Development of an ADAMTS13-based Therapy for Thrombosis

Abstract

Recombinant ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13)-based therapies were approved by the FDA to treat congenital thrombotic thrombocytopenic purpura (TTP), and preclinical studies have demonstrated its efficacy in mice. However, ADAMTS13 is susceptible to degradation in thrombo-inflammatory settings, which may reduce its therapeutic capacity. Therefore, we developed a protease-resistant (PR)-ADAMTS13 variant. Protease-sensitive sites on ADAMTS13 were mapped, replaced with glycine-serine linkers, and assessed for its resistance to coagulation- and neutrophil-mediated degradation. However, whether PR-ADAMTS13 maintains its functional activity in vivo is unknown. To continue the therapeutic development of PR-ADAMTS13, this thesis aims to validate thromboinflammation-mediated ADAMTS13 degradation, understand the potential mechanisms contributing to ADAMTS13 degradation in the context of TTP, and assess the therapeutic activity of PR-ADAMTS13 activity in vitro and in vivo. After incubating wild-type (WT) ADAMT13 with activated neutrophils in plasma, ADAMTS13 was readily degraded. Plasminogen activation was confirmed to be elevated in acute episodes of TTP but was independent of ADAMTS13 activity. Plasminogen activation exacerbated hemolytic anemia in TTP mice. Whereas tPA-mediated plasminogen activation was attenuated, uPA retained its capacity to stimulate plasminogen activation during platelet agglutination in plasma. Finally, PR-ADAMTS13 retained its functional activity, demonstrating the capacity to cleave vWF-platelet structures on endothelial cells under flow. A short half-life of 1.64 hours was observed, but PR-ADAMTS13 retained sufficient activity to reduce hemolytic anemia in a TTP mouse model. Overall, this thesis demonstrates thromboinflammation-mediated degradation of WT ADAMTS13, explores a uPA-driven mechanism of plasminogen activation in TTP, and validates the functional activity of a PR-ADAMTS13 variant both in vitro and in vivo. Future studies will focus on further characterizing the degradation resistance of PR-ADAMTS13 in vivo, as well as evaluating its safety profile and therapeutic efficacy in additional mouse models of thrombosis.