Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation

Summary

Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipo-protein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the principal initiator of thrombolysis in human physiology, an effect that is mediated directly via localised activation of the plasmin zymogen plasminogen at the surface of fibrin clots in the vascular lumen. Here, we sought to identify a ligand to tPA capable of inhibiting the relevant LDL family receptors without interfering with the fibrinolytic activity of tPA. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to isolate tPA-binding RNA aptamers, which were characterised in biochemical assays of tPA association to low density lipoprotein receptor-related protein-1 (LRP-1, an LDL receptor family member); tPA-mediated in vitro and ex vivo clot lysis; and tPA-mediated plasminogen activation in the absence and presence of a stimulating soluble fibrin fragment. Two aptamers, K18 and K32, had minimal effects on clot lysis, but were able to efficiently inhibit tPA-LRP-1 association and LDL receptor family-mediated endocytosis in human vascular endothelial cells and astrocytes. These observations suggest that coadministration alongside tPA may be a viable strategy to improve the safety of thrombolytic treatment of cerebral ischaemic stroke by restricting tPA activity to the vascular lumen.

Keywords

Fibrin clot lysis time - low density lipoprotein receptor-related protein-1 - nucleic acid aptamer - stroke - tissue-type plasminogen activator

• References

• 1 Astrup T. The Biological Significance of Fibrinolysis. Lancet 1956; 268: 565-568.
• 2 Collen D. The plasminogen (fibrinolytic) system. Thromb Haemost 1999; 82: 259-270.
• 3 Murray CJ, Lopez AD. Mortality by cause for eight regions of the world: Global Burden of Disease Study. Lancet 1997; 349: 1269-1276.
• 4 Yepes M, Sandkvist M, Moore EG. et al. Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor – related protein. J Clin Invest 2003; 112: 1533-1540.
• 5 Gliemann J, Nykjaer A, Petersen CM. et al. The multiligand alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/ LRP). Binding and endocytosis of fluid phase and membrane-associated ligands. Ann NY Acad Sci 1994; 737: 20-38.
• 6 Herz J. LRP: a multifunctional scavenger and signaling receptor. J Clin Invest 2001; 108: 779-784.
• 7 Bassel-Duby R, Jiang NY, Bittick T. et al. Tyrosine 67 in the epidermal growth factor-like domain of tissue-type plasminogen activator is important for clearance by a specific hepatic receptor. J Biol Chem 1992; 267: 9668-9677.
• 8 Bu G, Warshawsky I, Schwartz AL. Cellular receptors for the plasminogen activators. Blood 1994; 83: 3427-3436.
• 9 Cassé F, Bardou I, Danglot L. et al. Glutamate controls tPA recycling by astrocytes, which in turn influences glutamatergic signals. J Neurosci 2012; 32: 5186-5199.
• 10 Rathore YS, Rehan M, Pandey K. et al. First structural model of full-length human tissue-plasminogen activator: a SAXS data-based modeling study. J Phys Chem B 2012; 116: 496-502.
• 11 Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Hae-most 2005; 03: 1894-1904.
• 12 Medved L, Nieuwenhuizen W. Molecular mechanisms of initiation of fibrinolysis by fibrin. Thromb Haemost 2003; 89: 409-419.
• 13 Wang YF, Tsirka SE, Strickland S. et al. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischaemia in wild-type and tPA-deficient mice. Nat Med 1998; 04: 228-231.
• 14 Yepes M, Sandkvist M, Wong MK. et al. Neuroserpin reduces cerebral infarct volume and protects neurons from ischaemia-induced apoptosis. Blood 2000; 96: 569-576.
• 15 Tsirka SE, Gualandris A, Amaral DG. et al. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature 1995; 377: 340-344.
• 16 Nicole O, Docagne F, Ali C. et al. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med 2001; 07: 59-64.
• 17 Indyk JA, Chen ZL, Tsirka SE. et al. Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury. Neuroscience 2003; 116: 359-371.
• 18 Junge CE, Sugawara T, Mannaioni G. et al. The contribution of protease-activated receptor 1 to neuronal damage caused by transient focal cerebral ischaemia. Proc Natl Acad Sci USA 2003; 100: 13019-13024.
• 19 Wang X, Lee S-RS-R, Arai K. et al. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med 2003; 09: 1313-1317.
• 20 Melchor JP, Strickland S. Tissue plasminogen activator in central nervous system physiology and pathology. Thromb Haemost 2005; 93: 655-660.
• 21 Rogove AD, Siao C, Keyt B. et al. Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system. J Cell Sci 1999; 112: 4007-4016.
• 22 Jin R, Yang G, Li G. Molecular insights and therapeutic targets for blood-brain barrier disruption in ischaemic stroke: critical role of matrix metalloproteinases and tissue-type plasminogen activator. Neurobiol Dis Elsevier Inc.; 2010; 38: 376-385.
