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Human α-thrombin is a trypsin-like serine protease that has the unique ability to convert soluble fibrinogen in insoluble fibrin clot. In addition to the active site, this enzyme owns two electropositive regions, (exosite I and II), located at opposite sides of its globular shape and playing an essential role in the recognition of many cofactors and substrates.1 A special class of thrombin synthetic ligands is represented by aptamers, which are short single stranded DNA or RNA oligonucleotides that bind their targets with very high affinity and specificity by adopting defined and stable three dimensional structural motifs.2 In particular, anti-thrombin aptamers adopt a G-quadruplex structure.3-5 Recently, the researchers interest has moved on a new class of oligonucleotides in which the addition of a duplex-forming sequences to a G-quadruplex module results in an improvement of the antithrombotic action. One of these oligonucleotides is NU172 that possesses an high anticoagulant activity with an IC50 value of 5–10 μg/mL in plasma6 and is currently the only thrombin binding aptamer evaluated in Phase II clinical trials (ClinicalTrials.gov identifier NCT00808964) for anticoagulation in heart disease treatments by ARCA Biopharma, Inc.7 The crystallographic characterization of thrombin-NU172 complex in the presence of either potassium or sodium ions provides together with conformational solution studies on NU172 variants and anticoagulant assays have provided a reasonable interpretation of the peculiar antithrombotic properties of NU172. Details on our results will be discussed at the Meeting. References: 1. Di Cera, E. J Thromb Haemost. 2007, 5 Suppl 1, 196 – 202. 2. Woodruff, R.S.; Sullenger, B.A. Arterioscler Thromb Vasc Biol. 2015, 35, 2083 – 2091. 3. Russo Krauss, I; Pica, A.; Merlino, A.; Mazzarella, L.; Sica, F. Acta Crystallogr D Biol Crystallogr. 2013, 69, 2403 – 2411. 4. Russo Krauss, I.; Spiridonova, V.; Pica, A.; Napolitano, V.; Sica, F. Nucleic Acids Res. 2016, 44, 983 – 991. 5. Russo Krauss, I.; Napolitano, V.; Petraccone, L.; Troisi, R.; Spiridonava, V.; Mattia, C.A.; Sica, F. Int J Biol Macromol. 2018, 107, 1697 – 1705. 6. Keefe, A.D.; Pai, S.; Ellington, A. Nat Rev Drug Discov. 2010, 9, 537 – 550. 7. Zhou, J.; Rossi, J. Nat Rev Drug Discov. 2017, 16, 181 – 202.

Human α-thrombin (thrombin) is a coagulation factor that maintains blood hemostasis by balancing pro- and anti-coagulant actions. Thrombin capability to perform such different functions relies on the ability to recognize a large variety of substrates, inhibitors and cofactors. This ability is finely regulated by two distinct regions on the protein surface, known as exosites I and II, which allosterically modulate the function, thus providing specificity to the proteolytic activity of the protein [1]. Oligonucleotide aptamers, short, single-stranded DNA or RNA sequences that bind to their target with high affinity and specificity through their tridimensional structures, are extensively used to modulate the function of most of the factors involved in the coagulation pathway [2]. Particular attention has been focused on the study of anti-thrombin aptamers which adopt a G-quadruplex structure [3-5]. Nowdays, the researchers interest moves on a new class of oligonucleotides in which the addition of a duplex-forming sequences to a G-quadruplex module results in an improvement of the antithrombotic action. One of these oligonucleotides is NU172 that possesses an high anticoagulant activity with an IC50 value of 5–10 μg/mL in plasma [6] and is currently the only thrombin binding aptamer evaluated in Phase II clinical trials (ClinicalTrials.gov identifier NCT00808964) for anticoagulation in heart disease treatments by ARCA Biopharma, Inc [7]. Here we present the crystallographic structure of the thrombin-NU172 complexes in the presence of either potassium or sodium ions, which provide a reasonable interpretation of the peculiar antithrombotic properties of this oligonucleotide. Moreover, in order to investigate the role of quadruplex loops and the duplex region on the aptamer folding, conformational solution studies on NU172 variants have been performed. Finally, the anticoagulant activities of NU172 and its variants have been evaluated following the conversion of fibrinogen in fibrin promoted by thrombin by means of dynamic light scattering experiments and compared with those of TBA. Details will be discussed at the Meeting. [1] E. Di Cera J Thromb Haemost. 2007, 5 Suppl 1, 196. [2] R.S. Woodruff, B.A. Sullenger Arterioscler Thromb Vasc Biol. 2015, 35, 2083. [3] I. Russo Krauss, A. Pica, A. Merlino, L. Mazzarella, F. Sica Acta Crystallogr D Biol Crystallogr. 2013, 69, 2403. [4] I. Russo Krauss, V. Spiridonova, A. Pica, V. Napolitano, F. Sica Nucleic Acids Res. 2016, 44, 983. [5] I. Russo Krauss, V. Napolitano, L. Petraccone, R. Troisi, V. Spiridonava, C.A. Mattia, F. Sica Int J Biol Macromol. 2018, 107, 1697. [6] A.D. Keefe, S. Pai, A. Ellington Nat Rev Drug Discov. 2010, 9, 537. [7] J. Zhou, J. Rossi Nat Rev Drug Discov. 2017, 16, 181.

Determination of the chemical composition of different parts of the wall paintings (pigments, mortars and binders) allows extracting information about technology of preparation of the artefact. Herein we present a multimethodological characterization of wall paintings from a Roman Archeological sites from Cuma, focusing on differences between indoor (domus) and outdoor (Temple) fabrication. Both pigments, binders and mortars were studied via a combination of destructive/microdestructutive (mass spectrometry, ionic chromatography, ICP-based techniques) and non-destructive (Raman microscopy, X-ray diffraction and SANS) methodologies. Particularly, the systematic presence of dolomite only in temple mortars suggests an intentional use of such limestone for outdoor fabrication. Differences between temple and domus extends also to binder composition: proteinaceous binders in domus (possibly egg [3] and animal glue), whereas in TCP no protein was detected but some fat acids (probably from animal fat). Ultimately, the multimethodological study allowed an overall picture of the components and technology of the paintings from fabrications with different use, proposing distinct engineering choices for indoor and outdoor walls. References [1] Cartechini L., Pucci P., Sgamellotti A., Marino G., Birolo L., “Proteomic strategies for the identification of proteinaceous binders in paintings” Analytical & Bioanalytical Chemistry 395(7), 2009, 2269-2280. [2] Gelzo M., Grimaldi M., Vergara A., Severino V., Chambery A., Dello Russo A., Piccioli C., Corso G,. Arcari P. "Comparison of binder composition in Pompeian wall painting styles from Insula Occidentalis" Chemistry Central J. 8(65), 2014, 1-10. [3] Vinciguerra R, Galano E, Vallone F, Greco G, Vergara A, Bonaduce I, Marino G, Pucci P, Amoresano A, Birolo L, “Deglycosylation Step to Improve the Identification of Egg Proteins in Art Samples” Analytical Chemistry 87 (20), 2015, 10178-10182. Acknowledgements This work was financially supported by Distretto ad Alta Tecnologia dei Beni Culturali (DATABENC scarl), progetto SNECS. “Soprintendenza Speciale per i Beni Archeologici di Napoli e Pompei” – Ufficio Archeologico di Cuma - Dr Paolo Caputo is also acknowledged.

The intriguing crystal organization of the domain-swapped dimer of a human pancreatic ribonuclease variant indicates that the protein can form fibrils in solution. They were observed and characterized by atomic force microscopy. The importance of domain swapping in inducing native-like fibril formation is highlighted.