Anticoagulant thrombins in vitro and in vivo

体外和体内抗凝凝血酶

基本信息

批准号：
7120523
负责人：
Enrico Di Cera
金额：
$ 49.87万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-15 至 2007-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Our laboratory has discovered that Na+ binding is required for the procoagulant, but not the anticoagulant activity of thrombin, and has elucidated the structural basis of this effect. This discovery has in turn enabled the rational engineering of thrombin mutants selectively compromised in their procoagulant properties. Recently, one of these mutants was tested in a primate model of platelet-dependent thrombosis and has shown safe and potent anticoagulant and antithrombotic effects, consistent with the predictions from in vitro studies. We are now in a position to bring the basic knowledge garnered from our in vitro studies and the exciting new developments of our preliminary in vivo studies to an entirely new level. In the proposed research project, we plan to rationally engineer new thrombin mutants with exclusive activity toward the anticoagulant protein C. We will thoroughly characterize these mutants in vitro and structurally. We will then use these mutants, together with the one already tested in vivo, to assess their effects on thrombosis and hemostasis in baboons and how in vivo properties correlate to those predicted from in vitro studies. We will also compare the effects of these mutants with the direct administration of activated protein C and other antithrombotic agents. We will finally use these mutants as tools to selectively deplete the plasma protein C pool and to assess the consequences of this depletion on the homeostatic balance between coagulation and anticoagulation. Developments from the proposed research plan will reveal new and rational strategies to re-engineer thrombin specificity and will have an enormous impact on molecular enzymology in general. In addition, they will reveal the true pharmacologic potential of engineered thrombin mutants in the control of clinical disorders that involve coagulation and thrombosis. Finally, these studies will produce valuable reagents to further elucidate the role of protein C in balancing the response of thrombin to vascular injury

描述（由申请人提供）：我们的实验室发现Na+结合是促凝剂所必需的，但凝血酶的抗凝活性不需要，并且已经阐明了这种作用的结构基础。这一发现反过来使凝血酶突变体的合理工程成为可能，选择性地损害其促凝血特性。最近，其中一种突变体在血小板依赖性血栓形成的灵长类动物模型中进行了测试，并显示出安全有效的抗凝和抗血栓作用，与体外研究的预测一致。我们现在能够将从体外研究中获得的基础知识和初步体内研究令人兴奋的新进展提升到一个全新的水平。在拟议的研究项目中，我们计划合理设计新的凝血酶突变体，使其对抗凝血蛋白C具有独特的活性。我们将在体外和结构上彻底表征这些突变体。然后，我们将使用这些突变体以及已经在体内测试的突变体，评估它们对狒狒的血栓形成和止血的影响，以及体内特性与体外研究预测的特性如何相关。我们还将比较这些突变体与直接施用活化蛋白 C 和其他抗血栓药物的效果。我们最终将使用这些突变体作为工具来选择性地消耗血浆蛋白 C 库，并评估这种消耗对凝血和抗凝之间稳态平衡的影响。拟议研究计划的进展将揭示重新设计凝血酶特异性的新的合理策略，并将对分子酶学产生巨大影响。此外，他们还将揭示工程凝血酶突变体在控制涉及凝血和血栓形成的临床疾病方面的真正药理学潜力。最后，这些研究将产生有价值的试剂，以进一步阐明蛋白 C 在平衡凝血酶对血管损伤的反应中的作用