiting peptide,RIP)是葡萄球菌一种有效的全面抑制剂，作用机制与传统抗菌素明显不同，有着良好的应用前景。通过实验评价聚乳酸乙醇酸(polyaiticglycolic acid,PLGA)/RIP缓释微球的血液相容性。 方法: 实验于2005-10/2007-10在解放军总医院临床药理研究所及医学动物实验中心完成。选择成年健康新西兰大白兔30只，按随机数字表法分组，每组6只。药品及试剂: PLGA，二甲基亚砜、MTT，DMEM，二硝基氟苯。实验方法：①PLGA/RIP微球制备：采用Fmoc法由C端至N端先合成粗品肽；采用反相液相色谱法对RIP粗品进行纯化分析,按紫外吸收峰收集组分,冷冻干燥,得到RIP纯品。再采用液相复乳法制备直径50～70 mm的PLGA/RIP微球。②洗提液制备：PLGA/RIP微球粉末按1 g/L在37 ℃无菌条件下用生理盐水洗提72 h，制得洗提液原液；加入同体积的无菌生理盐水制得0.5 g/L的稀释液。③溶血实验：以蒸馏水和生理盐水分别为阳性、阴性对照，观察PLGA/RIP洗提液原液和0.5 g/L洗提液的溶血率。④凝血实验及PLGA/RIP对凝血酶原时间和活化部分凝血酶时间的影响实验：以生理盐水为阴性对照，观察PLGA/RIP洗提液原液和0.5 g/L洗提液对兔凝血时间的影响和凝血酶原时间和活化部分凝血酶时间的影响。⑤PLGA/RIP对兔白细胞、红细胞和血小板及血小板聚集的影响实验：观察PLGA/RIP洗提液原液和0.5 g/L洗提液对兔白细胞、红细胞和血小板及血小板聚集的影响。 结果：纳入动物30只, 均进入结果分析。①溶血实验结果显示PLGA/RIP洗提液原液和0.5 g/L洗提液的溶血率分别为3.24%和2.67%，两者的溶血率均 < 5%，符合医用生物材料的溶血实验要求。②凝血实验结果表明PLGA/RIP洗提液原液和0.5 g/L洗提液对兔凝血时间无明显影响。③PLGA/RIP对各时间点兔凝血酶原时间和活化部分凝血酶时间均无明显作用。④PLGA/RIP对兔白细胞、红细胞和血小板无明显影响。⑤PLGA/RIP对兔血小板聚集无明显影响。 结论: PLGA/RIP缓释微球具有良好的血液相容性。     

不同透析膜材料在维持性血液透析过程中的生物相容性

摘要 目的：对聚砜膜、血仿膜和醋酸纤维膜在维持性血液透析中的应用进行综合评价，以选择适宜的透析膜材料进行血液透析。 方法：电子检索中国期刊全文数据库(CNKI：1990/2008)，以“透析膜，相容性，并发症”为检索词收集关于不同透析膜材料的临床应用文献共50篇，排除综述类文献、实验研究及透析膜清洗等文献，共纳入22篇关于不同透析膜溶质清除率及生物相容性评价的文献，对不同材料的血液透析膜的临床应用情况进行全面总结。 结果：通过对聚砜膜、血仿膜和醋酸纤维膜在维持性血液透析中的溶质清除率的分析得出，聚砜膜、血仿膜对全段甲状旁腺激素、血磷、血钙的清除效果优于醋酸纤维膜，对于B细胞活化的血清CD23清除效果优于醋酸纤维膜，对血小板活化程度的影响弱于醋酸纤维膜。高通透量的聚砜膜不增加炎症反应，醋酸纤维膜透析的P选择素的水平高于聚砜膜和血仿膜，3种透析膜的血清胱抑素C清除率无差异。 结论：聚砜膜、血仿膜和醋酸纤维膜对血清CD23浓度、血小板活化程度、炎症反应、出凝血并发症、血清P选择素水平均的影响与其生物相容性有关:生物相容性越好，对血清CD23浓度水平、血小板活化程度、炎症反应、出凝血并发症、血清P选择素水平的影响越小，反之则大，聚砜膜和血仿膜生物相容性好于醋酸纤维膜，血仿膜的血液透析并发症最少。 关键词：聚砜膜；血仿膜；醋酸纤维膜；血液透析；溶质清除率；生物相容性；并发症 doi:10.3969/j.issn.1673-8225.2010.34.045     

