整合素αvβ3在血管紧张素Ⅱ诱导人脐静脉内皮细胞衰老中的变化

目的观察整合素αvβ3在血管紧张素Ⅱ(AngⅡ)诱导的人脐静脉内皮细胞(HUVECs)衰老中的变化。方法体外培养HUVECs,采用CCK-8法检测细胞存活率,用AngⅡ(终浓度10-6mol/L)干预,分为实验对照组、AngⅡ诱导组。以衰老相关β-半乳糖苷酶活性和细胞增殖能力两种衰老标志物为主要观察指标。其中衰老相关β-半乳糖苷酶活性采用免疫化学染色方法,流式细胞术检测细胞周期来反应细胞的增殖能力;利用Western印迹法分析AngⅡ诱导HUVECs 0、12、24、36、48 h的整合素αvβ3表达的时间效应关系。结果与对照组相比,10-6 mol/L AngⅡ诱导组存活的细胞数为对照组的(77.15±6.83)%;(81.80±0.92)%的细胞呈现β-半乳糖苷酶阳性染色,流式细胞仪检测细胞周期停滞于G0～G1,证实细胞衰老;AngⅡ呈时间依赖性上调整合素αvβ3表达。结论 AngⅡ可以诱导HUVECs衰老,其机制可能与上调衰老细胞整合素αvβ3表达有关。     

前列腺腺癌中整合素αvβ3、基质金属蛋白酶-2和-9表达及关系

目的检测前列腺腺癌中整合素αvβ3、基质金属蛋白酶(MMP)-2和-9表达,分析其相关性及在不同临床病理特征中的意义。方法观察组为58例前列腺腺癌患者,对照组为31例前列腺增生患者,均留取术后新鲜组织,两组中整合素αvβ3、MMP-2和MMP-9表达应用流式细胞术检测。结果整合素αvβ3、MMP-2和MMP-9在观察组中表达量明显高于对照组。观察组中整合素αvβ3、MMP-2和MMP-9表达量均与增殖指数、Gleason评分和转移相关。观察组中整合素αvβ3和MMP-2、整合素αvβ3和MMP-9的表达呈正相关。结论前列腺腺癌中存在着明显的整合素αvβ3、MMP-2和MMP-9的高表达,三种蛋白在肿瘤发生中可能有一定促进作用。整合素αvβ3可能通过对细胞外基质进行降解促进肿瘤进展。     

整合素αvβ3小干扰RNA载体构建及其对人视网膜血管内皮细胞的干扰效应检测

目的设计和构建整合素αvβ3（integrin αvβ3）特异性的小干扰RNA表达载体，并初步验证其对靶基因的抑制作用j方法设计靶点特异性的寡核苷酸，连接到经BamH 1-Hind Ⅲ酶切线性化的pGCsilencer2．0-U6质粒上，转染重组质粒到人视网膜血管内皮细胞，通过免疫印迹（Western印迹）及逆转录（RT-PCR）实验检测靶基因的抑制情况。结果成功构建pGCU6-ITGαv和pGCU6-ITGβ3 siRNAs重组质粒，转染人视网膜血管内皮细胞，Western印迹，RT-PCR结果证实重组质粒在蛋白及mRNA水平均能抑制整合素αvβ3的表达。结论成功构建了针对整合素αvβ3的siRNA质粒，该质粒可抑制整合素αvβ3在人视网膜血管内皮细胞中的表达。     

