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Rivaroxaban (RXB), an oral direct factor Xa inhibitor, presents innovative therapeutic profile. However, RXB has shown adverse effects, mainly due to pharmacokinetic limitations, highlighting the importance of developing more effective formulations. Therefore, this work aims at the preparation, physicochemical characterization and in vitro evaluation of time-dependent anticoagulant activity and toxicology profile of RXB-loaded poly(lactic-co-glycolic acid) (PLGA)/poloxamer nanoparticles (RXBNps). RXBNp were produced by nanoprecipitation method and physicochemical characteristics were evaluated. In vitro analysis of time-dependent anticoagulant activity was performed by prothrombin time test and toxicological profile was assessed by hemolysis and MTT reduction assays. The developed RXBNp present spherical morphology with average diameter of 205.5 ± 16.95 nm (PdI 0.096 ± 0.04), negative zeta potential (-26.28 ± 0.77 mV), entrapment efficiency of 91.35 ± 2.40%, yield of 41.81 ± 1.68% and 3.72 ± 0.07% of drug loading. Drug release was characterized by an initial fast release followed by a sustained release with 28.34 ± 2.82% of RXB available in 72 h. RXBNp showed an expressive time-dependent anticoagulant activity in human and rat blood plasma and non-toxic profile. Based on the results presented, it is possible to consider that RXBNp may be able to assist in the development of promising new therapies for treatment of thrombotic disorders.

Recently the literature described the binding of Haptoglobin (HP) with ecotin, a fold-specific serine-proteases inhibitor with an anticoagulant profile and produced by Escherichia coli. In this work, we used some in silico and in vitro techniques to evaluate HP 3D-fold and its interaction with wild-type ecotin and two variants. Our data showed HP models conserved trypsin fold, in agreement to the in vitro immunological recognition of HP by trypsin antibodies. The analysis of the three ecotin-HP complexes using the mutants RR and TSRR/R besides the wild type revealed several hydrogen bonds between HP and ecotin secondary site. These data are in agreement with the in vitro PAGE assays that showed the HP-RR complex in native gel conditions. Interestingly, the ternary complex interactions varied depending on the inhibitor structure and site-directed mutation. The interaction of HP with TSRR/R involved new residues compared to wild type, which infers a binding energy increase caused by the mutation.