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Amino acid sequence and inhibitory activity of rhesus monkey tissue factor pathway inhibitor (TFPI): comparison with
S Kamei1, Y Kamikubo, T Hamuro
1Chemo-Sero-Therapeutic Research Institute, Kumamoto.
Abstract:
Rhesus monkey cDNA for tissue factor pathway inhibitor (TFPI) was cloned by means of the reverse transcriptase-polymerase chain reaction, using liver mRNA, and its nucleotide sequence was determined by sequencing five independent clones. Monkey TFPI was found to have a signal peptide of 28 amino acid residues and to be a mature protein of 276 amino acid residues, in which three and seventeen amino acid residue substitutions compared to human TFPI were found, respectively. All the cysteine residues, three putative carbohydrate-linked asparagine residues, and the P1 amino acid residues of each of the three Kunitz inhibitor domains were conserved in the two species. Recombinant monkey TFPI (rTFPI) was isolated from the culture medium of transformed Chinese hamster ovary cells. Amino acid sequence analysis and immunoblotting analysis, using polyclonal and monoclonal antibodies, showed that the carboxyl-terminal basic part of Rhesus monkey rTFPI had been truncated. The inhibitory activity of monkey rTFPI was compared with that of human rTFPI without the carboxyl-terminal basic part. The prothrombin time of human plasma was slightly more prolonged by the addition of monkey rTFPI than by that of human rTFPI. However, no significant differences were found between the potencies of human and monkey rTFPI as to the inhibition of factor Xa and tissue factor-factor VIIa complex.
Insights
Researchers cloned Rhesus monkey tissue factor pathway inhibitor (TFPI) cDNA, finding conserved domains but some substitutions compared to human TFPI. Recombinant monkey TFPI showed similar inhibitory activity to human TFPI, despite a truncated carboxyl-terminal region.
Area of Science:
- Biochemistry
- Molecular Biology
- Primate Genetics
Background:
- Tissue Factor Pathway Inhibitor (TFPI) is a key regulator of the extrinsic coagulation pathway.
- Understanding TFPI structure and function across species aids in developing anticoagulants.
- Rhesus monkey TFPI has not been extensively characterized compared to human TFPI.
Purpose of the Study:
- To clone and sequence Rhesus monkey TFPI cDNA.
- To characterize the recombinant monkey TFPI (rTFPI) and compare its inhibitory activity with human TFPI.
Main Methods:
- Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone monkey TFPI cDNA from liver mRNA.
- Nucleotide sequencing was performed on five independent clones.
- Recombinant monkey TFPI was expressed in Chinese hamster ovary (CHO) cells.
- Amino acid sequence analysis, immunoblotting, and functional assays were conducted.
Main Results:
- Rhesus monkey TFPI cDNA was successfully cloned and sequenced, revealing a 28-amino acid signal peptide and a 276-amino acid mature protein.
- Three and seventeen amino acid substitutions were noted compared to human TFPI, though critical domains (cysteine residues, N-linked glycosylation sites, Kunitz inhibitor domains) were conserved.
- Recombinant monkey TFPI exhibited a truncated carboxyl-terminal basic region.
- Monkey rTFPI showed slightly greater prolongation of prothrombin time in human plasma compared to human rTFPI.
- No significant differences in the inhibition of Factor Xa and the tissue factor-factor VIIa complex were observed between human and monkey rTFPI.
Conclusions:
- Rhesus monkey TFPI shares significant structural homology with human TFPI, particularly in critical functional domains.
- The truncated carboxyl-terminal region of monkey rTFPI does not substantially impair its inhibitory potency against Factor Xa and the TF-FVIIa complex.
- These findings provide valuable insights into TFPI evolution and potential cross-species applications in anticoagulant research.