眼睑基底细胞癌患者术后组织SOX18、整合素αvβ3表达与血管生成的关系

目的分析眼睑基底细胞癌患者术后组织SOX18、整合素αvβ3和CD34的表达及对血管生成的作用。方法 105例眼睑基底细胞癌术后组织为研究组,55例基底细胞乳头状瘤术后组织为对照组。应用免疫组化SABC法检测两组SOX18、整合素αvβ3和CD34表达。结果研究组SOX18、整合素αvβ3蛋白表达阳性率和CD34标记的微血管密度(MVD)均明显高于对照组(P<0.000 1),研究组SOX18、整合素αvβ3和MVD表达均与肿瘤最大径、脉管累犯、TNM分期和增殖细胞核抗原(PCNA)指数密切相关。SOX18和整合素αvβ3均与MVD具有正相关性。结论眼睑基底细胞癌患者术后肿瘤组织SOX18和整合素αvβ3高表达,间质中MVD增高,三者具有一定的协同作用,对促进肿瘤的发生和进展有重要作用。     

整合素β3胞浆段尾部序列对αvβ3介导的细胞行为的影响

目的:探讨在与整合素αv复合的模式下,整合素β3亚基胞浆段序列在肿瘤细胞的黏附、伸展和迁移中所起的作用,从而为寻找干扰肿瘤进展的分子靶点提供思路。方法:利用中国仓鼠卵巢(CHO)细胞模型,建立共表达整合素αv与整合素β3全长及其T758、Y759位截短体的稳定细胞株,检测各稳定细胞株在固相化的αvβ3配体玻璃黏连蛋白上的黏附、伸展功能,运用transwell观察各细胞株的迁移能力。结果:稳定表达整合素αvβ3全长的CHO细胞具有在固相化玻璃黏连蛋白上的黏附、伸展和迁移能力。相较于CHO-αvβ3全长细胞株,CHO-αvβ3△758截短体细胞株的黏附、伸展以及迁移能力均明显受损;而CHO-αvβ3△759截短体细胞株仍保留着黏附能力,但伸展能力减弱,并且迁移能力受损明显。结论:整合素β3胞浆尾端YRGT和RGT氨基酸序列在αvβ3介导的细胞黏附和迁移方面发挥不同的作用,提示β3亚基胞内段参与调控肿瘤细胞的重要细胞行为,是潜在的肿瘤治疗靶点。     

黄芩素对人乳腺癌细胞系MDA-MB-231侵袭、迁移、上皮间充质转化的调控作用及其机制

目的 观察黄岑素对人乳腺癌细胞系MDA-MB-231的侵袭、迁移及上皮间充质转化(EMT)的调控作用,探讨其可能作用机制。方法 取对数生长期MDA-MB-231细胞分为一组、二组、三组及对照组,一组、二组、三组分别加入2.5、5、10μmol/L的黄岑素,对照组不做任何处理。培养48 h时采用划痕修复实验观察四组细胞迁移能力、采用Transwell侵袭实验观察四组细胞侵袭能力,采用Western Blotting法检测细胞EMT标志物波形蛋白(vimentin)及E-钙黏蛋白(E-cadherin)、整合素αv、β3、磷酸化黏着斑激酶(p-FAK)、磷酸化磷脂酰肌醇3激酶(整合素p-PI3K)。结果 与对照组相比,黄岑素组细胞迁移率降低、侵袭细胞数少,细胞E-cadherin相对表达量高,vimentin、整合素αv、整合素β3、p-FAK、p-PI3K蛋白相对表达量低,且呈剂量依赖性(P均<0.05)。结论 黄芩素抑制MDA-MB-231细胞的侵袭、迁移及EMT。黄岑素可能通过抑制整合素αv、整合素β₃表达,进一步抑制p-FAK、p-PI3K蛋白表达,抑制...     

