整合素αⅡb/β3在血小板双向信号转导中的作用

整合素αⅡb／β3是血小板表面表达最丰富的膜糖蛋白，不仅通过与配体的结合介导血小板的聚集、黏附、伸展，而且通过信号转导参与血小板的活化，在止血和血栓形成的过程中起重要作用。对整合素αⅡb／β3的结构及其参与双向信号转导的认识近几年有了较大进展。     

血小板活化标志物的研究进展

正常情况下血小板以分散状态在血管内运行,处于静息状态;当血管损伤、血流改变或受到化学物质刺激时血小板被活化而发生一系列改变。血小板活化是许多疾病的发病机制之一,包括：动脉硬化症、冠状动脉疾病、脑血管疾病及糖尿病等,吸烟、高血压及口服避孕药等都可诱发血小板活化。血小板活化包括血小板形态的改变、血小板聚集及血小板成分的释放。现就血小板活化时释放的分子标志物进行综述。     

利用显性阴性模型对整合素αⅡbβ3介导的信号转导的初步研究

本研究目的是明确整合素β3亚单位胞浆段序列在整合素αⅡbβ3介导的信号转导中的重要作用,并进一步了解在无αⅡb亚单位存在的条件下其对信号转导及特异性的影响程度。运用由IL-2R胞外段和跨膜段（Tac）与整合素β3胞浆段组成的融合蛋白（Tac／β3）,使其在表达GPIbIX、整合素αⅡbβ3的CHO细胞株（1bIX／ⅡbⅢa—CHO细胞株）中稳定表达（即得到IbIX／ⅡbⅢa—CHO／Tac-762细胞）,并进行多项试验,包括在固相纤维蛋白原的伸展、稳定黏附试验、细胞纤维蛋白凝块回缩功能试验以及游离纤维蛋白原结合试验（分别代表了外向内及内向外信号转导事件）。结果表明,在稳定表达有Tac／133的IbIX／ⅡbⅢa—CHO／Tac-762细胞中αⅡbβ3介导的双向信号转导严重受到抑制。结论：Tac／β3可以在IbIX／ⅡbⅢa—CHO／Tac-762细胞中发挥显著有效的显性阴性作用,并且证实整合素p3亚单位胞浆段的单独存在即可影响由αⅡbβ3介导的双向信号转导。     

血小板膜糖蛋白Ⅱb／Ⅲa基因多态性与血小板输注疗效的关系

目的通过对98例患者血小板膜糖蛋白（GP）Ⅱb／Ⅲa基因多态性及血小板抗体的检测,探讨其与血小板输注疗效的关系。方法采用聚合酶链反应（PCR）扩增结合测序的方法检测患者GPⅡb／Ⅲa基因中5个外显子的9个多态性位点,采用固相凝集法检测患者血小板抗体。结果98例患者中只检测到GP11b基因的第26外显子存在T18809G变异,导致Ile843Ser多态性,产生HPA-3aa／ab／bb血型的3种基因型。此3种基因型均与血小板抗体产生相关,造成血小板输注无效。98例患者的血小板抗体阳性率为52．0％,1468袋血小板输注总有效率为77．8％,其中65袋配合性血小板输注的有效率为83．1％。3种基因型之间均无明显差异。结论血小板特异性及相关性抗体的存在是造成血小板输注无效的主要免疫性因素,GP11b基因上的HPA-3多态性与血小板输注无效有关。     

