Chen Younan, Tan Weidong, Lu Xiaofeng, Lu Yanrong, Qin Shengfang, Li Shengfu, Zeng Yangzhi, Bu Hong, Li Youping, Cheng Jingqiu
Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, PR China.
Blood Cells Mol Dis. 2007 Mar-Apr;38(2):93-9. doi: 10.1016/j.bcmd.2006.10.010. Epub 2006 Dec 22.
Prothrombin is a vitamin K-dependent serine protease and plays pivotal roles in both procoagulant and anticoagulant pathway of hemostasis. In this study, we cloned the full-length cDNA of porcine prothrombin by cDNA library screening and SMART RACE technique. The full-length cDNA is 2027 bp, with a 1869 bp Open Reading Frame (ORF) coding 623 amino acids. The deduced protein of porcine prothrombin contains signal peptide, propeptide, Gla domain, two kringle domains and trypsin domain. Porcine prothrombin shares 86.15% nucleotide similarity and 83% amino acid similarity with human prothrombin. The trypsin domain is highly conserved between the two species with 92.1% amino acid identity. Macromolecular interaction sites comparison between porcine and human prothrombin suggests that the Gla domain in porcine prothrombin contains an additional potential gamma-carboxyglutamic acid site. However, a thrombin cleavage site (Arg284-Thr285) in its light chain is lost. When thrombin heavy chain is concerned, the most important functional sites such as catalytic triad DHS, RGD site, Na+ binding site and anion-binding exosite-I and II are highly conserved. However, great differences have been observed between residues 145 and 158 of heavy chain which is associated with thrombomodulin binding. Two important limited proteolysis sites at Ala150 and Lys154 were lost in porcine sequence, which would affect epsilon-thrombin and gammaT-thrombin generation. Comparison on 3-D protein models demonstrates that these proteins are obviously different in autolysis loop (Lys145 to Gly155). Compared with that of human prothrombin, variation at critical recognition sites would likely alter its binding affinity and reaction velocity, which would contribute to coagulation disorder when porcine liver is transplanted into human body.
凝血酶原是一种维生素K依赖的丝氨酸蛋白酶，在止血的促凝和抗凝途径中都起着关键作用。在本研究中，我们通过cDNA文库筛选和SMART RACE技术克隆了猪凝血酶原的全长cDNA。全长cDNA为2027 bp，具有1869 bp的开放阅读框（ORF），编码623个氨基酸。推导的猪凝血酶原蛋白包含信号肽、前肽、Gla结构域、两个kringle结构域和胰蛋白酶结构域。猪凝血酶原与人类凝血酶原的核苷酸相似性为86.15%，氨基酸相似性为83%。两种物种的胰蛋白酶结构域高度保守，氨基酸同一性为92.1%。猪和人类凝血酶原的大分子相互作用位点比较表明，猪凝血酶原的Gla结构域含有一个额外的潜在γ-羧基谷氨酸位点。然而，其轻链中的一个凝血酶切割位点（Arg284-Thr285）缺失。当涉及凝血酶重链时，最重要的功能位点如催化三联体DHS、RGD位点、Na+结合位点以及阴离子结合外位点I和II高度保守。然而，在与血栓调节蛋白结合相关的重链145和158位残基之间观察到很大差异。猪序列中Ala150和Lys154处的两个重要的有限蛋白水解位点缺失，这将影响ε-凝血酶和γT-凝血酶的产生。三维蛋白质模型比较表明，这些蛋白质在自溶环（Lys145至Gly155）上明显不同。与人类凝血酶原相比，关键识别位点的变异可能会改变其结合亲和力和反应速度，这将导致猪肝移植到人体时出现凝血障碍。
