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Abstract Anticoagulants are commonly utilized during surgeries and to treat thrombotic diseases like stroke and deep vein thrombosis. However, conventional anticoagulants have serious side‐effects, narrow therapeutic windows, and lack safe reversal agents (antidotes). Here, an alternative RNA origami displaying RNA aptamers as target‐specific anticoagulant is described. Improved design and construction techniques for self‐folding, single‐molecule RNA origami as a platform for displaying pre‐selected RNA aptamers with precise orientational and spatial control are reported. Nuclease resistance is added using 2′‐fluoro‐modified pyrimidines during in vitro transcription. When four aptamers are displayed on the RNA origami platform, the measured thrombin inhibition and anticoagulation activity is higher than observed for free aptamers, ssRNA‐linked RNA aptamers, and RNA origami displaying fewer aptamers. Importantly, thrombin inhibition is immediately switched off by addition of specific reversal agents. Results for single‐stranded DNA (ssDNA) and single‐stranded peptide nucleic acid (PNA) antidotes show restoration of 63% and 95% coagulation activity, respectively. To demonstrate potential for practical, long‐term storage for clinical use, RNA origami is freeze‐dried, and stored at room temperature. Freshly produced and freeze‐dried RNA show identical levels of activity in coagulation assays. Compared to current commercial intravenous anticoagulants, RNA origami‐based molecules show promise as safer alternatives with rapid activity switching for future therapeutic applications. 

Abstract Native platelets are crucial players in wound healing. Key to their role is the ability of their surface receptor GPIIb/IIIa to bind fibrin at injury sites, thereby promoting clotting. When platelet activity is impaired as a result of traumatic injury or certain diseases, uncontrolled bleeding can result. To aid clotting and tissue repair in cases of poor platelet activity, synthetic platelet‐like particles capable of promoting clotting and improving wound healing responses have been previously developed in the lab. These are constructed by functionalizing highly deformable hydrogel microparticles (microgels) with fibrin‐binding ligands including a fibrin‐specific whole antibody or a single‐domain variable fragment. To improve the translational potential of these clotting materials, the use of fibrin‐binding peptides as cost‐effective, robust, high‐specificity alternatives to antibodies are explored. Herein, the development and characterization of soft microgels decorated with the peptide AHRPYAAK that mimics fibrin knob “B” and targets fibrin hole “b” are presented. These “fibrin‐affine microgels with clotting yield” (FAMCY) are found to significantly increase clot density in vitro and decrease bleeding in a rodent trauma model in vivo. These results indicate that FAMCYs are capable of recapitulating the platelet‐mimetic properties of previous designs while utilizing a less costly, more translational design.