Anticoagulant thrombins in vitro and in vivo

描述(申请人提供)：我们的实验室发现促凝血剂需要Na+结合，但凝血酶的抗凝血活性不需要，并阐明了这种作用的结构基础。这一发现反过来又使凝血酶突变体的合理工程得以实现，这些突变体选择性地损害了它们的促凝血特性。最近，这些突变体中的一个在灵长类动物的血小板依赖性血栓形成模型中进行了测试，并显示出安全而有效的抗凝和抗血栓作用，与体外研究的预测一致。我们现在能够将从我们的体外研究中获得的基本知识和我们体内初步研究的令人兴奋的新发展提升到一个全新的水平。在拟议的研究项目中，我们计划合理地设计出对抗凝蛋白C具有专属活性的新的凝血酶突变体。我们将在体外和结构上对这些突变体进行彻底的表征。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，来评估它们对狒狒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之间的关系。我们还将比较这些突变体与直接使用活化蛋白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耗尽血浆蛋白C池，并评估这种耗竭对凝血和抗凝之间的动态平衡的影响。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和合理的策略，并将对一般的分子酶学产生巨大的影响。此外，它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

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