Analysis of Functional Domains of Tissue Factor Pathway Inhibitor

(1)凝血酶对TFPI的降解。研究表明，TFPI在血浆中以自由形式和脂蛋白相关形式存在，也以蛋白水解引起的截短形式存在。我们研究了多种蛋白酶对重组TFPI的作用，发现凝血酶通过特异性裂解TFPI的三个肽键使TFPI的功能失活。(2)TFPI与肝素的相互作用。我们通过特异性去除n -磺酸盐、2-0-硫酸盐或6-0-硫酸盐残基的改性肝素来分析TEPI与肝素的相互作用。通过使用更短的多糖链。结果表明，所有硫酸盐残基都是相互作用所必需的，具有14个单位的糖链与肝素具有几乎相同的与TFPI相互作用的能力。我们还研究了TFPI的两个肝素结合位点与肝素的相互作用。我们发现Arg257和Arg259是通过合成具有c端碱基部分的肽与肝素相互作用所必需的。利用Baculo病毒系统分离重组K3结构域，并检测其与肝素的相互作用。

(1) Degradation of TFPI by thrombinIt has been shown that TFPI exists in plasma as a free-form and liporptoein-associated forms and also as truncated forms which were caused by proteolysis. We investigated the actions of various proteases on recombinant TFPI and found that thrombin inactivated the functions of TFPI by secific cleavage of three peptide bonds of TFPI(2) Interaction of TFPI with heparinWe analyzed the interaction of TEPI with heparin by using modified heparin in which N-sulafate, 2-0-sulfate or 6-0-sulfate residues were specifically removed.and by using shorter chains of polysaccharide. The results indicate that all sulfate residues are essential for the interaction and that sugar chain with 14 units has almost the same ability to interact with TFPI as heparin. We also investigated the interaction of two heparin binding sites of TFPI with heparin. We found that Arg257 and Arg259 were essential for the interaction with heparin by using synthetic peptides with C-terminal basic part. We isolated recombinant K3 domain by Baculo virus system and examined the interaction with heparin.