STUDIES ON ANTITHROMBIN III-THROMBIN-HEPARIN REACTIONS

该项目的长期目标是阐明 通过研究抗凝血酶III的化学性质， 肝素结合位点和蛋白酶结合的结构关系 在这个蛋白质中。 具体而言，目标将是：（1） 通过鉴定肝素结合位点的性质， 与肝素相互作用的特异性赖氨酸残基。 这部分 研究将涉及抗凝血酶III的赖氨酸修饰， 使用三硝基苯磺酸和还原剂， 甲基化（HCHO/NaBH 4）。 被肝素掩盖的特异性赖氨酸残基 将通过放射性同位素和氰的分析来鉴定 溴化物和蛋白水解片段。 (2)为了阐明 蛋白酶结合位点的动力学和构象的调查 凝血酶和Xa因子与重折叠抗凝血酶结合的方面 III -抗凝血酶III的中间形式，已失去60%肝素 辅因子活性和42%的α-螺旋已知存在于 蛋白质，但仍具有结合凝血酶的能力。 这部分 该研究将使用构象敏感工具如圆二色性， 荧光和吸收差光谱。 第一个目标 旨在测试肝素结合位点模型（肽）的有效性 段281-289）由主要研究者预测。 第二 目的是为了证明是否独立， 抗凝血酶中存在非相互作用的蛋白酶和肝素结合位点 三. 预计本项目中提出的调查， 将利用抗凝血酶III的改变形式将提供有用的 一个模型，通过它我们可以了解抗凝血酶III的复杂功能 并最终开发出生理调节抗凝血酶的方法 三. 拟议的研究，以确定赖氨酸残基的必要性， 肝素结合有望为更好地理解 抗凝血酶III影响血液凝固的机制。 在这项研究的其他成果中， 鉴定可能被认为是 血浆蛋白酶-蛋白酶抑制剂相互作用的一般特征。 是 预期与分子构象的细节一起揭示了 通过这些研究中提出的各种生物物理方法，相关性可以 在抗凝血酶III的分子顺序和其生物学性质之间进行 活动

The long term goal of the proposed project is to elucidate the mechanism of ation of antithrombin III by investigation of the chemical nature and structural relationships of the heparin binding site and protease binding site in this protein. Specifically, the objectives will be: (1) to elucidate the nature of the heparin binding site by identifying the specific lysine residues which interact with heparin. This part of the study will involve lysine modification of antithrombin III in the presence and absence of heparin using trinitrobenzenesulfonic acid and reductive methylation (HCHO/NaBH4). The specific lysine residues masked by heparin will be identified by using radioactive isotopes and analysis of cyanogen bromide and proteolytic fragments. (2) to elucidate the nature of the protease binding site by investigation of the kinetics and conformational aspects of the binding of thrombin and Factor Xa with refolded antithrombin III - an intermediate form of antithrombin III which has lost 60% heparin cofactor activity and 42% of the Alpha-helix known to be present in the protein but still possesses the capacity to bind thrombin. This part of the study will use conformation-sensitive tools such as circular dichroism, fluorescence and absorption difference spectroscopy. The first objective intends to test the validity of the heparin binding site model (peptide segment 281-289) predicted by the principal investigator. The second objective is intended to prove whether or not independent and non-interacting protease and heparin binding sites exist in antithrombin III. It is expected that the investigations proposed in this project which will utilize an altered form of antithrombin III will provide a useful model by which we can understand the complex function of antithrombin III and ultimately develop methods for physiological regulation of antithrombin III. The proposed study to identify the lysine residues essential for heparin binding is expected to provide information for better understanding of the mechanism by which antithrombin III affects blood coagulation. Among other outcomes visualized from this research is the possible identification of a conformational mechanism which might be considered a general feature of plasma protease-proteinase inhibitor interaction. It is expected that together with the details of molecular conformation revealed by various biophysical methods proposed in these studies, correlation can be made between the molecular order of antithrombin III and its biological activities.