Analysis of Functional Domains of Tissue Factor Pathway Inhibitor

(1)凝血酶对TFPI的降解研究表明，TFPI在血浆中以游离形式和脂蛋白结合形式存在，也以蛋白水解引起的截短形式存在。我们研究了各种蛋白酶对重组TFPI的作用，发现凝血酶通过特异性地切割TFPI的三个肽键而使TFPI失活。（2）TFPI与肝素的相互作用我们用特异性地去除N-硫酸酯、2-O-硫酸酯和6-O-硫酸酯残基的修饰肝素和用较短的多糖链分析了TEPI与肝素的相互作用。结果表明，所有的硫酸酯残基是必需的相互作用和糖链的14个单位几乎有相同的能力与肝素TFPI的相互作用。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，Arg 257和Arg 259是必需的与肝素的相互作用，通过使用合成肽与C-末端碱性部分。我们利用杆状病毒系统分离重组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.