Efficacy of Kunitz Domain 1 variants of TFPI-2 as novel antifibrinolytic agents

描述（由申请方提供）：本提案的目的仍然是测试一种新型独特的纤溶酶抑制剂，该抑制剂有可能减少大手术和凝血病中的严重出血。直到最近，另一种纤溶酶抑制剂，称为抑肽酶，被用作抗纤维蛋白溶解剂，以减少心脏，器官移植，整形外科和血管手术中的失血量。事实上，抑肽酶是FDA批准的唯一一种减少心肺转流心脏手术输血的药物。1999年报道的三项抑肽酶荟萃分析表明，抑肽酶降低了死亡率（2倍）、手术再探查的发生率以及输血。然而，在最近的一项多机构研究中，据报道抑肽酶可显著增加肾、心血管和神经损伤的风险，这是基于其抑制激肽释放酶并因此抑制激肽形成的能力，从而导致器官血流的自动调节紊乱。使用晶体学，分子建模和生物化学方法，我们产生了两个变体的第一个Kunitz域（KD 1）的人组织因子途径抑制剂-2（TFPI-2），这是显着更有效的纤溶酶抑制剂相比，野生型KD 1或全长TFPI-2。初步数据揭示了KD 1变体强烈抑制人和小鼠纤溶酶的独特能力，而对激肽释放酶的抑制最小或没有抑制。基于KD 1变体的这一基本性质，其提供了优于抑肽酶的明显优势，我们假设KD 1变体将是有效的抗纤维蛋白溶解剂，而不会引起与激肽释放酶抑制相关的显著器官损伤。拟议的研究旨在使用两个特定目标确定KD 1变体的疗效和毒性。在具体目标1中，我们将在E.大肠杆菌，进一步表征它们与纤溶酶、组织和血浆激肽释放酶以及参与凝血级联的其他蛋白酶的结合。此外，我们将获得晶体结构，以验证KD 1变体和纤溶酶之间的分子相互作用。在具体目标2中，我们将在动物模型中比较KD 1变体与抑肽酶在减少大血管和小血管出血方面的功效。此外，我们将在动物模型中研究KD 1变体对重要器官的毒性，并将其与抑肽酶进行比较。我们预计，KD 1变体将减少总失血量，而不会对器官功能产生不良影响。本提案的长期目标是研究KD 1变体在体外循环心脏手术动物模型中的疗效和毒性。公共卫生相关性：在心脏直视手术和器官移植的患者中，由于纤溶酶分解纤维蛋白而导致大量失血。抑肽酶是一种用于抑制纤溶酶的抗纤维蛋白溶解药物，2007年停止使用，因为发现它会导致严重的器官损伤。在这里，我们建议测试一种新药，我们的实验室设计用于减少高风险心脏手术和移植患者的出血。

DESCRIPTION (provided by applicant): The goal of this proposal remains to test a novel and unique inhibitor of plasmin that has the potential to reduce severe bleeding in major surgery as well as in coagulopathy. Until recently, another inhibitor of plasmin, called aprotinin was used as an antifibrinolytic agent to reduce blood loss in cardiac, organ transplant, orthopedic and vascular surgery. In fact, aprotinin was the only FDA-approved drug to reduce blood transfusion in cardiac surgery with cardiopulmonary bypass. Three meta-analyses of aprotinin reported in 1999 indicated that aprotinin decreased mortality (by two fold), the incidence of surgical reexploration, as well as blood transfusions. However, in a recent multi-institutional study, aprotinin was reported to significantly increase the risk of renal, cardiovascular and neurological injury, based on its ability to inhibit kallikrein and therefore kinin formation, leading to disturbed autoregulation of organ blood flow. Using crystallography, molecular modeling and biochemical approaches, we generated two variants of the first Kunitz domain (KD1) of human tissue factor pathway inhibitor-2 (TFPI-2) that are significantly more potent inhibitors of plasmin as compared to the wild-type KD1 or to the full-length TFPI-2. Preliminary data revealed the unique ability of KD1 variants to strongly inhibit human and mouse plasmin with minimal or no inhibition of kallikrein. Based upon this fundamental property of the KD1 variants, which provides a distinct advantage over aprotinin, we hypothesize that KD1 variants will be effective antifibrinolytic agents without causing significant organ injury related to the inhibition of kallikreins. The proposed studies are designed to determine the efficacy and toxicity of KD1 variants using two specific aims. In specific aim 1, we will express wild type KD1 and the two variants (one modified at P2' residue and the second at P1 and P2' residues) in E. coli, further characterize their binding to plasmin, tissue and plasma kallikreins and other proteases involved in the coagulation cascade. Further, we will obtain crystal structures to validate the molecular interactions between the KD1 variants and plasmin. In specific aim 2, we will compare the efficacy of the KD1 variants to aprotinin in reducing bleeding from large and small vessels in animal models. Further, we will study the toxicity of the KD1 variants to the vital organs in the animal models and compare it to aprotinin. We anticipate that KD1 variants will reduce total blood loss without having an adverse effect on organ function. The long term goals of this proposal are to study the efficacy and toxicity of KD1 variants in animal models of cardiac surgery with cardiopulmonary bypass. PUBLIC HEALTH RELEVANCE: In patients with open-heart surgery and organ transplant, there is significant blood loss due to breakdown of fibrin by plasmin. Aprotinin, an antifibrinolytic drug that has been used to inhibit plasmin was discontinued in 2007 as it was found to cause significant organ injury. Here, we are proposing to test a new drug that our laboratory has designed to reduce bleeding in high-risk cardiac surgery and transplant patients.