选择性5-羟色胺再摄取抑制剂艾司西肽普兰对血小板功能的影响

目的 观察选择性5-羟色胺再摄取抑制剂艾司西肽普兰对血小板功能的影响。方法 选取抑郁症患者61例,随机分为治疗组(艾司西酞普兰10 mg/日)和安慰剂对照组,测定治疗前后的血小板计数(PLT)、血小板平均体积(MPV)、血小板分布宽度(PDW)、血小板聚集率(PAR)、血小板膜糖蛋白CD62P和CD63表达。结果 治疗组在治疗前后的PLT(10⁹/L),MPV(fl),PDW(fl),PAR(%),CD62P(%)和CD63(%)分别为175.8±59.1 vs 169.1±60.5,11.8±1.2 vs 10.3±0.9,15.6±2.1 vs 16.7±1.9,60.1±11.5 vs 59.3±14.9,23.8±5.6 vs25.1±3.7,20.6±7.2 vs 22.4±4.7,对照组在治疗前后的PLT(10⁹/L),MPV(fl),PDW(fl),PAR(%),CD62P(%)和CD63(%)分别为198.6±76.5 vs 203.2±49.8,9.4±1.1 vs 10.2±0.8,12.5±1.8 vs 13.3±2.9,62.7±13.6 vs 66.4±10.7,27.9±7.1 vs 24.8±4.9,26.9±8.1 vs 24.1±6.4,差异均无统计学意义(均P>0.05)。结论 艾司西肽普兰对血小板功能无明显影响。     

一例GPⅡb基因突变导致血小板无力症的诊断

目的对1例血小板无力症(GT)患者进行实验诊断,分析引起血小板膜糖蛋白(GP)缺陷的突变基因。方法通过对先证者凝血常规、血小板计数、血小板聚集功能、出血时间、GPⅡb/Ⅲa(CD41/CD61)含量等项目的检测和血涂片中血小板形态及分布的观察,进行临床表型诊断。通过聚合酶链反应(PCR)扩增结合测序的方法,分析先证者GPⅡb/Ⅲa基因的所有外显子区及其侧翼序列,家系成员仅在相应突变区域进行PCR扩增和测序。同时以100名健康人作为对照进行分析,以排除基因多态性。结果先证者凝血常规检测和血小板计数正常,但出血时间延长,血小板聚集功能异常;表现为对多种生理性诱聚剂反应低下,但对瑞斯托霉素反应正常,血块退缩不良;流式细胞术检测CD41、CD61含量显著降低;血涂片中血小板散在,无聚集现象。经测序,发现先证者GPⅡb基因23号外显子存在18183A>C杂合变异,导致GPⅡb蛋白Q778P替换。与100名健康对照者测序比较,排除基因多态性,证实其为基因突变。结论 GPⅡb基因的Q778P杂合突变是导致该先证者患GT的原因之一。     

三个遗传性血小板无力症家系临床特性和基因分析

目的 对三个遗传性血小板无力症(GT)家系进行整合素αⅡbβ3临床特性分析和基因突变检测.方法 经BPC、血涂片、出血时间(BT)、凝血常规检查、血小板聚集试验和流式细胞术进行实验检测;用PCR法对先证者αⅡbβ3基因所有外显子及其侧翼序列进行扩增;PCR产物纯化后直接测序,检测其基因突变.突变位点经直接测序排除基因多态性,103名健康人作对照.结果 三个家系的先证者BPC正常,血小板不聚集,BT延长,凝血常规指标检查正常,对ADP、凝血酶、肾上腺素、胶原、花生四烯酸等多种诱聚剂反应低下,而对瑞斯托霉素反应基本正常;家系1和家系3先证者的血小板膜表面αⅡbβ3的含量极度降低,家系2先证者的血小板膜表面αⅡb阳性血小板为63%,β3阳性血小板为76%.家系1先证者αⅡb基因存在G10A纯合突变,β3基因存在G1412T纯合突变.家系2先证者β3基因存在G1199A和1525delC复合杂合突变.家系3先证者在αⅡbβ3基因未检测到突变.结论 G10A和G1412T复合纯合突变是导致家系1先证者发生GT的原因,G1199A和1525delC复合杂合突变是导致家系2先证者发生GT的原因.G10A、G1412T和1525delC为国际首次报道的突变.     

