ANTICOAGULANT PROTEIN COMPLEX STRUCTURE AND FUNCTION

描述（申请人的逐字描述）： 抗凝系统通过蛋白水解消化起作用， 促凝血辅因子因子VIIIa和Va的下调 酶激活蛋白C（APC）。这个系统的遗传缺陷导致了 静脉血栓形成的先天性倾向。该路径在以下情况下启动 凝血酶（T）与内皮上的血栓调节蛋白（TM）结合， 辅因子依赖的变构构象变化。在TM界 构象，凝血酶失去其促凝血活性，并获得 可选的底物选择性。T-TM复合体现在识别酶原 蛋白C作为其底物，并非常有效地激活它。因此，主要 促凝血酶具有抗凝活性，并催化其自身的 作为该辅因子诱导的变构调节的结果的下调。的 这种转变的结构性质是完全未知的。 如果我们能在结构基础上理解这种特异性开关， 开始设计治疗剂， 从促凝血剂转变为抗凝血剂结果的基础上 感染性休克中APC输注，这种治疗方法导致更宽的窗口 而不会增加出血。我们建议研究其结构， T-TM复合物的功能与X射线晶体学，以了解 凝血酶抗凝血活性的结构起源。我们已经成长 衍射晶体的一种形式的这种复杂的，目前正在解决其 结构我们计划在这一初步成功的基础上， 相关的复合物，每一个都被设计成显示这种活性的另一个方面， 并将我们的发现应用于解释一些小的 分子和凝血酶点突变，以实现部分抗凝 活动最终，我们将进行基于结构的药物设计， 这些局部效应。

DESCRIPTION (Applicant's Description Verbatim): The enzymatic phase of the anticoagulation system functions by the proteolytic digestion and down-regulation of the procoagulant cofactors factors VIIla and Va by the enzyme activated protain C(APC). Hereditary defects in this system lead to a congenital predisposition to venous thrombosis. The pathway is initiated when thrombin (T) binds to thrombomodulin (TM) on the endothelium and undergoes a cofactor dependent allosteric conformational change. In the TM bound conformation, thrombin loses its procoagulant activity, and gains an alternative substrate selectivity. The T-TM complex now recognizes the zymogen protein C as its substrate and very efficiently activates it. Thus, the major procoagulant enzyme takes on an anticoagulant activity and catalyzes its own down-regulation as a result of this cofactor induced allosteric modulation. The structural nature of this transition is completely unknown. If we could understand this specificity switch on a structural basis, we might begin to design therapeutic agents which could bind to thrombin and shift it from a procoagulant towards an anticoagulant activity. Based on the results of APC infusion in septic shock, this therapeutic approach leads to a wider window of efficacy without increased bleeding. We propose to study the structure and function of the T-TM complex with X-ray crystallography to understand the structural origin of thrombin's anticoagulant activity. We have grown diffracting crystals of one form of this complex and are currently solving its structure. We plan to build on this initial success to solve the structures of related complexes, each designed to show an additional aspect of this activity, and to apply our findings towards explaining the ability of some small molecules and thrombin point mutations to achieve partial anti-coagulant activity. Ultimately we will undertake structure based drug design to improve these partial effects.