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

XIIIA在人细胞系中的表达在细胞增殖过程中呈下降趋势，在细胞生长处于明显的稳定状态时呈上升趋势。在细胞传代过程中，XIIIA在U937细胞中逐渐减少，而在Meg-01细胞中增加。免疫荧光显微镜显示XIIIA在MEG-01细胞中有三种典型的定位模式：(1)胞浆内弥漫分布；(2)核周围丝状结构；(3)核内弥漫分布。在PMA处理的THP-1细胞中也发现了XIIIA的核定位。XIIIA基因部分与中间纤维蛋白波形蛋白共定位，酵母双杂交实验证实了XIIIA基因与波形蛋白的直接相互作用。在XIIIA缺乏症患者基因组DNA中检测到XIIIA基因突变。通过分子模拟和力学方法预测了Arg260的氨基酸被Cys取代，从而导致XIIIA分子不稳定。在酵母中的表达研究证实了该突变体的快速降解。一个新的Tyr283-Cys突变体的快速降解也归因于它的不稳定性，其特征是使用内源性合成XIIIA的巨核母细胞MEGO1细胞的表达系统。为了了解XIII缺乏症在体内的分子病理学，对XIIIA基因敲除(KO)小鼠进行了功能分析。虽然纯合子XIIIA雌性KO小鼠能够受孕，但大多数小鼠因妊娠期间阴道出血过多而死亡。腹部切开显示，死亡小鼠的子宫里充满了血液，而且在一个子宫内，一些胚胎比其他胚胎小得多。对怀孕动物的一系列组织学检查表明，在怀孕第10天，XIIIA KO小鼠的子宫内出现了大量的胎盘出血和随后的坏死。无论胎儿的基因类型如何，这一点都是正确的。

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.