造血干细胞移植建立血小板整合素β3胞内段截短体转基因小鼠模型

本研究通过逆转录病毒感染整合素β3缺陷小鼠（β3-/-）骨髓造血干细胞再进行骨髓移植建立β3及其胞内段不同截短体的转基因小鼠模型,为进一步研究整合素β3胞内段在血小板双向信号转导途径中的作用提供基础。将整合素β3野生型、β3-Δ759（缺失R760GT762氨基酸片段）和β3-Δ754（缺失T755NITYRGT762氨基酸片段）的cDNA分别克隆至带有GFP编码基因的MSCV MigR1逆转录病毒载体质粒中,用质粒转染BOSC23包装细胞株包装出带有β3全长及不同突变体的逆转录病毒,再感染β3-/-供体小鼠骨髓细胞,尾静脉注射移植入经致死剂量辐照的野生型C57BL/6小鼠体内,移植后6-8周流式细胞术检测GFP和β3的表达率以评估转基因效率。结果表明,成功建立了空载（Vector）、β3野生型和截短突变β3-Δ759、β3-Δ754等4种小鼠模型,血小板转基因成功的比率（即GFP阳性率）为18%-66%,转基因血小板β3表达可达到β3＋/-水平。结论：利用逆转录病毒感染的造血干细胞移植建立血小板转基因小鼠模型是一种高通量快速有效的建立转基因小鼠模型的方法,这为进一步研究整合素β3胞内不同区段对血小板整合素信号转导途径的影响及体内对血小板进行基因操作和基因治疗奠定了基础。     

益气养血方联合西医治疗难治性血小板减少症疗效及对血小板膜糖蛋白CD41、CD61水平影响

[目的]探讨中西医结合治疗难治性免疫性血小板减少症(RITP)的临床疗效及治疗前后患者血小板膜糖蛋白CD41、CD61水平变化特点.[方法]选取2010年5月至2014年12月本院收治的78例RITP患者,随机分为对照组与观察组,每组39例,对照组给予强的松片口服,观察组给予强的松片+益气养血方联合治疗.治疗6个月后,比较两组临床疗效,治疗前后血小板计数,血小板膜糖蛋白CD41、CD61及T细胞亚群变化.[结果]治疗后观察组总有效率显著高于对照组,差异具有统计学差异(x2 =20.146,P＜0.05);治疗后两组PLT、PCT、CD41、CD61均明显高于治疗前水平(P＜0.05),且观察组升高幅度高于对照组(P＜0.05);治疗后两组CD3+、CD4+、CD4+/CD8+均明显高于治疗前,CD8+明显低于治疗前(P＜0.05),且观察组变化幅度大于对照组(P＜0.05);与对照组比较,观察组患者糖皮质激素副作用有明显改善(P＜0.05).[结论]中西医结合治疗RITP临床疗效优于单纯西药治疗,可改善患者细胞免疫功能及降低糖皮质激素副作用,提高血小板膜糖蛋白CD41、CD61水平.     

整合素αⅡbβ3在血小板中作用的研究现状

近年, 心脑血管血栓性疾病的发生率逐年增高, 而引起血栓性疾病的首要原因是病理性血栓的形成。血小板在止血和血栓形成中发挥重要作用。整合素αⅡbβ3是血小板表面表达最丰富的受体, 且是血小板活化聚集的终末通路。整合素αⅡbβ3拮抗剂, 如阿昔单抗、依替巴肽及替罗非班, 具有抑制血栓形成作用, 但其在抗血栓的同时可导致严重出血, 因此如何在抗血栓的同时降低出血发生率是相关研究热点。整合素αⅡbβ3可介导血小板双向信号转导, 因此研发新型拮抗剂, 选择性地阻断细胞外向细胞内信号转导而不影响细胞内向细胞外信号转导, 可发挥抗血栓作用而不导致出血, 是新的抗血栓策略。笔者拟重点对整合素αⅡbβ3及其在血小板相关信号通路中的作用, 以及各种靶向整合素αⅡbβ3的抗血栓药物进行介绍, 为临床抗血栓治疗提供新思路。     

