Platelet-Targeted Pro-urokinase for Thrombolysis

在出血风险高的环境中，预防术后深静脉血栓形成（DVT）和其他血栓性并发症仍然是一个重大的临床挑战。这项A1提案的重点是一种新的血栓预防策略，使用可凝血酶激活的尿激酶，前药通过allb/B3 （PLT/uPA-T）结合到血小板表面，选择性地靶向新生血栓，同时保留成熟血栓。我们正在寻求在其n端抗allb抗体可变区不同的结构，并且与激活的allb/B3 （LIB）与未激活的受体结合具有相当或更大的亲和力。我们的试点数据表明，PLT/uPA-Ts靶向预期受体，被凝血酶特异性激活，其纤溶活性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、安全的、有效的血栓预防方法，提供了前所未有的时空活性控制。我们现在希望通过动物模型的原理验证，将这些结构中的一种或多种推进到临床开发中，重点关注深静脉血栓预防。目的1：利用hallb*小鼠对PLT/uPA-T前药进行表征。这些小鼠是人类基因的转基因小鼠，缺乏小鼠基因。我们将利用创新的“双损伤”模型，研究药物分布及其对血小板生物学的影响。在该模型中，允许初始凝块（尾巴剪断）成熟不同时间，然后在该动物的下腔静脉诱导急性新生DVT。目的2：人血小板异种输血小鼠模型的体内研究。我们将确认关键的药理学和血栓预防功效研究，将人血小板输注到NOD/SCID/y-干扰素缺陷小鼠中，使用DVT模型。此外，一种新的微动脉和小静脉激光损伤模型可能为我们提出的血栓预防前药提供机制见解。目的3：狒狒（Pap/o anubis）模型的体内研究。人uPA在小鼠体内的纤溶酶原激活剂作用弱于人体内。为了更接近地模拟它们预期的临床应用，类似于Aim 1的关键研究将在狒狒身上进行。这些研究将包括一个成熟/新生的狒狒髂静脉血管闭塞模型的双损伤变异，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中提出的研究将延续本项目先前在allb/B3受体和尿激酶研究方面的研究成果，并将涉及与项目1在allb/B3生物学方面的相互作用和共享试剂、模型和见解，项目3在血小板活化方面，项目4了解血栓形成的细节。这些相互作用应该最大限度地提高我们成功开发这种新型血栓预防前药的可能性。

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.