氨基酸序列突变对TFPI生物半衰期和生物活性的影响

通过对个别氨基酸突变的研究,获得了保持良好生物活性的长半衰期组织因子途径抑制因子(tissue factor pathwayinhibitor,TFPI)重组蛋白的有效途径.采用定点诱变和基因重组技术,首先在TFPI cDNA特定位点形成一个位点的沉默突变,以提高TFPI在毕赤酵母细胞内的表达量,此cDNA称为mTFPI.在此基础上,通过系列位点突变,形成3个羧基端突变体:m0TFPI、m1TFPI和m2TFPI.将上述4种TFPI cDNA与表达质粒pPic9连接,转染大肠杆菌,通过PCR和DNA测序确认重组质粒,转染酵母细胞GS115,甲醇诱导表达重组蛋白.采用层析方法纯化TFPI重组蛋白,用125I标记重组蛋白,静脉注射给药,比较四者在SD大鼠体内血浆代谢清除速度.用底物显色法测定重组蛋白抑制凝血因子Xa(Fxa)的活性,比较各株TFPI重组蛋白突变体在体内、体外对FXa的抑制作用及肝素对各株TFPI重组蛋白功能的影响.结果显示,相比野生型TFPI重组蛋(mTFPI)而言,3株羧基端突变体m0TFPI、m1TFPI、m2TFPI在SD大鼠体内血浆代谢清除时间均有不同程度延长,其生物代谢半衰期分别是mTFPI的1.5倍、1.9倍和大于2倍,与m-TFPI相比,3个rTFPI突变体在体内、体外抑制FXa的作用无明显减弱,与肝素的结合能力及协同能力也无明显减弱.结果表明,m0TFPI、m1TFPI和m2TFPI在生物半衰期得到明显延长的同时,仍保持良好的抑制Fxa的生物活性.

Decreasing the Clearance Rate of Tissue Factor Pathway Inhibitor From Plasma by Selectively Mutating The Amino Acid Residues in Its C-Terminal Region

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation system. Recent studies showed that TFPI could also inhibit the proliferation of vascular smooth muscle cells. Studies has suggested that TFPI may find wide applications for the therapy of various diseases such as sepsis, DIC, vascular stenosis, atherosclersis, and miocardial infarction, etc. However, the rapid clearance of TFPI from the circulation remains to be one of major drawbacks to its clinical application. The aim of the present study is to prolong the half-life of recombinant TFPI while maintaining its biological activities by reducing the interaction between TFPI and cell surface, which is mediated by the negative charges on the membrane of the cells and the positive charge on the molecule of TFPI. For this purpose, three rTFPI mutants, m₀TFPI, m₁TFPI, and m₂TFPI, were generated by introducing mutations in the DNA sequences coding for the C-terminal amino acid sequences. The clearance rate of the experimental group (three rTFPI mutants) and the control group (wild-type TFPI, namely mTFPI) in rats was investigated. The inhibitory function on FXa and the heparion binding capacity of the TFPI mutants were also studied. It was shown that at 10 min after intravenous injection, (59.06 ± 13.54) % of the injected mTFPI remained in the circulation, while (71.14 ± 13.04) % of the m₀TFPI, (77.99 ± 2.53) % of the m₁TFPI, and (82.95 ±11.36) % of the m₂TFPI were still in the circulation. The relative half-life of m₀TFPI, m₁TFPI and m₂TFPI were prolonged 0.5, 0.9, and more than 1 times respectively in comparison with that of mTFPI, while their FXa inhibiting activity and heparin-binding capacity were little changed.