Developing Novel Platelet-Based Targeting Strategies for Thrombolytics

Use of plasminogen activators (PAs) as thrombolytic drugs is restricted to life threatening thrombotic settings because these therapies are associated with a high risk of bleeding. We hypothesize that platelet-delivered PAs would preferentially lyse nascent, pathological clots that are actively recruiting platelets, while sparing preformed hemostatic clots. Two potential approaches were pursued: 1) PA-loaded platelets that release the thrombolytic from its granular stores upon activation, and 2) a thrombolytic chimeric protein that specifically binds to human platelets and activated when the platelets are incorporated into a growing thrombus. In our first approach, we desired to develop a strategy for producing platelets ex-vivo from cultured megakaryocytes that ectopically expressed urokinase-PA (uPA). No group had successfully produced sufficient ex-vivo generated platelets before, to side step this issue we infused ex-vivo generated megakaryocytes and showed that we can achieve a significant number of donor-derived platelets from these infused megakaryocytes in a murine model. The resulting platelets were normal in size, surface markers, circulating half-life, and were functional. Infused megakaryocytes localized to the pulmonary vasculature to shed platelets. We demonstrated, beginning with megakaryocytes derived from a transgenic mouse that ectopically express and store uPA in their alpha-granules that we can interfere with thrombosis by platelets generated from these megakaryocytes. In the second approach we produced a chimeric protein by fusing a single chain variable fragment (scFv) directed to the human-αIIb (hαIIb) platelet receptor subunit, with a human thrombin activatable pro-urokinase (uPA-T). The fusion protein (anti-PLT scFv/uPA-T) bound specifically to human and to transgenic mice platelets that expressed hαIIb, termed hαIIb+ mice, but did not bind to wildtype (WT) mouse platelets. Anti-PLT scFv/uPA-T retained its zymogenic properties until activated by thrombin. HαIIb+ mice were protected from forming occlusive thrombi for at least 10 hrs post anti-PLT/uPA-T treatment in contrast to the short functional half-life of soluble uPA-T. Thus this dissertation presents two distinct strategies that in proof of principle studies are each promising as approaches for effective and targeted platelet directed thrombolytic, which merit further study to test clinical applicability. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Dr. Mortimer Poncz