Anticoagulant thrombins in vitro and in vivo

描述（由申请人提供）：我们实验室发现促凝剂需要Na+结合，而凝血酶的抗凝活性不需要Na+结合，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选择性地损害了它们的促凝特性。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血和抗血栓作用，与体外研究的预测一致。我们现在处于一个位置，将我们从体外研究中获得的基本知识和我们初步体内研究的令人兴奋的新发展带到一个全新的水平。在本研究项目中，我们计划合理设计具有抗凝血蛋白c专属活性的新型凝血酶突变体。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，以及体内特性如何与体外研究预测的特性相关联。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。最后，我们将使用这些突变体作为工具，选择性地消耗血浆蛋白C池，并评估这种消耗对凝血和抗凝血之间稳态平衡的影响。该研究计划的发展将揭示新的、合理的策略来重新设计凝血酶的特异性，并将对分子酶学产生巨大的影响。此外，它们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，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