FUNCTIONAL MAPPING AND PROTEIN ENGINEERING OF THROMBIN

凝块结合凝血酶具有功能活性，相对免受肝素- atiii抑制。它可能有助于溶栓后的再血栓形成和深静脉血栓的传播。利用丙氨酸扫描诱变产生的凝血酶突变体文库绘制凝血酶的纤维蛋白结合位点。用凝血酶突变体与125i野生型（WT）凝血酶与纤维蛋白凝块相竞争。6个涉及8个残基的突变体在竞争中相对缺陷，表明这些残基对与纤维蛋白的相互作用很重要。将这些残基映射到凝血酶表面显示，它们聚集在一个不同于外源位点I和II的表面上，支持纤维蛋白结合位点由一个离散结构域介导的假设。具体目标1。选定的取代WT凝血酶的能力显著降低的突变体将被表达并直接与纤维蛋白凝块结合。其他涉及邻近残基的丙氨酸突变体将被制成更精确地定义纤维蛋白结合位点。具体目标2。通过确定特定的纤维蛋白结合位点，我们将合成能够取代纤维蛋白凝块中的凝血酶的肽。线性肽和环化肽都将被测试，最有效的肽的三级结构将被核磁共振光谱确定。作为一种新型的抗血栓药物，它们没有内在的直接或间接的凝血酶抑制作用，而是在肝素存在或不存在的情况下利用血浆ATIII来抑制移位的凝血酶，它们的有效性将在兔体内血栓模型中进行测试。具体目标3。最近，Sandra Degen博士利用胚胎干细胞/基因靶向技术成功培育出半合子和纯合子凝血酶原缺陷小鼠。与Degen博士合作，我们将创建表达纤维蛋白结合缺陷凝血酶原突变（II+/+, TG+/-）的转基因小鼠，并与半合子凝血酶原缺陷（II+/-）小鼠杂交，获得内源性凝血酶原基因缺失但携带突变基因（II-/-, TG+/-）的小鼠。这些小鼠将在全身和局部静脉血栓模型中进行测试。这些研究将提供凝血酶纤维蛋白结合位点的重要结构和功能信息，并有助于确定凝块结合凝血酶在体内的作用。这也可能导致新的抗血栓药物和新的小鼠血栓模型的发展。

Clot-bound thrombin is functionally active and relatively protected from heparin-ATIII inhibition. It may contribute to re-thrombosis after thrombolysis and the propagation of deep venous thrombosis. A library of thrombin mutants generated by alanine scanning mutagenesis was used to map thrombin's fibrin binding site. Thrombin mutants were used to compete with 125I-wild type (WT) thrombin binding to fibrin clot. Six mutants involving eight residues were relatively defective in competition, suggesting that these residues are important for interaction with fibrin. Mapping these residues onto thrombin's surface shows that they are clustered together on a surface distinct from the exosites I and II, supporting the hypothesis that the fibrin-binding site is mediated by a discrete domain. Specific Aim number 1. Mapping the fibrin-binding site on thrombin Selected mutants with significantly diminished capability to displace WT thrombin will be expressed and direct binding to fibrin clot characterized. Additional alanine mutants involving neighboring residues will be made to more precisely define the fibrin-binding site. Specific Aim number 2. Development of peptides that will displace thrombin from fibrin clot By identifying the specific fibrin-binding site, we will synthesize peptides that will displace thrombin from fibrin clot. Both linear and cyclized peptides will be tested and the tertiary structure of the most potent peptide determined by NMR spectroscopy. Their efficacy as a new class of antithrombotic which has no intrinsic direct or indirect thrombin inhibition but will utilize plasma ATIII in the presence or absence of heparin to inhibit the displaced thrombin will be tested in a rabbit thrombosis model in vivo. Specific Aim number 3. Testing the fibrin-binding deficient thrombin in a mouse model Recently Dr. Sandra Degen has successfully generated hemizygous and homozygous prothrombin deficient mice using ES cell/gene targeting technology. In collaboration with Dr. Degen, we will create transgenic mice expressing the fibrin-binding deficient prothrombin mutant (II+/+, TG+/-) and crossing with hemizygous prothrombin deficient (II+/-) mice to obtain mice null for the endogenous prothrombin gene but carrying the mutant transgene (II-/-, TG+/-). These mice will be tested in systemic as well as local venous thrombosis models. These studies will provide important structural and functional information on thrombin's fibrin binding site and help to define the role of clot bound thrombin in vivo. It may also lead to the development of novel antithrombotic agents and new thrombosis models in the mouse.