tPA-binding RNA Aptamers

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

  • Nils Bjerregaard
The serine protease Tissue-type Plasminogen Activator (tPA) is the principal initiator of fibrinolysis in mammalian physiology. Recombinant tPA is employed in the pharmacological resolution of vessel occlusions caused by pathological thrombosis, the originating cause of cerebral ischaemic strokes. Globally, stroke is the leading cause of disability and the third leading cause of mortality. Fibrinolytic therapy with recombinant tPA rescues suboptimally perfused penumbral nervous tissue, but has also been implicated in detrimental neurotoxic effects, which are chiefly mediated by the Low-density lipoprotein receptor Related Protein-1 (LRP-1). Here, we describe the selection and characterisation of structured RNA ligands (“RNA aptamers”) to tPA, K18 and K32. Both aptamers were truncated to minimal 32-nucleotide constructs (v2) with improved or unchanged activities, and were shown to bind tPA with low nanomolar affinities and efficiently inhibit tPA-LRP-1 binding and LRP-1 mediated cellular endocytosis. Both aptamers minimally affected the fibrinolytic properties of tPA despite efficiently inhibiting plasminogen activation stimulated by a soluble fibrin fragment. K18v2 additionally inhibited plasminogen activation in solution, and inhibition of tPA by PAI-1, the endogenous regulator of tPA activity. PAI-1 binds via the 37-loop residues of the serine protease domain of tPA, which were also found to mediate the K18v2 interaction. K18v2 and K32v2 retained their activites in physiologically relevant conditions, and upon conjugation to serum albumin. K18v2 was able to inhibit tPA-induced fibrinogen depletion in vitro, which may provide additional benefits in stroke treatment. A conjugate of both aptamers separated by a linker encompassed the activities of both constituent sequences, and additionally possessed equivalent activity at concentrations reduced by approximately two orders of magnitude. The present results suggests beneficial effects of coadministration of K18v2 or K32v2 in the fibrinolytic treatment of iscahemic stroke, and potential applications of the conjugate aptamer in the management of bleeding disorders.
Antal sider161
Rekvirerende organGraduate School of Science and Technology
StatusUdgivet - 9 dec. 2015

Note vedr. afhandling

Cerebral ischaemic stroke is the leading cause of disability and the third leading cause of mortality in the world. Currently, intravenous administration of recombinant tissue-type plasminogen activator (tPA), a fibrinolytic serine protease, is the only pharmacological treatment of cerebral ischaemic stroke approved by the European Medicines Agency and the American Food and Drug Administration. However, several lines of evidence implicate circulating tPA in detrimental effects, including neurotoxicity and haemorrhagic transformation, especially in an ischaemic context. The low-density lipoprotein receptor related protein-1 (LRP-1) is a central mediator of the adverse effects ascribed to tPA.

During his PhD studies, Nils Bjerregaard has characterised structured synthetic RNA ligands (“aptamers”) to tPA capable of inhibiting the interaction of tPA with LRP-1 while minimally affecting the crucial fibrinolytic properties of the protease. These aptamers may improve the safety and efficacy of fibrinolytic therapy with tPA. A conjugate construct combining two different aptamers in a single molecule possessed activity two orders of magnitude higher than each constituent aptamer, and was an efficient inhibitor of fibrinolysis with potential applications in the treatment of bleeding disorders or the management of bleeding during surgical procedures.

The PhD degree was completed at the Department of Molecular Biology and Genetics, Faculty of Science and Technology, Aarhus University.

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