Modulating Fibrinolysis Dynamics by Leveraging Multivalent Avidity to Control Enzyme Activity

项目摘要 血凝块比任何其他单一致病原因都要杀死更多的人。新的或复发性静脉炎诊断 血栓栓塞（VTE），包括肺栓塞（PE）和深静脉血栓形成 (DVT)仅在美国，每年就有超过90万人受到影响。目前的PE标准治疗包括 急性血凝块消化酶，如重组组织纤溶酶原激活剂（tPA），静脉内给药或 通过导管。所有目前FDA批准的凝块消化酶都依赖于患者内源性凝血因子的激活。 纤溶酶原几乎将其全部转化为活性凝块消化纤溶酶。这种广泛的激活 耗尽纤溶酶调节蛋白，导致病原体和有益血液的不受控制的消化 血栓导致5-10%的严重出血并发症，包括颅内出血。由于 安全性问题和禁忌症，近50%的患者将受益于主动血凝块消化 干预措施不符合接受当前治疗的条件。直接输注外源性活性纤溶酶， 由于循环α2-抗纤溶酶（丝氨酸蛋白酶抑制剂）几乎瞬间失活， 蛋白）和α2-巨球蛋白（空间抑制剂）。为了解决目前纤溶酶输注作为一种治疗方法的局限性， 直接纤维蛋白溶解治疗干预，一种利用多价 酶控制是必要的。初步数据表明，可逆的竞争性抑制剂可以 用于递送活性纤溶酶，其在体外减少了调节蛋白的失活，同时仍然允许 有效的凝块消化;然而，迫切需要确定负责的潜在特征 用于保护和递送纤溶酶作为体内直接的纤溶酶。该提案将确定关键的 有助于体内活性酶的多价控制以用于新的临床翻译的关系 治疗性干预措施，以消化血块，提高安全性。目标1：确定同性恋- 和异源多价抑制剂影响纤溶酶的抑制及其与调节蛋白的相互作用， 体外;目的2：评价可逆多价酶抑制对体外培养的人血凝块消化效率的影响。 剪切下的标准化离体血凝块;以及，目标3：检查多价抑制剂的影响 递送的纤溶酶对凝块的靶向和体内消化。总体而言，这些实验将确定关键 多价酶的传递原理，并提供了先导化合物，以进一步发展。更安全的直接 纤维蛋白溶解治疗剂将具有扩展的使用适应症，包括：缺血性中风、心肌梗塞、心肌梗塞、脑梗塞、脑梗塞和脑梗塞。 梗死、肢体缺血、PE、DVT和血管导管堵塞。

PROJECT SUMMARY Blood clots kill more humans than any other single pathogenic cause. A new or recurrent diagnosis of venous thromboembolism (VTE), which encompasses both pulmonary embolism (PE) and deep vein thrombosis (DVT), affects >900,000 people each year in the US alone. Current PE standard of care includes the delivery of acute clot digesting enzymes such as recombinant tissue Plasminogen Activator (tPA), administered IV or by catheter. All currently FDA cleared clot digesting enzymes rely on activation of the patient’s endogenous plasminogen converting nearly all of it to active clot digesting plasmin. This widespread activation massively depletes plasmin regulatory proteins, causing unchecked digestion of both pathogenic and beneficial blood clots resulting in a 5-10% rate of major bleeding complications, including intracranial hemorrhage. Due to safety concerns and contraindications nearly 50% of patients that would benefit from active clot digesting interventions are not eligible to receive the current therapy. Direct infusion of exogenous active plasmin to digest blood clots is limited due to the nearly instantaneous inactivation by circulating α2-antiplasmin (serpin protein) and α2-macroglobulin (steric inhibitor). To address the current limitations of plasmin infusion as a direct fibrinolytic therapeutic intervention, a novel clot digesting delivery strategy leveraging multivalent enzymatic control is necessary. Preliminary data demonstrates that reversible competitive inhibitors can be used to deliver active plasmin with reduced inactivation from regulatory proteins in-vitro while still allowing efficient clot digestion; however, there is critical need to determine the underlying characteristics responsible for protection and delivery of plasmin as a direct fibrinolytic in-vivo. This proposal will identify critical relationships that contribute to multivalent control of active enzymes in-vivo for the novel clinical translation of therapeutic interventions to digest blood clots with an improved safety profile. Aim 1: Determine how homo- and hetero-multivalent inhibitors impact inhibition of plasmin and its interactions with regulatory proteins in- vitro; Aim 2: Evaluate the impact of reversible multivalent enzyme inhibition on clot digestion efficiency of standardized ex-vivo blood clots under shear; and, Aim 3: Examine the impact of multivalent inhibitor delivered plasmin on clot targeting and digestion in-vivo. Overall, these experiments will identify key multivalent enzyme delivery principles and provide lead compounds to be further developed. A safer direct fibrinolytic therapeutic will have expanded indications for use that include: ischemic stroke, myocardial infarction, limb ischemia, PE, DVT, and occluded vascular catheters.