糖尿病大鼠肾脏局部α3、β1整合素与血管紧张素Ⅱ水平的相关性研究

目的探讨糖尿病大鼠肾脏局部α3、β1整合素与血管紧张素Ⅱ(ATⅡ)的相关性及ATⅡ1型受体(AT1)拮抗剂对其的影响。方法 40只雄性Wistar大鼠随机分为正常对照组(NC)、糖尿病组(DM)、糖尿病肼屈嗪干预组(Hyd)、糖尿病厄贝沙坦干预组(Irb),每组各10只。肼屈嗪及厄贝沙坦干预8周后进行PAS染色,观察肾脏病理变化。免疫组织化学法检测肾小球足细胞密度,ELISA及RTPCR检测肾脏局部ATⅡ水平、UAER、肾脏α3/β1整合素mRNA的表达。结果 (1)与NC组比较,DM组肾小球足细胞密度下降、蛋白尿增加、α3及β1整合素mRNA的表达下降、ATⅡ升高(P0.05)。予肼屈嗪及厄贝沙坦干预后,上述指标改善(P0.05)。(2)除外血压影响,与Hyd组比较,Irb组蛋白尿下降,足细胞密度升高(P0.05);ATⅡ下降(P0.05);α3及β1整合素mRNA的表达进一步改善(P0.05)。(3)肾小球足细胞密度与肾皮质α3、β1整合素mRNA表达呈正相关(rα3=0.883,rβ1=0.853,P0.01);α3及β1整合素mRNA的表达与ATⅡ呈负相关(rα3=-0.527,rβ1=-0.421,P0.01)。结论糖尿病大鼠肾脏局部α3、β1整合素表达变化与ATⅡ水平相关,可能是AT1拮抗剂除降压外,对糖尿病大鼠肾脏的保护机制之一。     

应用RNA聚合酶Ⅰ反向遗传操作技术拯救汉滩病毒84FLi株微基因组

目的 建立基于汉滩病毒(HTNV)84FLi株L片段的RNA聚合酶Ⅰ微基因组拯救体系.方法 将含有氯霉素乙酰转移酶(CAT)编码区的cDNA插入含有HTNV 84FLi株L片段5'端和3'端非编码区的质粒内的两个非编码区之间,将此eDNA嵌合体(polⅠ表达盒)克隆人人polⅠ肩动子和终止子之间,分别获得正义和反义方向的RNA polⅠ转录报告质粒.用报告质粒转染293T细胞或等量293T和HTNV感染的Vero混合培养细胞,在HTNV的L蛋白和核衣壳蛋白表达质粒共转染后48 h收获细胞,检测CAT活性.用上清液感染293T细胞,了解CAT活性的传代能力.结果 构建了正义和反义方向的HTNV 84FLi株L片段微基因组RNA聚合酶Ⅰ报告质粒pLvRNA-CAT和pLcRNA-CAT,用此质粒转染细胞后能检测到CAT的表达,且CAT活性能在拯救病毒微基因组中传代.结论 应用RNA聚合酶Ⅰ反向遗传操作技术成功拯救了HTNV 84FLi株微基冈组.     

内皮抑素30肽抑制HepG2细胞侵袭转移的机制

目的探讨RGD修饰的人内皮抑素30肽抑制HepG2细胞侵袭转移的机制。方法以人内皮抑素27肽作为对照,采用细胞增殖实验和Transwell实验分别检测30肽对HepG2细胞增殖和侵袭能力的影响,同时筛选与侵袭作用相关的整合素;免疫荧光检测30肽对HepG2细胞表面αvβ3整合素的聚集作用;RT-PCR及Western印迹检测30肽对细胞诱导型环氧合酶(iCOX-2)、基质金属蛋白酶(MMP)-2和MMP-9 mRNA和蛋白表达的影响。结果 30肽能明显抑制HepG2细胞增殖、侵袭,对侵袭作用的影响与整合素αvβ3相关,同时30肽能下调Hep G2细胞中i Cox-2、MMP-2和MMP-9 mRNA及蛋白表达,30肽加入αvβ3抗体后,上述作用明显增强。结论 30肽可通过整合素αvβ3发挥其抗肿瘤侵袭转移作用。     

整合素αvβ3、黏着斑激酶在卵巢上皮性癌中的表达及意义

目的 整合素αvβ3、黏着斑激酶(FAK)在卵巢上皮性癌(OEC)组织中的表达及其与临床病理特征的关系.方法 采用免疫组织化学SP法检测49例卵巢上皮恶性肿瘤和15例卵巢上皮性良性肿瘤组织中整合素αvβ3和FAK的表达情况.结果 整合素αvβ3、FAK在OEC中的表达明显高于卵巢良性肿瘤组织;整合素αvβ3的表达与OEC组织的分化程度及临床分期有关;FAK的表达与OEC组织的临床分期有关;在OEC组织中整合素αvβ3、FAK的表达呈正相关.结论 整合素αvβ3、FAK共同参与OEC的侵袭和转移.     