聚乳酸乙醇酸/RNAⅢ抑制肽缓释微球的血液相容性*☆

目的：研究表明RNA Ⅲ抑制肽(RNA Ⅲ inhibiting peptide,RIP)是葡萄球菌一种有效的全面抑制剂,作用机制与传统抗菌素明显不同,有着良好的应用前景。通过实验评价聚乳酸乙醇酸(polyaiticglycolic acid,PLGA)/RIP缓释微球的血液相容性。 方法: 实验于2005-10/2007-10在解放军总医院临床药理研究所及医学动物实验中心完成。选择成年健康新西兰大白兔30只,按随机数字表法分组,每组6只。药品及试剂: PLGA,二甲基亚砜、MTT,DMEM,二硝基氟苯。实验方法：①PLGA/RIP微球制备：采用Fmoc法由C端至N端先合成粗品肽;采用反相液相色谱法对RIP粗品进行纯化分析,按紫外吸收峰收集组分,冷冻干燥,得到RIP纯品。再采用液相复乳法制备直径50～70 mm的PLGA/RIP微球。②洗提液制备：PLGA/RIP微球粉末按1 g/L在37 ℃无菌条件下用生理盐水洗提72 h,制得洗提液原液;加入同体积的无菌生理盐水制得0.5 g/L的稀释液。③溶血实验：以蒸馏水和生理盐水分别为阳性、阴性对照,观察PLGA/RIP洗提液原液和0.5 g/L洗提液的溶血率。④凝血实验及PLGA/RIP对凝血酶原时间和活化部分凝血酶时间的影响实验：以生理盐水为阴性对照,观察PLGA/RIP洗提液原液和0.5 g/L洗提液对兔凝血时间的影响和凝血酶原时间和活化部分凝血酶时间的影响。⑤PLGA/RIP对兔白细胞、红细胞和血小板及血小板聚集的影响实验：观察PLGA/RIP洗提液原液和0.5 g/L洗提液对兔白细胞、红细胞和血小板及血小板聚集的影响。 结果：纳入动物30只, 均进入结果分析。①溶血实验结果显示PLGA/RIP洗提液原液和0.5 g/L洗提液的溶血率分别为3.24%和2.67%,两者的溶血率均 < 5%,符合医用生物材料的溶血实验要求。②凝血实验结果表明PLGA/RIP洗提液原液和0.5 g/L洗提液对兔凝血时间无明显影响。③PLGA/RIP对各时间点兔凝血酶原时间和活化部分凝血酶时间均无明显作用。④PLGA/RIP对兔白细胞、红细胞和血小板无明显影响。⑤PLGA/RIP对兔血小板聚集无明显影响。 结论: PLGA/RIP缓释微球具有良好的血液相容性。     

高分子材料聚砜膜滤器临床应用及相容性评价

摘要 目的：评价高分子材料聚砜膜滤器的性能以及置入体内与宿主的生物相容性,为其在临床上的应用提供参考依据。 方法：以“血液透析;透析膜材料;组织相容性;血液相容性;聚醚砜”为中文关键词;以“hemodialysis, dialysis membrane materials;histocompatibility;blood compatibility;polyethersulfone”为英文关键词,采用计算机检索2006-01/2010-03相关文章。纳入与有关生物材料与宿主相容性的文章;排除重复研究或Meta分析类文章。以22篇文献为重点进行探讨聚砜膜滤器材料的性能及应用前景。 结果：目前各种新型的膜材料在不断地研发,部分已在临床使用或即将问世,如维生素E 修饰的透析膜、多黏菌素B 修饰的透析膜、甲壳素膜、以及人肾单位透析器和生物活性膜等。聚砜膜滤器由于具有较好的生物相容性和封堵效果,在临床广泛应用。 结论：聚砜膜滤器通过高分子材料滤器强大的对流、黏附作用,高效清除组织损伤过程中产生的炎症递质和毒性代谢产物,排除机体内潴留的多余水分,维持水、电解质、酸碱平衡,实现内环境的稳定,改善重要脏器功能,调节免疫系统。聚砜膜滤器目前虽广泛应用,但不可避免存在着相容不良,引发附近组织发炎,产生病变,造成溶血或凝血现象等。理想的膜滤器材料无论材料本身还是其降解产物都不能产生炎症和毒性反应,所以良好生物相容性的封堵器需进一步开发研制。 关键词：血液透析;透析膜材料;组织相容性;血液相容性;聚醚砜 doi:10.3969/j.issn.1673-8225.2010.42.032     

