FUNCTIONAL MAPPING AND PROTEIN ENGINEERING OF THROMBIN

凝血酶在功能上是活性的，相对不受肝素-ATIII的抑制。可能与溶栓后再血栓形成和深静脉血栓形成的扩散有关。丙氨酸扫描突变产生的凝血酶突变体库被用来定位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)凝血酶结合纤维蛋白凝块竞争。涉及8个残基的6个突变体在竞争中相对缺陷，这表明这些残基对与纤维蛋白的相互作用是重要的。将这些残基映射到凝血酶的表面上显示，它们聚集在与外切酶I和II不同的表面上，支持纤维蛋白结合部位由离散结构域介导的假设。在凝血酶上定位纤维蛋白结合位点将被表达出来，并直接与纤维蛋白凝块结合。更多涉及邻近残基的丙氨酸突变体将被用来更精确地定义纤维蛋白结合部位。通过鉴定特定的纤维蛋白结合部位，我们将合成取代纤维蛋白凝块中的凝血酶的多肽。将测试线状和环状多肽，并通过核磁共振波谱确定最有效的多肽的三级结构。它们作为一种新的抗血栓药物的有效性将在兔体内血栓形成模型中进行测试，这种药物没有内在的直接或间接凝血酶抑制作用，但在肝素存在或不存在的情况下利用血浆ATIII来抑制置换的凝血酶。具体目标3.在小鼠模型中测试纤维蛋白结合缺陷凝血酶最近，Sandra Degen博士利用ES细胞/基因打靶技术成功地培育出了半合子和纯合子凝血酶原缺陷小鼠。与Degen博士合作，我们将创造表达纤维蛋白结合缺陷凝血酶原突变体(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为零但携带突变转基因(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。这些研究将为凝血酶的纤维蛋白结合部位提供重要的结构和功能信息，并有助于确定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开发。

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.