Platelet-Targeted Pro-urokinase for Thrombolysis

预防术后深静脉血栓形成（DVT）和其他血栓性并发症，发生在出血风险高的设置仍然是一个重大的临床挑战。该A1提案集中于使用凝血酶可激活的尿激酶的新型血栓预防策略，所述尿激酶是通过allb/B3（PLT/uPA-T）与血小板表面结合的前药，其选择性靶向新生血栓，同时保留成熟血栓。我们正在寻求构建体，其N-末端抗allb抗体可变区不同，并且与活化的allb/B3（LIB）相比，与未活化的受体具有相当或更大的亲和力。我们的试验数据显示，PLT/uPA-Ts靶向预期的受体，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、安全的和有效的血栓预防方法，提供了前所未有的时空活性控制。我们现在希望通过原理验证动物模型将这些结构中的一种或多种推向临床开发，重点是DVT的血栓预防。目的1：使用hallb* 小鼠表征PLT/uPA-T前药。这些小鼠是人类allb的转基因小鼠，缺乏小鼠allb。我们将研究药物分布和对血小板生物学的影响，利用一个创新的“双损伤”模型。在该模型中，允许初始凝块（剪尾）成熟不同的时间，随后在该动物的下腔静脉中诱导急性新生DVT。目的2：人血小板异种输血小鼠模型的体内研究。我们将使用目的1中的DVT模型，使用人血小板输注到NOD/SCID/γ-干扰素缺陷小鼠中，确认关键药理学和血栓预防有效性研究。此外，一种新的提睾肌小动脉和小静脉激光损伤模型可能提供我们提出的血栓预防前药的机制的见解。目的3：狒狒（Pap/o anubis）模型的体内研究。人uPA在小鼠中是比在人中效力更低的纤溶酶原激活剂。为了更接近地模拟其预期临床应用，将在狒狒中进行与目标1中相似的关键研究。这些研究将包括狒狒髂静脉血管闭塞模型的成熟/新生双损伤变体，以确定PLT/uPA-T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中关于allb/B3受体和尿激酶研究的先前研究成果，并将涉及与项目1关于allb/B3生物学、项目3关于血小板活化和项目4了解血栓形成细节的相互作用和共享试剂、模型和见解。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.