Characterization of Antitissue Factor Antibodies Developed in Patients After Liver Surgery : Analysis of Epitope and Inhibitory Effects

肝脏手术后，两名患者开发的IgG抗体型直接抗凝血剂对兔和牛组织因子（TF），没有交叉反应，无论是人或猴TF。在显色试验中，患者IgG引起的Km和Vmax的TF-因子VIIa（VIIa）复合物的因子X激活的减少。从牛TF的cDNA中克隆了编码牛TF胞外区（氨基酸残基1-213）的DNA，命名为重组牛可溶性TF（recombinant bovine soluble TF，rsBTF）。该蛋白在酵母菌株AH 22中表达，并使用CM-Sepharose柱从培养基中纯化。rsTF在Arg-129的NH_2侧被胰蛋白酶部分消化，并被分成两个结构域（NH_2端结构域和COOH端结构域 ...更多信息 ain）进行SDS-聚丙烯酰胺凝胶电泳。免疫印迹分析显示，患者IgG与两个结构域均反应，用KNCO处理rsBTF既降低了其对VIIa的酰胺分解活性的增强作用，又降低了其对VIIa的亲和力。重组rsBTF经胰蛋白酶消化后，用反相高效液相色谱（HPLC）分离纯化，证明赖氨酸17的ε-氨基被氨甲酰化，使其与VIIa的结合亲和力丧失。最近报道，TF分子上的VII（a）结合位点包括NH_2端和COOH端两个结构域，每个结构域由约100个氨基酸残基组成。第81-90位和第197-218位的氨基酸序列在人、牛、兔和小鼠四种动物中有很大的差异，因此，在肝脏手术后患者体内产生的抗TF抗体与这两个结构域上的VII（a）结合位点反应，抑制TF-VIIa复合物的形成。少

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 … More 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