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

在分子水平上：表达全长重组因子XIIIB亚基（rXIIIB）和具有不同数目Sushi结构域的截短的人XIIIB（rXIIIBx-y）。rXIIIB为二聚体形式，并与XIIIA产生异源四聚体复合物。凝胶过滤和XIIIA结合分析表明，第一个Sushi结构域负责XIIIB与XIIIA的结合，第4和第9个Sushi结构域参与XIIIB同源二聚体组装。通过添加rXIIIB加速XIIIB缺陷血浆中的纤维蛋白原交联，而在使用纯化纤维蛋白原的重构系统中没有观察到效果，这意味着存在介导血浆中纤维蛋白原交联的XIIIB依赖性修饰的未知因子。通过免疫共沉淀和共聚焦激光显微镜证实了XIIIA与肌动蛋白或三聚体G蛋白β亚基（G_<β2>）之间的相互作用。检测到胺底物掺入核蛋白中，并确认为转氨酶反应。G_β 2与XIIIA共定位于细胞表面的脂筏中，表明细胞质中新合成的XIIIA被转运到细胞膜外。这是该研究领域一个划时代的新发现。在个体水平上：我们产生了缺乏XIIIA或XIIIB的小鼠。XIIIA基因缺失雄性的存活率在出生后10个月时降至约50%。4只XIIIA缺失雄性动物死于严重胸腔内出血，在其心脏中发现大血肿。在其他死亡的XIIIA缺失雄性动物的心脏中观察到出血、含铁血黄素沉积和/或纤维化。在出生后6-8个月处死的XIIIA缺失和XIIIB缺失雄性动物的心脏中也发现了纤维化和含铁血黄素沉积，因此检查先天性XIII缺乏症人类患者可能存在的心脏并发症非常重要。

At the molecular level: Full-length recombinant factor XIIIB subunit (rXIIIB) and truncated human XIIIBs with various numbers of Sushi domains (rXIIIBx-y) were expressed. The rXIIIB was in dimer form and produced a heterotetramer complex with XIIIA. Gel-filtration and XIIIA-binding analysis revealed that the first Sushi domain was responsible for the binding of XIIIB to XIIIA, and that the 4th and 9th Sushi domains were involved in the XIIIB homodimer assembly. Fibrin crosslinking in XIIIB-deficient plasma was accelerated by the addition of rXIIIB, while no effect was observed in a reconstitution system using purified fibrinogen, implying the presence of an unknown factor(s) that mediate the XIIIB-dependent modificaton of fibrin crosslinking in plasma.At the cellular level: The interaction between XIIIA and actin or a trimeric G protein β-subunit (G_<β2>)was confirmed by co-immunoprecipitation and by con-focal laser microscopy. The incorporation of amine substrates into nuclear proteins was detected and confirmed as a transglutaminase reaction. G_<β2> was found to be co-localized with XIIIA in lipid rafts on the cell surface, implying that the newly synthesized XIIIA in the cytoplasm was transported outside the cell membrane. This is an epoch-making new finding in this research field.At the individual level: We generated mice lacking either XIIIA or XIIIB. Survival rates of XIIIA null males decreased to approximately 50% at 10 months from birth. Four XIIIA null males died of severe intra-thoracic hemorrhage, and a large hematoma was found in their hearts. Hemorrhage, hemosiderin deposition, and/or fibrosis were observed in the hearts of other dead XIIIA null males. Fibrosis together with hemosiderin deposition was also found in the hearts of XIIIA null and XIIIB null males sacrificed at 6-8 months from birth, it is important therefore to examine the possible existence of cardiac complications in human patients with congenital XIII deficiency.