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

肝手术后，2例患者出现针对兔和牛组织因子(Tf)的Ig G-lambda-型即刻抗凝剂，但不与人或猴的Tf发生交叉反应。在显色试验中，患者的Ig G引起Tf-F VIIa(VIIa)复合体激活因子X的Km和Vmax均降低。通过对胶原剂Triton提取物的免疫印迹分析，提出了一种术中暴露于局部止血胶原剂中的牛TF的医源性免疫方法。在酵母菌AH22中表达了该蛋白，并用CM-Sepharose柱纯化了该蛋白。重组转移因子在精氨酸129NH_2侧被胰酶部分消化，并分离出两个结构域(NH_2末端结构域和COOH末端DOM…更多的AIN)在SDS-聚丙烯酰胺凝胶电泳上。免疫印迹分析表明，患者的免疫球蛋白与这两个结构域均有反应。用KNCO处理rsBTF后，其对VIIa氨解活性的增强作用和与VIIa的亲和力均减弱。对修饰的rsBTF进行胰酶消化，并用反相高效液相色谱分离，结果表明Lys-17的epsilon氨基的氨甲基化导致了与VIIi的结合亲和力的丧失。最近的报道表明，在Tf分子上的VII(A)结合位置涉及来自NH_2末端和COOH末端的残基，每个残基由大约100个氨基酸残基组成。人、牛、兔和小鼠四个物种的第81-90位和197-218位氨基酸序列差异很大，因此推测肝手术后患者产生的抗TF抗体与这两个结构域上的VII(A)结合部位发生反应，从而抑制了TF-VIIa复合体的形成。较少

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