Comparative Study Of Macaca Mulatta Coagulation Function And Partial Coagulation Genes Molecular Heredity

Purpose: Macaca mulatta (rhesus) is the most important animal model in pre-clinical study of the coagulation drugs and allotransplantation and xenotransplantation. To explore the efficacy of new coagulant, and provide some clues of the etiopathogenisis of coagulopathy and the strategy of prevention and cure in post-transplantation of pig to non-human primate, and predict the possible coagulation results of the pig-to-human xenotransplantation, we primarily studied the rhesus coagulation system.Methods: TEG(Thromboelastometry) was adopted to measure the whole blood coagulation and fibrolysis function of the rhesus; The PT(Prothrombin time) and APTT(Activated partial thromboplastin time) were determined with clotting test, and the activities of the CFII(Coagulation factor II), CFVII(Coagulation factor VII), CFX(Coagulation factor X) with the one-stage assays, and the activities of ATIII(Antithrombin III) by amidolytic assay with chromogenic substrates; The platelet was counted by automatic hematology analyzer; Totallab 2.0 software was used to analyze the CFII, CFVII, CFX and ATIII mRNA expression in rhesus, human and pig liver tissue after detected with RT-PCR (Reverse transcription polymerase chain reaction) method; The data above was analyzed and compared with those of human and pig by SPSS 12.0 statistics software; PCR method was adopted to screen the positive clones from the cDNA library, which constructed withλZAP Express vector from rhesus liver tissue, or combined RACE (Rapid amplification of cDNA ends)technology to obtain the full-length cDNA sequences of the rhesus CFII, CFVII, CFX and ATIII genes. Bioinformatics methods were used to deduce amino acid sequences of the four genes and analyze the differences among rhesus, human and pig. SWISS-MODEL program was used to model the 3-D (Three-dimension) protein structure of prothrombin of rhesus, and compare its variations in some important macromolecule interaction sites with those of human and pig.Results: TEG detection is shown that rhesus coagulation function is stronger than that of human, but the fibrolysis function is weaker than that of human, and have statistical significance(P<0.05); PT in rhesus is longer than that of human PT, and APTT is shorter than that of pig but longer than that of human(P<0.05). The activities of rhesus CFII and CFVII are stronger than those of human and pig, but the activities of CFX and ATIII are weaker than those of human and pig(P<0.05); The amount of platelet of rhesus is more than that of human(P<0.05); The mRNA expressions of CFII, CFX and ATIII in rhesus liver are lower than those of pig(P<0.05), and mRNA of CFVII is lower than those of human and pig(P<0.05). At the first time, the full-length cDNA sequences of rhesus four coagulation related genes were obtained. The comparison of the rhesus four coagulation genes with those of human and pig has shown huge differences, the biggest and the smallest difference is CFVII and ATIII, respectively. The rhesus prothrombin protein sequence deduced from full-length cDNA sequence contains signal peptide, propeptide, Gamma Carboxyglutamate (Gla) domain, two kringle domains and Trypsin domain. Some variations are observed in some important macromolecule binding sites with those of human and pig.Conclusions: The comparative results above showed that huge differences existed in blood coagulation of rhesus with human and porcine, although the highly similarity of them confirmed the great value of rhesus as an animal model. Thus, we should pay more attention to the diversity including the structure and function among them, which would affect seriously the authenticity of the coagulant drugs in clinical appliance biased from pre-clinical study, and influence the reliability of pig-to-human xenotransplantation prediction results deduced from pig-to-rhesus xenotransplantation.