Characterization of porcine factor VII, X and comparison with human factor VII, X
Characterization of porcine factor VII, X and comparison with human factor VII, X
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猪因子 VII、X 的表征以及与人因子 VII、X 的比较。
DOI：
10.1016/j.bcmd.2009.02.004
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发表时间：
2009-07-01
影响因子：
2.3
通讯作者：
Cheng, Jingqiu
中科院分区：
文献类型：
--
作者：
Chen, Younan;Qiao, Jianlin;Cheng, Jingqiu
目的：凝血因子VII（FVII）和凝血因子X（FX）是凝血级联反应的两种主要分子.猪FVII和FX能否在人体循环中有效发挥作用，对猪-人肝移植的成功至关重要。我们比较了猪和人FVII和FX的遗传特性和凝血活性，以深入研究异种移植中猪FVII、FX和人源性促凝剂和抗凝剂之间潜在的种间分子不相容性。通过5' RACE和3' RACE得到全长cDNA。用Swiss-Model程序模拟蛋白质的三维结构。用凝血自动分析仪测定猪和人血浆的凝血酶原时间（PT）。结果：克隆了猪FVII和FX的全长cDNA，分别为1416 bp和1856 bp，编码445和479个氨基酸。猪FVII和FX与人FVII和FX的氨基酸同源性分别为74.08%和73.1%。序列比对结果表明，猪FVII的Gla结构域可能含有额外的γ-羧基谷氨酸，轻链可能含有Lys 62-Glu的一个重要变异。在重链TF结合区没有观察到显著差异，而在负责蛋白水解活性的重要功能残基中鉴定出4个变异，即Gln 217-Glu、Thr 151-Lys、Glu 154-瓦尔和Gln 40-Leu。然而，在猪FVII重链的3-D模型中没有显示出明显的变化。当分析猪FX时，在活性肽（Ser 143至Arg 194）处发现了很大的变化，仅11.6%的同一性。在γ-羧基谷氨酸和钙离子结合位点的一些重要的变化被确定，而在其他功能位点的高度保守性被发现。三维蛋白质模型的比较表明，猪和人FX的蛋白质骨架是高度保守的，在抗凝剂结合位点S2和S3的分子表面上的差异不大。结论：猪FVII和FX与人FVII和FX在核苷酸、氨基酸序列和三维结构上具有较高的同源性。然而，异种肝移植中由于基因差异而导致的对重要大分子的亲和力不同，可能是导致异种肝移植中分子不相容的原因之一。(C)2009 Elsevier Inc. All rights reserved.
Objective: Factor VII (FVII) and factor X (FX) are two predominant molecules of coagulation cascade. Whether porcine FVII and FX could efficiently work in human circulation is important for successful pig to human liver transplantation. We compared the genetic characterizations and coagulation activities of porcine and human FVII and FX to shed insight into the further investigation of potential inter-species molecular incompatibility between porcine FVII, FX and human derived procoagulants and anticoagulants in xenotransplantation.Methods: Multiple rounds of PCR were used to screen the positive clones from a porcine liver tissue cDNA library. 5' RACE and 3' RACE were conducted to get the full-length cDNA. The three-dimensional structure of protein was modeled by Swiss-Model program. Prothrombin Time (PT) of porcine and human plasma was determined by coagulation autoanalyzer. Activities of porcine FVII and FX were detected by adding the porcine plasma into FVII or FX-deficient human plasma.Results: We cloned the full-length cDNA of porcine FVII and FX, which contained 1416 bp and 1856 bp, coding 445 and 479 amino acids, respectively. Porcine FVII and FX shared 74.08% and 73.1% amino acid identities with human FVII and FX. Sequence alignments showed that porcine FVII might have additional gamma-carboxyglutamic acid in Gla domain, and one important variation of Lys62-Glu in light chain. No significant difference was observed in TF binding region of heavy chain, while 4 variations were identified in the important functional residues responsible for proteolysis activity, as Gln217-Glu, Thr151-Lys, Glu154-Val and Gln40-Leu. However, no apparent change was displayed in the 3-D model of the heavy chain of porcine FVII. When porcine FX was analyzed, great variations have been found at active peptide (Ser143 to Arg194) with only 11.6% identity. Some important variations at gamma-carboxyglutamic acids and Ca2+ binding sites were identified, while high conservations were discovered at other functional sites. Comparisons on 3-D protein models demonstrated that the protein backbones of porcine and human FX were highly conserved, and little difference was shown at the molecular surface of anticoagulant binding sites S2 and S3. PT detection of porcine and human plasma showed similar results, while coagulation activities of porcine FVII and FX were remarkably higher than that of human.Conclusion: Porcine FVII and FX showed relatively high homology with human FVII and FX in nucleotide, amino acid sequences and three-dimensional structure. However, the different affinities to important macromolecules caused by genetic differences might contribute to the molecular incompatibilities in liver xenotransplantation. (C) 2009 Elsevier Inc. All rights reserved.