肝素化纳米材料P(LLA-CL)的制备及其表征和抗凝血性特征

目的：采用等离子体技术引发表面接枝肝素方法，使纳米材料P（LLA-CL）表面肝素化，以提高材料的抗凝血性。 方法：制备静电纺高聚物P(LLA-CL)纳米纤维，采用等离子体技术引发纳米材料P(LLA-CL)表面肝素化，对其表面进行改性，并测试其力学性能，同时对肝素化材料进行了接触角测试观察其表面亲水性，傅里叶全反射红外光谱分析进行表面分析，扫描电镜观察纤维形态，EDS能谱对表面元素组成进行表征，血液相容性实验评价肝素化P（LLA-CL）材料表面的血液相容性。 结果：采用等离子体技术能够成功将肝素接枝到P(LLA-CL)表面。等离子体处理后P(LLA-CL)材料表面接触角显著减小，亲水性增强；等离子引发肝素化后，材料仍能保持良好的机械力学性能；红外光谱、扫描电镜和EDS结果表明等离子体引发肝素成功接枝到P(LLA-CL)表面；血液相容性实验显示肝素化材料复钙时间全血凝血时间明显延长。 结论：材料在等离子体肝素化改性处理后抗凝血性能改善，血液相容性提高。     

Platelet membrane early activation markers during prolonged storage

The relationship between platelet aging and early markers of membrane activation have not been defined clearly. Activation markers expressed during prolonged storage are similar if not identical to those that appear after exposure to thrombin. Using flow cytometry, we investigated platelet membrane expression of CD62P, CD63, and annexin V binding (i.e., loss of membrane asymmetry) in platelets stored for up to 11 days under standard blood banking conditions. We compared five apheresis platelets to two random donor platelet concentrates, and to one pooled platelet preparation from six single platelet concentrates before and after exposure to thrombin. CD62P, CD63 expression, and annexin V binding increased during storage albeit with different kinetics. The differential increments observed between resting and thrombin (1 unit/ml) activated platelets showed an inverse correlation to storage time for CD62P, CD63, and annexin V binding, which was identical to published survival curves. A difference between apheresis platelets and platelet concentrates was observed only on day 1. Our data indicate that the in vitro platelet reserve activity to thrombin activation mirrors that of radiolabeled platelet survival in vivo and that platelet cross-sectional residual life span can explain their diminished capacity to respond to thrombin as a function of viability.     

Multicolor flow cytometry for evaluation of platelet surface antigens and activation markers

Introduction

Flow cytometry allows the analysis of multiple antigens in a single tube at a single cell level. We present a rapid and sensitive two tube flow cytometric protocol for the detection of multiple platelet antigens and activation markers gated on a pure platelet population.

Materials and methods

The presence of platelet specific antigens was analyzed in citrated whole blood of normal platelets and from patients diagnosed with platelet abnormalities. Quiescent platelets as well as stimulated platelets were analyzed using a gating strategy based on ubiquitously expressed platelet membrane markers.A ubiquitously expressed platelet marker was combined with antibodies against the activated alpha2b-beta3 (PAC-1), Lysosomal Activated Membrane Protein (CD63) and P-selectin (CD62P).

Results

We were able to detect the platelet antigens CD36, CD41, CD42a, CD42b and CD61 in one single tube. Our approach allowed the single tube determination of PAC-1, CD63 and CD62P after activation of platelets by thrombin, collagen, ADP and PAR-1, and determination of platelet abnormalities.

Conclusions

Our two tube multi-parameter screening protocol is suited for the analysis of platelet antigens expressed on quiescent and activated platelets and allows the detection of aberrancies as found in blood of patients with thrombocytopathy such as Glanzmann Thrombasthenia, storage pool disease with diminished granule content and patients treated with clopidogrel and acetylsalicylic acid.     

