| Project/Area Number |
05670915
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Hematology
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
TSUDA Hiroko Kyushu Univ., Faculty of Medicine, Research Associate, 医学部, 助手 (30180003)
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| Co-Investigator(Kenkyū-buntansha) |
IWANAGA Sadaaki Kyushu Univ., Faculty of Biology Professor, 理学部, 教授 (90029942)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | Tissue Factor / Fator VII / Inhibitory Antibody |
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| Research Abstract |
After liver surgery, two patients developed IgG lambda-type immediate anticoagulants directed against both rabbit and bovine tisue factor (TF) that did not cross react with either human or monkey TF.In a chromogenic assay, the patient IgG caused a decrease in both the Km and the Vmax of the factor X activation by TF-factor VIIa (VIIa) complex. The iatrogenic immunization by intraoperative exposure of bovine TF retained in the topical hemostatic collagen agent was suggested by immunoblotting analysis using the Triton extract of the collagen agent.
The DNA encoding the extracellular domain of bovine TF (amino acid reisues 1-213, named recombinant bovine soluble TF,rsBTF) was constructed from the cDNA of bovine TF previously isolated. The protein was expressed in yeast strain AH22 and purified from the culture medium using a CM-sepharose column. The rsTF was partially digested with trypsin at the NH_2 side of Arg-129 and separated into two domins (NH_2 terminal domain and COOH terminal dom
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ain) on SDS-polyacrylamide gel electrophoresis. Immunoblotting analysis revealed that the patient IgG reacted with both domains.
The treatment of rsBTF with KNCO diminished both its enhancing effect on the amidolytic activity of VIIa and its affinity for VIIa determined by ligand blotting assay. The modified rsBTF were subjectd to tryptic digestion, and the digests were separated by reversed-phase HPLC,which demonstrated that carbamylation of the epsilon-amino group of Lys-17 resulted in the loss of binding affinity for VIIa.
It was recently reported that the binding site for VII(a) on TF molecule involves residues from both NH_2 terminal and COOH terminal domains each of which consists of about 100 amino acid residues. The amino acid sequences of position 81-90 and of position 197-218 are highly variable among four species, human, bovine, rabiit and mouse.Therefore, it is sugested that the anti-TF antibodies developed in patients after liver surgery react with the VII(a)-binding sites on both domains and inhibit the TF-VIIa complex formation. Less
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