Platelet-Targeted Pro-urokinase for Thrombolysis

预防术后深静脉血栓形成(DVT)和发生在出血风险高的环境中的其他血栓并发症仍然是一个重大的临床挑战。这份A1提案关注的是一种新的血栓预防策略，该策略使用凝血酶激活的尿激酶，前药通过Al1b/B3(PLT/uPA-T)结合到血小板表面，选择性地针对新生的血栓，同时保留成熟的血栓。我们正在寻找在其N端抗Allb抗体可变区不同，并与激活的Allb/B3(Lib)与未激活的受体具有类似或更大亲和力的结合结构。我们的初步数据显示，PLT/uPA-ts针对预期的受体，可被凝血酶特异性激活，其纤溶活性可被临床使用的赖氨酸类似物氨甲环酸阻止。基于这些数据和小鼠止血模型，我们认为PLT/uPA-ts代表了一种新的、安全和有效的血栓预防方法，提供了前所未有的时空活动控制。我们现在希望通过原理验证的动物模型，重点研究DVT的血栓预防，将这些结构中的一个或多个推向临床开发。目的1：用Hallb*小鼠鉴定PLT/uPA-T前药。这些小鼠是人类Allb转基因小鼠，缺乏小鼠Allb。我们将利用创新的“双损伤”模型，研究药物在体内的分布及其对血小板生物学的影响。在这个模型中，最初的凝块(尾部剪断)被允许成熟不同的时间，然后在该动物的下腔静脉诱导急性新生DVT。目的2：人血小板异种输注小鼠模型的体内研究。我们将使用与目标1相同的DVT模型，使用NOD/SCID/y干扰素缺陷小鼠体内的人血小板，确认关键的药理和血栓预防效果研究。此外，一种新的提睾肌小动脉和小静脉激光损伤模型可能为我们建议的血栓预防前药物提供机制方面的见解。目的3：在恒河猴(Pap/o Anubis)模型中进行体内研究。人uPA在老鼠体内的纤溶酶原激活剂比在人类体内更弱。为了更好地模拟其预期的临床应用，将在狒狒身上进行类似于目标1中的关键研究。这些研究将包括一个成熟的/新生的两次损伤变种的狒狒髂静脉血管闭塞模型，以确定PLT/uPA-TS在大型动物模型中的有效性和安全性。项目2中提议的研究将继承本计划项目中关于Allb/B3受体和尿激酶研究的先前的研究工作，并将涉及与项目1关于Allb/B3生物学的相互作用和共享试剂、模型和见解，与项目3关于血小板激活的研究，以及与项目4了解血栓形成的细节。这些相互作用将最大限度地提高我们成功开发这种用于预防血栓的新型前体药物的可能性。

Prevention of post-operative deep vein thrombosis (DVT) and other thrombotic complications that occur in settings where the risk of bleeding is high remains a significant clinical challenge. This A1 proposal focuses on a novel thromboprophylactic strategy using a thrombin-activatable urokinase, prodrug bound to the platelet surface through allb/B3 (PLT/uPA-T) that selectively targets nascent thrombi, while sparing mature clots. We are pursuing constructs that differ in their N-terminal anti-allb antibody variable regions and bind with comparable or with greater affinity to activated allb/B3 (LIB) vs. unactivated receptor. Our pilot data show that-the PLT/uPA-Ts target the expected receptor, are specifically activated by thrombin, and their fibrinolytic activity is prevented by the clinically employed lysine analogue, tranexamic acid. Based on these data and murine hemostatic models, we believe that PLT/uPA-Ts represent a new, safe, and effective approach to thromboprophylaxis affording unprecedented spatiotemporal control of activity. We now wish to advance one or more of these constructs towards clinical development through proof-of-principle animal models, focusing on thromboprophylaxis of DVT. Aim 1: Characterization of PLT/uPA-T prodrugs using hallb* mice. These mice are transgenic for human allb and lack mouse allb. We will study drug distribution and effects on platelet biology, making use of an innovative "two-injury" models. In this model, an initial clot (tail snip) is allowed to mature for varying times followed by induction of an acute nascent DVT in the inferior venae cava in that animal. Aim 2: In vivo studies in a human platelet xenotransfusion mice model. We will confirm key pharmacologic and thromboprophylaxis efficacy studies using human platelets infused into NOD/SCID/y-interferon-deficient mice using DVT models as in Aim 1. In addition, a novel cremaster arteriole and venule laser injury model may provide mechanistic insights underlying our proposed thromboprophylactic prodrugs. Aim 3: In vivo studies in a baboon (Pap/o anubis) model. Human uPA is a less potent plasminogen activator in mice than in humans. To more closely simulate their intended clinical application, key studies similar to those in Aim 1 will be conducted in baboons. These studies will include a mature/nascent two-injury variant of the baboon iliac vein vascular occlusion model to define the efficacy and safety of the PLT/ uPA-Ts in a large animal model. The studies proposed in Project 2 will carry forward prior research efforts in this Program Project on the allb/B3 receptor and on studies of urokinase, and will involve interactions and shared reagents, models and insights with Project 1 on the biology of allb/B3, Project 3 on platelet activation and with Project 4 understanding the details of thrombus development. These interactions should maximize the likelihood of our successful development of this novel prodrug for thromboprophylaxis.