Structural Mechanisms for the Inhibition of Thrombosis

描述(由申请人提供)：本研究的长期目标是了解控制凝血酶活性的三个自然机制：1)通过抑制上游凝血因子Xa来防止凝血酶的形成；2)通过蛇抗凝血酶(AT)和肝素辅因子II(HCII)直接抑制凝血酶；以及3)通过辅因子结合和变构调节来减弱凝血酶的促凝活性。这将通过完成以下五个特定目标(SA)来实现：SA1-确定AT和凝血因子Xa之间的Michaelis络合物的晶体结构将确定AT识别凝血因子Xa的分子基础，凝血酶是负责形成凝血酶的蛋白酶。我们将通过创造AT和Xa因子的变体来解决AT和Xa因子之间的米氏复合体的晶体结构，这些变体旨在改善结晶或增加复合体的稳定性，以提高成功的可能性。SA2-确定AT的肝素结合机制，通过确定：a)AT如何与低亲和力肝素相互作用；b)AT与低亲和力肝素和五糖结合的高分辨率晶体结构；c)单体天然和激活AT的晶体结构；d)铰链区排出在肝素结合中的作用；以及e)静电在传播构象变化中的作用，来解决AT肝素结合特异性的基础问题。SA3-确定天然和寡糖激活的HCII的结构将定义肝素如何激活HCII对凝血酶的抑制。这一目标的成功完成将有助于阐明HCII激活的分子机制，并将有助于开发一类新型的抗凝血酶药物HCII激动剂。SA4-为了确定凝血酶变构的分子基础，研究了凝血酶活性如何通过与TM或与单价阳离子Na+结合而引起的构象变化来改变，方法是通过测定TM结合(活性部位无)凝血酶的晶体结构和无Na+(慢)形式的凝血酶。将使用2D核磁共振技术在溶液中验证结构。结晶学和溶液核磁共振相结合的方法将一劳永逸地建立凝血酶变构的分子基础。SA5-测定凝血酶的Na+结合特性研究了Na+变构的潜在相关性，这取决于生理条件下凝血酶对Na+的亲和力。滴定研究将准确地确定温度、离子强度、离子类型、pH和白蛋白浓度对凝血酶对Na+的表观Kd的影响，以建立Na+变构在凝血中的潜在相关性。成功完成这五个目标将大大提高我们对限制凝血酶活性的自然机制的了解，并可能导致开发新的治疗方法来预防和治疗血栓--血栓是美国和其他发达国家发病率和死亡的主要原因。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the three natural mechanisms which control the activity of thrombin, the final protease of the blood coagulation cascade: 1) prevention of thrombin formation through inhibition of upstream coagulation factor Xa; 2) direct inhibition of thrombin by serpins antithrombin (AT) and heparin cofactor II (HCII); and, 3) attenuation of thrombin's procoagulant activity through cofactor binding and allosteric modulation. This will be achieved by completing the following five specific aims (SA): SA1-To determine the crystal structures of the Michaelis complexes between AT and factor Xa will define the molecular basis of AT recognition of factor Xa, the protease responsible for thrombin formation. We will solve the crystal structures of the Michaelis complexes between AT and factor Xa in the absence and presence of heparin by creating variants of AT and factor Xa designed to improve crystallization or to increase the stability of the complex to improve the likelihood of success. SA2-To determine the heparin binding mechanism of AT addresses fundamental questions concerning the basis of heparin binding specificity of AT, by determining: a) how AT interacts with low-affinity heparin; b) high resolution crystal structures of AT bound to low and high- affinity heparin and pentasaccharides; c) crystal structures of monomeric native and activated AT; d) the role of hinge region expulsion in heparin binding; and, e) the role of electrostatics in propagating the conformational change. SA3-To determine the structures of native and oligosaccharide-activated HCII will define how heparin activates thrombin inhibition by HCII. Successful completion of this aim will elucidate the molecular mechanism of HCII activation, and will aid in the development of a novel class of anti-thrombin agents HCII agonists. SA4-To determine the molecular basis of thrombin allostery investigates how thrombin activity can be altered by conformational changes induced by binding to TM or to the monovalent cation Na+, by determining the crystallographic structures of TM-bound (active site-free) thrombin, and the Na+-free ('slow') form of thrombin. Structures will be validated in solution using 2D NMR techniques. The combined crystallographic and solution NMR approach will establish, once-and-for-all, the molecular basis of thrombin allostery. SA5-To determine the Na+ binding properties of thrombin investigates the potential relevance of Na+-allostery, which depends on the affinity of thrombin for Na+ under physiological conditions. Titration studies will accurately determine the effect of temperature, ionic strength, ion type, pH, and albumin concentration on the apparent Kd of thrombin for Na+, in order to establish the potential relevance of Na+ allostery in blood coagulation. Successful completion of these five aims will significantly improve our understanding of the natural mechanisms which limit the activity of thrombin, and could lead to the development of novel therapeutic approaches for the prevention and treatment of thrombosis-a leading cause of morbidity and death in the United States, and the rest of the developed world.