Characterization of porcine factor VII, X and comparison with human factor VII, X.
ABSTRACT 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.
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.
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 Ca(2+) 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.
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.
- SourceAvailable from: Jianlin Qiao[show abstract] [hide abstract]
ABSTRACT: Glycoprotein Ib-IX-V (GPIb-IX-V) is a platelet adhesion receptor complex that initiates platelet aggregation. Glycoprotein Ibα (GPIbα) is the central component of the GPIb-IX-V complex, anchoring the complex to the cytoskeleton and harboring the binding site for von Willebrand factor (vWF). Previous studies suggest that the coagulation function in pigs differs from that in humans, especially with respect to the interaction between vWF and platelets. However, we have little knowledge about the function of porcine platelets, which is important with regard to studies of cardiovascular disease, clotting, and surgery that use pigs as animal models. To extend this information, we cloned and analyzed the porcine GPIbα sequence. Porcine GPIbα contains 1891 nucleotides and includes an open reading frame that encodes 627 amino acids. The nucleotide sequence showed 67% identity with human GPIbα, whereas the deduced amino acid sequences were 59% identical. The vWF binding domain shares the highest identity among different species, whereas the PEST domain shows variations. Evaluation of platelet function by using ristocetin-induced platelet aggregation revealed remarkably lower levels of aggregation in porcine than human platelets. According to the sequence analysis and platelet aggregation tests, we propose that the function of GPIbα, especially regarding the ristocetin-vWF-GPIbα interaction, differs between pigs and humans. This characterization of porcine GPIbα will enhance our knowledge of the porcine coagulation system.Comparative medicine 01/2012; 62(4):291-7. · 1.12 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.Hybridoma (2005) 12/2012; 31(6):443-8. · 0.33 Impact Factor
Article: Porcine model of hemophilia a.[show abstract] [hide abstract]
ABSTRACT: Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.PLoS ONE 01/2012; 7(11):e49450. · 3.73 Impact Factor