Upregulation of osteoclast alpha2beta1 integrin compensates for lack of alphavbeta3 vitronectin receptor in Iraqi-Jewish-type Glanzmann thrombasthenia

Osteoclasts utilize alphavbeta3 integrin adhesion to bone matrix during bone resorption. We have generated osteoclasts from the peripheral blood of Iraqi-Jewish patients with Glanzmann thrombasthenia (GT) who are completely deficient in beta3 integrin and exhibit a haemorrhagic diathesis resulting from the absence of platelet alphaIIbbeta3. We show that, in contrast to osteoclasts generated from normal subjects or patients with alphaIIb integrin deficiency, GT osteoclasts lack alphavbeta3. These osteoclasts exhibited a two- to fourfold increase in alpha2 and beta1 integrin expression, whereas other alphav integrins, including alphavbeta5, were not significantly affected. An accompanying decrease in bone resorption was observed, with 44% and 59% declines in pit number and depth, respectively, and resorption lacunae showed abnormal morphology on scanning electron microscopy. However, osteoclasts from GT developed in similar numbers to controls and exhibited an otherwise 'normal' phenotype. We conclude that the observed rise in alpha2beta1 expression compensates for the chronic genetic deficiency of alphavbeta3 in osteoclasts from patients with GT and is sufficient to enable bone resorption to proceed, albeit to a submaximal extent. This explains why Iraqi-Jewish patients with GT do not have osteopetrosis.     

Major mutations in calf-1 and calf-2 domains of glycoprotein IIb in patients with Glanzmann thrombasthenia enable GPIIb/IIIa complex formation,but impair its transport from the endoplasmic reticulum to the Golgi apparatus

The crystal structure of integrin alphavbeta3 comprises 3 regions of contact between alphav and beta3. The main contact on alphav is located in the beta-propeller while calf-1 and calf-2 domains contribute minor interfaces. Whether or not contacts between calf-1 and calf-2 domains of glycoprotein (GP) IIb (alphaIIb) and GPIIIa (beta3) play a role in GPIIb/IIIa complex formation has not been established. In this study we analyzed the effects of 2 naturally occurring mutations in calf-1 and calf-2 domains on GPIIb/IIIa complex formation, its processing, and transport to the cell membrane. The mutations investigated were a deletion-insertion in exon 25 located in calf-2 and an in-frame skipping of exon 20 located in calf-1. Mutated GPIIb cDNAs were cotransfected in baby hamster kidney cells with normal GPIIIa (beta3) cDNA. Analysis by flow cytometry failed to demonstrate detectable amounts of GPIIb or GPIIb/IIIa complex on the surface of cells transfected with each mutation, but immunohistochemical staining revealed their intracellular presence. GPIIb was mainly demonstrable as pro-GPIIb by immunoprecipitation of cell lysates expressing each mutation. Differential immunofluorescence staining of GPIIb and cellular organelles suggested that most altered complexes were located in the endoplasmic reticulum. Homology modeling of normal GPIIb based on the alphavbeta3 crystal structure revealed similar contacts between alphav and beta3 and between alphaIIb and beta3. Introduction of the mutations into the model yielded partial disruption of the normal contacts in the corresponding domains. These data suggest that despite partial disruption of calf-1 or calf-2 domain, GPIIb/IIIa complex is formed but its transport from the endoplasmic reticulum is impaired.     