Changes of Platelet Surface Antigens in Patients Suffering from Abdominal Septic Shock

Sepsis and related syndromes account for a high morbidity and mortality caused by the development of multiorgan failure. Pathogenesis of sepsis is complex, involving humoral as well as cellular factors. Since the role of platelets is still undefined in this concern, we investigated CD63, CD62P, CD36, and CD31 expression on platelets of patients in septic shock (n=18) using a flow cytometric assay in whole blood. Samples were drawn within 24 hours of onset. We found thrombocytopenia accompanied by a significantly higher expression of CD63, CD62P, and CD31 and a significant downregulation of CD36 in comparison to healthy volunteers (n=18). Changes in CD63 and CD62P expression indicates platelet activation. Because CD62P, CD36, and CD31 mediate interaction of platelets with leukocytes, subendothelial matrix and probably endothelial cells as well as platelet adhesion/aggregation, our findings suggest an involvement of platelets in leukocyte/endothelial cell interaction in septic shock. We suspect that thrombocytopenia is not due to bone marrow depression, but rather is due to consumption of highly activated platelets in the microcirculation. We feel that our observations may offer a rationale for potentially beneficial effects of antiplatelet therapy in sepsis; however, further studies have to evaluate its beneficial impact as well as its potential risk for bleeding complications.     

冰冻单采血小板在外周血干细胞移植中的应用：与输注新鲜单采血小板

背景：由于实行义务献血后血小板来源紧张,输注新鲜单采血小板有时难以保证。而冰冻单采血小板有较好的即刻止血效果,可用于各种低血小板患者的止血。 目的：探讨冰冻单采血小板输注在外周血干细胞移植中的替代疗效。 方法：44例血液病或淋巴瘤患者均接受外周血干细胞移植,血小板计数低于40×109 L-1,随机分为新鲜血小板组、冰冻血小板组,22例/组。外周血干细胞移植后0~14 d,新鲜血小板组患者直接输注经专用白细胞滤器过滤后的新鲜单采血小板;冰冻血小板组患者将同型冰冻单采血小板取出后,放入37 ℃水浴迅速融化,用白细胞滤器过滤后,于40 min内输注。一般每3 d输注1次,10 U/次,共输入3~16次,输入次数根据患者出血情况酌情增减。 结果与结论：与新鲜血小板组比较,冰冻血小板组输注后24 h血小板及凝血四项指标无明显变化(P > 0.05);输注后48 h血小板计数显著降低(P < 0.01);输注后72 h出血时间、凝血酶原时间、活化部分凝血活酶时间及血小板计数均显著降低(P < 0.05或 P < 0.01),凝血酶时间及纤维蛋白原无明显差异(P > 0.05)。建议在外周血干细胞移植中,应用冰冻单采血小板防止出血输注时间为2 d输注1次。     

CD63 associates with the alphaIIb beta3 integrin-CD9 complex on the surface of activated platelets

The tetraspanins are integral membrane proteins expressed on cell surface and granular membranes of hematopoietic cells and have been identified in multi-molecular complexes with specific integrins. In resting platelets, CD63, a member of the tetraspanin superfamily, is present in dense granule and lysosomal membranes and, following platelet activation, translocates to the plasma membrane. In the present study, platelet activation by thrombin leads to incorporation of CD63 into the Triton-insoluble actin cytoskeletal fraction. This incorporation was inhibited by preincubation of platelets with RGDS or EGTA and did not occur in platelets from a patient with Glanzmann's thrombasthenia, suggesting that it was dependent upon alphaIIbbeta3. In activated platelets, the anti-CD63 MoAb, D545, co-immunoprecipitated CD63 with other surface-labeled proteins, including alphaIIbbeta3 and another tetraspanin, CD9. The association of CD63 with CD9 and alphaIIbbeta3, was not inhibited by preincubation of platelets with RGDS or EGTA. D545 did not inhibit the adhesion of activated platelets to purified extracellular matrix proteins, but significantly decreased adhesion of thrombin-activated platelets to neutrophils in a rosetting assay. D545 also caused disaggregation of platelets stimulated by ADP, but had no effect on aggregation induced by other agonists. These results are consistent with the proposal that CD63 becomes part of an alphaIIbbeta3-CD9-CD63 integrin-tetraspanin complex in activated platelets--an association that may modulate the function of alphaIIbbeta3-dependent interaction with other cells such as neutrophils.     

