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.

Public Health Relevance

Project 2 focuses on a novel platelet-targeting, thrombin-activated thrombolytic prodrug that we believe offers enhanced safety and efficacy in preventing thrombosis. Using a number of informative and clinically-relevant animal models, we focus on demonstrating the utility of this therapy in preventing new deep vein thrombosis in the post-surgical setting, an important clinical challenge that still needs better therapeutic intervention. In the context of this Program Project, these studies should advance this novel therapy towards clinical application.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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University of Pennsylvania
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Tomaiuolo, Maurizio; Stalker, Timothy J; Welsh, John D et al. (2014) A systems approach to hemostasis: 2. Computational analysis of molecular transport in the thrombus microenvironment. Blood 124:1816-23
Welsh, John D; Stalker, Timothy J; Voronov, Roman et al. (2014) A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets. Blood 124:1808-15
Lian, Lurong; Suzuki, Aae; Hayes, Vincent et al. (2014) Loss of ATE1-mediated arginylation leads to impaired platelet myosin phosphorylation, clot retraction, and in vivo thrombosis formation. Haematologica 99:554-60
Stalker, Timothy J; Welsh, John D; Tomaiuolo, Maurizio et al. (2014) A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity. Blood 124:1824-31
Stalker, Timothy J; Welsh, John D; Brass, Lawrence F (2014) Shaping the platelet response to vascular injury. Curr Opin Hematol 21:410-7
Kowalska, M Anna; Zhao, Guohua; Zhai, Li et al. (2014) Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation. Arterioscler Thromb Vasc Biol 34:120-6
Min, Sang H; Suzuki, Aae; Stalker, Timothy J et al. (2014) Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice. Nat Commun 5:4691
Brass, Lawrence F; Tomaiuolo, Maurizio; Stalker, Timothy J (2013) Harnessing the platelet signaling network to produce an optimal hemostatic response. Hematol Oncol Clin North Am 27:381-409
Lu, Qiongyu; Dong, Ningzheng; Wang, Qi et al. (2013) Increased levels of plasma soluble Sema4D in patients with heart failure. PLoS One 8:e64265
Stalker, Timothy J; Traxler, Elizabeth A; Wu, Jie et al. (2013) Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood 121:1875-85

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