AlphaIIbG236E causes Glanzmann thrombasthenia by impairing association with beta3

Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of the platelet fibrinogen receptor, integrin alphaIIbbeta3. The N-terminal domain of the alphaIIb subunit is folded in a beta-propeller that plays the role of binding fibrinogen and associating with the ligand-binding region of beta3. Analysing the mutations of Italian GT patients we found that a patient had a alphaIIb G236E missense substitution that substitutes a glycine from the highly conserved PhiPhiGPhi motif of blade 4 of the beta-propeller. To verify experimentally the effect of the substitution of glycine 236 human embryonic kidney (HEK) cells were transfected with normal or mutated alphaIIb in conjunction with normal beta3. Using flow cytometry analysis we found the percentage of HEK cells transfected with alphaIIbG236Ebeta3 that reacted with anti alphaIIbbeta3 was very low. In HEK cells transfected with either alphaIIbbeta3 or alphaIIbG236Ebeta3 and lysed, when immunoblotting was done in non-reducing conditions a band reacting with an antibody against alphaIIb was present in both lysates, although less intense in cells transfected with alphaIIbG236Ebeta3. In reducing condition alphaIIb from cells transfected with alphaIIbbeta3 was nearly all mature, while in cells transfected with alphaIIbG236Ebeta3 the ratio pro-alphaIIb: alphaIIb was 1 : 1, with signs of degradation of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against alphaIIb and immunoblotted with an antibody reacting with beta3. While in immunoblots from cells transfected with alphaIIbbeta3 a band corresponding to beta3 was strongly detectable, in immunoblots originating from cells transfected with alphaIIbG236Ebeta3 no band at the same level of normal beta3 was detected. Immunofluorescence studies showed accumulation of alphaIIbG236Ebeta3 in the endoplasmic reticulum and minimal transport to the Golgi. In conclusion we demonstrated that the alphaIIbG236E mutation causes GT by impairing the association with beta3 during biogenesis of the receptor.     

Pathogenic hantaviruses bind plexin-semaphorin-integrin domains present at the apex of inactive, bent alphavbeta3 integrin conformers

The alphavbeta3 integrins are linked to human bleeding disorders, and pathogenic hantaviruses regulate the function of alphavbeta3 integrins and cause acute vascular diseases. alphavbeta3 integrins are present in either extended (active) or dramatically bent (inactive) structures, and interconversion of alphavbeta3 conformers dynamically regulates integrin functions. Here, we show that hantaviruses bind human alphavbeta3 integrins and that binding maps to the plexin-semaphorin-integrin (PSI) domain present at the apex of inactive, bent, alphavbeta3-integrin structures. Pathogenic hantaviruses [New York-1 virus (NY-1V) and Hantaan virus (HTNV)] bind immobilized beta3 polypeptides containing the PSI domain, and human (but not murine) beta3 polypeptides inhibit hantavirus infectivity. Substitution of human beta3 residues 1-39 for murine beta3 residues directed pathogenic hantavirus infection of nonpermissive CHO cells expressing chimeric alphavbeta3 receptors. Mutation of murine beta3 Asn-39 to Asp-39 present in human beta3 homologues (N39D) permitted hantavirus infection of cells and specified PSI domain residue interactions with pathogenic hantaviruses. In addition, cell-surface expression of alphavbeta3 locked in an inactive bent conformation conferred hantavirus infectivity of CHO cells. Our findings indicate that hantaviruses bind to a unique domain exposed on inactive integrins and, together with prior findings, suggest that this interaction restricts alphavbeta3 functions that regulate vascular permeability. Our findings suggest mechanisms for viruses to direct hemorrhagic or vascular diseases and provide a distinct target for modulating alphavbeta3-integrin functions.     