• 23 Benchenane K, Berezowski V, Ali C. et al. Tissue-type plasminogen activator crosses the intact blood-brain barrier by low-density lipoprotein receptor-related protein-mediated transcytosis. Circulation 2005; 111: 2241-2249.
• 24 Willnow TE, Rohlmann A, Horton J. et al. RAP, a specialized chaperone, prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors. EMBO J 1996; 15: 2632-2639.
• 25 Ballabh P, Braun A, Nedergaard M. The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiol Dis 2004; 16: 1-13.
• 26 Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science (80-) 1990; 249: 505-510.
• 27 Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature 1990; 346: 818-822.
• 28 Bouchard PR, Hutabarat RM, Thompson KM. Discovery and development of therapeutic aptamers. Annu Rev Pharmacol Toxicol 2010; 50: 237-257.
• 29 Blake CM, Sullenger BA, Lawrence DA. et al. Antimetastatic potential of PAI-1-specific RNA aptamers. Oligonucleotides 2009; 19: 117-128.
• 30 Madsen JB, Dupont DM, Andersen TB. et al. RNA aptamers as conformational probes and regulatory agents for plasminogen activator inhibitor-1. Biochemistry 2010; 49: 4103-4115.
• 31 Dupont DM, Madsen JB, Hartmann RK. et al. Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding. RNA 2010; 16: 2360-2369.
• 32 Botkjaer KA, Deryugina EI, Dupont DM. et al. Targeting tumor cell invasion and dissemination in vivo by an aptamer that inhibits urokinase-type plasminogen activator through a novel multifunctional mechanism. Mol Cancer Res 2012; 10: 1532-1543.
• 33 Damare J, Brandal S, Fortenberry YM. Inhibition of PAI-1 Antiproteolytic Activity Against tPA by RNA Aptamers. Nucleic Acid Ther 2014; 00: 1-11.
• 34 Dupont DM, Andersen LM, Botkjaer KA. et al. Nucleic acid aptamers against proteases. Curr Med Chem 2011; 18: 4139-4151.
• 35 Ng EWM, Shima DT, Calias P. et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov 2006; 05: 123-132.
• 36 Deutsch DG, Mertz ET. Plasminogen: Purification from Human Plasma by Affinity Chromatography. Science 1970; 170: 1095-1096.
• 37 Steffen P, Voss B, Rehmsmeier M. et al. RNAshapes: an integrated RNA analysis package based on abstract shapes. Bioinformatics 2006; 22: 500-503.
• 38 Wiborg Simonsen AC, Heegaard CW. et al. Very low density lipoprotein receptor from mammary gland and mammary epithelial cell lines binds and mediates endocytosis of Mr, 40,000 receptor associated protein. FEBS Lett 1994; 354: 279-283.
• 39 Andersen OM, Petersen HH, Jacobsen C. et al. Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein. Biochem J 2001; 357: 289-296.
• 40 Jensen P, Christensen E, Ebbesen P. et al. Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells. Cell Regul 1990; 01: 1043-1056.
• 41 Argraves KM, Battey FD, MacCalman CD. et al. The Very Low Density Lipoprotein Receptor Mediates the Cellular Catabolism of Lipoprotein Lipase and Urokinase-Plasminogen Activator Inhibitor Type I Complexes. J Biol Chem 1995; 270: 26550-26557.
• 42 Le Jan S, Le Meur N, Cazes A. et al. Characterization of the expression of the hypoxia-induced genes neuritin, TXNIP and IGFBP3 in cancer. FEBS Lett 2006; 580: 3395-3400.
• 43 Cederholm-Williams S. Concentration of plasminogen and antiplasmin in plasma and serum. J Clin Pathol 1981; 34: 979-981.
• 44 Yao Y, Tsirka SE. Truncation of monocyte chemoattractant protein 1 by plasmin promotes blood-brain barrier disruption. J Cell Sci 2011; 124: 1486-1495.
• 45 Niego B, Freeman R, Puschmann TB. et al. t-PA-specific modulation of a human blood-brain barrier model involves plasmin-mediated activation of the Rho kinase pathway in astrocytes. Blood 2012; 119: 4752-4761.
• 46 Samson AL, Nevin ST, Croucher D. et al. Tissue-type plasminogen activator requires a co-receptor to enhance NMDA receptor function. J Neurochem 2008; 107: 1091-1101.
• 47 Al Ahmad A, Taboada CB, Gassmann M. et al. Astrocytes and pericytes differentially modulate blood-brain barrier characteristics during development and hypoxic insult. J Cereb Blood Flow Metab 2011; 31: 693-705.
• 48 Cesarman GM, Guevara CA, Hajjar KA. An endothelial cell receptor for plasminogen/tissue plasminogen activator (t-PA). II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation. J Biol Chem 1994; 269: 21198-21203.
• 49 Hajjar KA, Guevara C A., Lev E. et al. Interaction of the fibrinolytic receptor, annexin II, with the endothelial cell surface. Essential role of endonexin repeat 2. J Biol Chem 1996; 271: 21652-21659.
• 50 Hembrough TA, Li L, Gonias SL. Cell-surface Cytokeratin 8 Is the Major Plasminogen Receptor on Breast Cancer Cells and Is Required for the Accelerated Activation of Cell-associated Plasminogen by Tissue-type Plasminogen Activator. J Biol Chem 1996; 271: 25684-25691.