CD63 modulates spreading and tyrosine phosphorylation of platelets on immobilized fibrinogen

CD63 is a member of the tetraspanin superfamily of integral membrane proteins. Present on a variety of cells, tetraspanins can form lateral associations with integrins and may act as 'organizers' of multimolecular networks that modulate integrinmediated signaling, cell morphology, motility and migration. In resting platelets, CD63 is present on the membranes of dense granules and lysosomes but relocates to the plasma membrane following platelet activation and exocytosis where it associates with the platelet integrin alphaIIBbeta3-CD9 complex and with the actin cytoskeleton in an alphaIIBbeta 3-dependent manner. D545, a monoclonal antibody directed at the second extracellular loop of CD63,was used to investigate the role of CD63 in platelet adhesion, spreading and tyrosine phosphorylation. Using immunofluorescence microscopy and confocal imaging, we have demonstrated that D545 does not alter adhesion of platelets to immobilized fibrinogen, but instead platelet spreading. In the presence of buffer or non-specific mouse IgG, activated platelets showed fully spread morphology, F-actin reorganization, redistribution of vinculin and extensive tyrosine phosphorylation, all of which were inhibited by D545. D545 also inhibited the phosphorylation of focal adhesion kinase in thrombin-activated adherent platelets. These results suggest that CD63 may modulate alphaIIBbeta3-dependent cytoskeletal reorganization. To identify signaling enzymes associated with CD63 that could affect this pathway, lipid kinase assays were performed on D545 immunoprecipitates. CD63 co-immunoprecipitated with a lipid kinase which, on the basis of enzymatic properties(stimulated by nonionic detergents, inhibited by adenosine), is consistent with PI 4-kinase type II. The CD63-PI 4-kinase complex was not activation-dependent as the constituents were co-purified from both resting and activated platelets. The linkage of CD63 with PI 4-kinase may result in the recruitment of this signaling enzyme to specific membrane locations in the platelet where it influences phosphoinositide-dependent signaling and platelet spreading.     

Platelet tetraspanin complexes and their association with lipid rafts

Tetraspanins are a superfamily of integral membrane proteins that facilitate the organization of membrane and intracellular signaling molecules into dynamic signaling microdomains, tetraspanin-enriched microdomains (TEMs). Four tetraspanin family members have been identified in platelets: CD9, CD151 and TSSC6, which are constitutively associated with alphaIIbbeta3, and CD63, which is present on granule membranes in resting platelets and associates with alphaIIbbeta3-CD9 following platelet activation. CD63 and CD9 associate with a type II phosphatidylinositol 4-kinase, PI4K55, in both resting and activated platelets. Immunoelectron microscopic studies showed co-localization of CD63 and PI4K55 on internal membranes of resting platelets and on the filopodia of thrombin-activated platelets. Because TEMs in malignant cell lines appear to be distinct from prototypic lipid rafts, this study examined whether CD63-PI4K55 and CD9-PI4K55 complexes were resident in platelet-lipid rafts, or formed distinct microdomains. CD63, CD9 and PI4K55 were recovered from low-density membrane fractions (LDMFs) of sucrose gradients following platelet lysis in Brij 35, but unlike lipid-raft proteins were not insoluble in Triton X-100, being absent from LDMFs of platelets lysed with Triton. Incubation of platelets with methyl-beta-cyclodextrin, to deplete cholesterol and disrupt lipid rafts, shifted the complexes to higher density sucrose gradient fractions, but did not disrupt the tetraspanin-PI4K55 complexes. These results demonstrate that tetraspanin complexes in platelets form cholesterol-associated microdomains that are distinct from lipid rafts. It is probable that TEMs and lipid rafts associate under certain conditions, resulting in the close proximity of distinct sets of signaling molecules, facilitating signal transduction.     

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。     

Activated platelets in transient global amnesia and TIA

To assess whether platelets are activated in transient global amnesia (TGA) and TIA, blood samples were analyzed by fluorescence-activated cytoscan using antibodies specific for platelet fibrinogen receptor (PAC1) and P-selectin (CD62P). Samples from TIA contained high levels of CD62P compared with age-matched control subjects, whereas those from TGA did not. The authors suggest that activated platelets are involved in brain ischemia, whereas ischemia appears not to be associated with most TGA.