A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the alphaIIbbeta3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin and a P53L mutation in glycoprotein (GP) Ibalpha. We show that GPIbalpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta3-H723 causes constitutive, albeit partial, activation of the alphaIIbbeta3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the alphaIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alphaIIbbeta3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alphaIIbbeta3-H723, but not wild-type alphaIIbbeta3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alphaIIbbeta3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree.     

A naturally occurring mutation near the amino terminus of alphaIIb defines a new region involved in ligand binding to alphaIIbbeta3

Decreased expression of functional alphaIIbbeta3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of alphaIIb(P145) in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P(145)A substitution, whereas a Chinese patient was doubly heterozygous for a P(145)L substitution. The mutations affect expression levels of surface alphaIIbbeta3 receptors on their platelets, which was confirmed by co-transfection of alphaIIb(P145A) and beta3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of alphaIIbbeta3 on affected platelets to interact with ligands. Moreover, when alphaIIb(P145A) and beta3 were stably coexpressed in Chinese hamster ovary cells, alphaIIbbeta3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate-fibrinogen after alphaIIbbeta3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to alphaIIb, induced PF4 secretion, indicating that the mutant alphaIIbbeta3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving alphaIIb(P145 )impair surface expression of alphaIIbbeta3 and that the alphaIIb(P145A) mutation abrogates ligand binding to the activated integrin. A comparative analysis of other alphaIIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the alphaIIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)     

A push-pull mechanism for regulating integrin function

Homomeric and heteromeric interactions between the alphaIIb and beta3 transmembrane domains are involved in the regulation of integrin alphaIIbbeta3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of alphaIIbbeta3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the alphaIIb transmembrane domain. One mutation that enhanced the dimerization of the alphaIIb transmembrane domain indeed induced constitutive alphaIIbbeta3 activation. However, a series of mutations that disrupted homodimerization also led to alphaIIbbeta3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the alphaIIb transmembrane domain, and that mutations that disrupt the alphaIIb/beta3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for alphaIIbbeta3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an alphaIIb/beta3 transmembrane domain heterodimer and by stabilizing the resulting alphaIIb and beta3 transmembrane domain homodimers.     

Evidence for alphavbeta3 and alphavbeta5 integrin-like vitronectin (VN) receptors in Candida albicans and their involvement in yeast cell adhesion to VN.

The expression of integrin vitronectin (VN) receptors on Candida albicans yeasts and their involvement in the adhesion to VN were investigated. By immunofluorescence and cytofluorimetric analysis, several antibodies directed against human alphav, beta3, beta5, alphavbeta3, or alphavbeta5 integrin positively stained C. albicans yeasts. Biochemical analysis on yeast lysates with anti-human alphav, beta3, or beta5 antibody revealed molecular species of 130, 110, 100, and 84 kDa. The 130-kDa band was identified as alphav, whereas the doublet of 110/100 kDa and the 84-kDa band likely correspond to the beta3 and beta5 subunits, respectively. Some 48%-54% of Candida yeasts specifically adhered to VN, and this binding was strongly inhibited by anti-human alphav, beta3, alphavbeta3, and alphavbeta5 antibodies and by RGD- but not RGE-containing peptides. In addition, VN inhibited C. albicans adherence to a human endothelial cell line. Thus, C. albicans in the yeast phase expresses VN receptors antigenically related to the vertebrate alphavbeta3 and alphavbeta5 integrins, which mediate its adhesion to VN.     

A Leu55 to Pro substitution in the integrin alphaIIb is responsible for a case of Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. A new mutation, a T to C substitution at base 258 in the alphaIIb gene, leading to the replacement of Leu55 with Pro, was found by sequence analysis of a patient's alphaIIb cDNA. In transfection experiments using COS7 cells, the cells co-transfected with the mutated alphaIIb cDNA containing C258 and wild-type beta3 cDNA scarcely expressed the alphaIIbbeta3 complex. The Leu55 to Pro substitution in the alphaIIb gene was found to be responsible for this case of Glanzmann's thrombasthenia.