Hemophilia A and B are inherited blood bleeding disorders due to insufficient blood coagulation factors. Individuals with hemophilia A have a deficiency in factor VIII, while individuals with hemophilia B have a deficiency in factor IXa. Both of these factors are important in the intrinsic pathway of blood coagulation. In the absence of these factors, hemophilia patients are prone to bleeding. Thrombin, the primary factor in the blood coagulation pathway, promotes blood clotting and prevents bleeding. Hemophilia patients develop insufficient thrombin, resulting in an imbalance between the procoagulant and anticoagulant systems. Thus, generating more thrombin activity in these patients is an ideal therapeutic approach. We propose to increase thrombin's activity by inhibiting the primary inhibitor of thrombin and the natural anticoagulant protein, antithrombin (AT), using RNA molecules (aptamers) that bind to their target protein with high affinity and specificity. Several aptamers are evaluated in clinical trials as therapies for various blood diseases. We hypothesized that AT-specific aptamers could inhibit AT activity by preventing AT from forming a high molecular weight complex with thrombin, resulting in an overall increase in thrombin activity and restoring hemostatic equilibrium. The main goal is to design novel therapeutic molecules that promote thrombus formation in hemophilia patients. Using Systematic evolution of ligands by exponential enrichment (SELEX), we developed a library of RNA aptamers molecules that bind to AT with high to moderate affinity. We assessed the individual aptamers' ability to inhibit AT and increase thrombin activity using various in vitro methods. Three individual aptamers inhibited AT activity by preventing AT from forming a complex with thrombin in the presence and absence of heparin, resulting in a significant increase in thrombin activity. We also showed a decrease in thrombin generation using an ex vivo thrombin generation assay in the presence of aptamers. Our data illustrate that AT-specific RNA aptamers can inhibit AT activity and increase thrombin activity. Thus, these molecules could potentially be used to treat patients with both hemophilia A and B. Additional studies are being conducted to assess their ability to increase thrombin activity in a pre-clinical model.