Molecular basis of platelet activation by an αIIbβ3-CHAMPS peptide

Summary.  Background:  A novel method, known as computed helical anti-membrane protein (CHAMP), for the design of peptides that bind with high affinity and selectivity to transmembrane helices was recently described and illustrated using peptides that bind αIIb- and αv-integrin subunits, which induce selective activation of integrins αIIbβ3 and αvβ3, respectively [ 1 ]. Objectives:  In the present study, we have investigated the ability of an αIIb-CHAMPS peptide (termed integrin-activatory-peptide or IAP) to stimulate protein tyrosine phosphorylation and aggregation in human and mouse platelets. Methods:  The ability of IAP to stimulate platelet aggregation and dense granule secretion was measured in washed preparations of human and mouse platelets. Samples were taken for measurement of tyrosine phosphorylation. Results:  IAP stimulates robust tyrosine phosphorylation of the tyrosine kinase Syk and the FcR γ-chain, but only weak phosphorylation of PLCγ2. Aggregation to low but not high concentrations of IAP is reduced in the presence of the Src kinase inhibitor, PP1, or by inhibitors of the two feedback agonists, ADP and thromboxane A₂ (TxA₂) suggesting that activation is reinforced by Src kinase-driven release of ADP and TxA₂. Unexpectedly, aggregation by IAP is only partially inhibited in human and mouse platelets deficient in integrin αIIbβ3. Further, IAP induces partial aggregation of formaldehyde-fixed platelets. Conclusions:  The present study demonstrates that the αIIb-CHAMPS peptide induces platelet activation through integrin αIIbβ3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR γ-chain and Syk. The use of the αIIb-CHAMPS peptide to study integrin αIIbβ3 function is compromised by non-integrin-mediated effects.     

Integrin-mediated Ras–Extracellular Regulated Kinase (ERK) Signaling Regulates Interferon γ Production in Human Natural Killer Cells

Recent evidence indicates that integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that β1 integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc–growth factor receptor–bound protein 2 (Grb2) and Shc–proline-rich tyrosine kinase 2–Grb2 complexes are the receptor-proximal events accompanying the β1 integrin–mediated Ras activation. In addition, we demonstrate that ligation of β1 integrins results in the stimulation of interferon γ (IFN-γ) production, which is under the control of extracellular signal–regulated kinase 2 activation. Overall, our data indicate that β1 integrins, by delivering signals capable of triggering IFN-γ production, may function as NK-activating receptors.     

Pharmacogenomics of β‐Adrenergic Receptors and Their Accessory Signaling Proteins in Heart Failure

β‐adrenergic receptors (βAR) are widely expressed on cardiovascular cells. Pharmacological stimulation or blockade of βAR signaling is the therapeutic mainstay in cardiogenic shock, hypertension, ischemia, arrhythmias, and heart failure. Interindividual variability in the response to βAR agonists and antagonists has prompted examination of variability in the genes encoding βAR signaling pathway members. Prominent among the genes that have been examined so far in heart failure are the β₁AR, β₂AR, and G‐protein‐coupled receptor kinase 5 (GRK5). Each has nonsynonymous polymorphisms that alter amino acid sequence and protein function and regulation in cell‐based systems, genetically altered mouse models, or human hearts. Here, we review these phenotypes and results from published clinical studies, with a focus on heart failure pharmacogenomics. Thus far, very few studies have utilized analogous protocols or drugs, and discrepancies in the clinical studies are apparent. A compelling approach is the use of multiple methods to understand the molecular, cellular, and organ phenotypes of a variant and couple these with clinical studies designed to specifically address the relevance of those phenotypes in humans. Undoubtedly, additional loci will be identified, and together, will provide for genetically driven, individualized treatments for heart failure.