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Analyses on Mechanisms for Biosynthesis and Release of Human Coagulation Factor XIII at Molecular, Cellular, and Individual Levels

人凝血因子XIII的分子、细胞和个体水平生物合成和释放机制分析

基本信息

批准号：
11470205
负责人：
ICHINOSE Akitada
金额：
$ 9.22万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2002
项目状态：
已结题

项目摘要

Expression of XIIIA in human cell lines decreased during cell proliferation and increased in an apparent steady state of cell growth. During cell passage, XIIIA gradually decreased in U937 cells, in contrast, it increased in MEG-01 cells. Immunofluorescence microscopy revealed three typical localization patterns of XIIIA in MEG-01 cells; (1) a diffuse pattern in cytoplasm; (2) a filamentous structure around the nucleus; and (3) a diffuse pattern in the nucleus. Nuclear localization of XIIIA was also found in PMA-treated THP-1 cells. XIIIA partly co-localized with an intermediate filament protein vimentin, and the direct interaction between XIIIA and vimentin was confirmed by an yeast Two-Hybrid assay.Mutations in the gene for XIIIA have been detected in genomic DNA samples obtained from patients with XIIIA deficiency. An amino acid substitution of Arg260 by Cys had been predicted by molecular modeling and mechanics to result in instability of the XIIIA molecule. Rapid degradation of this mutant has been confirmed by an expression study in yeast. Rapid degradation of a novel Tyr283-Cys mutant has also been ascribed to its instability characterized in an expression system employing megakaryoblastoid MEGO1 cells that endogenously synthesize XIIIA.In order to understand the molecular pathology of XIII deficiency in vivo, XIIIA-knockout (KO) mice were functionally analyzed. Although homozygous XIIIA female KO mice were capable of becoming pregnant, most of them died due to excessive vaginal bleeding during gestaion. Abdominal incisions revealed that the uteri of the dead mice were filled with blood and that some embryos were much smaller than others within a single uterus. A series of histological examinations of the pregnant animals suggested that massive placental hemorrhage and subsequent necrosis developed in the uteri of the XIIIA KO mice on day 10 of gestation. This was true regardless of the genotypes of fetuses.

XIIIA在人细胞系中的表达在细胞增殖期间降低，并且在细胞生长的表观稳定状态下增加。在细胞传代过程中，XIIIA在U937细胞中逐渐减少，而在MEG-01细胞中逐渐增加。免疫荧光显微镜检查显示MEG-01细胞中XIIIA的三种典型定位模式：（1）细胞质中的弥漫模式;（2）细胞核周围的丝状结构;和（3）细胞核中的弥漫模式。在PMA处理的THP-1细胞中也发现了XIIIA的核定位。XIIIA与中间丝蛋白波形蛋白部分共定位，并且XIIIA和波形蛋白之间的直接相互作用通过酵母双杂交测定证实。通过分子模拟和力学预测，Arg 260被Cys取代导致XIIIA分子不稳定。该突变体的快速降解已通过在酵母中的表达研究证实。一种新的Tyr283-Cys突变体的快速降解也被归因于其不稳定性，其特征在于在采用内源性合成XIIIA.To了解XIII缺乏症在体内的分子病理学的巨核细胞MEGO1细胞的表达系统中，XIIIA敲除（KO）小鼠进行了功能分析。虽然纯合子XIIIA雌性KO小鼠能够怀孕，但它们中的大多数由于妊娠期间阴道出血过多而死亡。腹部切口显示，死亡小鼠的子宫充满了血液，并且在一个子宫内，一些胚胎比其他胚胎小得多。妊娠动物的一系列组织学检查表明，妊娠第10天，XIIIA KO小鼠子宫中出现大量胎盘出血和随后的坏死。无论胎儿的基因型如何，这都是正确的。