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Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions., Competing Interests: Declaration of interests Duke has submitted patent applications on the Factor V/Va anticoagulant aptamer(s). E.E.S. and B.A.S. are inventors on such Duke Intellectual Property., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Biopharmaceuticals have become increasingly attractive therapeutic agents and are often PEGylated to enhance their pharmacokinetics and reduce their immunogenicity. However, recent human clinical trials have demonstrated that administration of PEGylated compounds can evoke anti-PEG antibodies. Considering the ubiquity of PEG in commercial products and the presence of pre-existing anti-PEG antibodies in patients in large clinical trials evaluating a PEG-modified aptamer, we investigated how anti-PEG antibodies effect the therapeutic activities of PEGylated RNA aptamers. We demonstrate that anti-PEG antibodies can directly bind to and inhibit anticoagulant aptamer function in vitro and in vivo. Moreover, in parallel studies we detected the presence of anti-PEG antibodies in nonhuman primates after a single administration of a PEGylated aptamer. Our results suggest that anti-PEG antibodies can limit the activity of PEGylated drugs and potentially compromise the activity of otherwise effective therapeutic agents., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia-associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer-drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.

Thrombosis is a necessary physiological process to protect the body from uncontrolled bleeding. Pathological thrombus formation can lead to devastating clinical events including heart attack, stroke, deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. Numerous drugs have been developed to inhibit thrombosis. These have been targeted to coagulation factors along with proteins and receptors that activate platelets. While these drugs are effective at preventing blood clotting, their major side effect is inadvertent hemorrhage that can result in significant morbidity and mortality. There exists a need for anticoagulants that are not only effective at preventing thrombosis but can also be readily reversed. Aptamers offer a potential solution, representing a new class of drug agents that can be isolated to any protein and where antidote oligonucleotides can be designed based on the sequence of the aptamer. We present a summary of the anticoagulant and antithrombotic aptamers that have been identified and their stage of development and comment on the future of aptamer-based drug development to treat thrombosis.

Potent and rapid-onset anticoagulation is required for several clinical settings, including cardiopulmonary bypass surgery. In addition, because anticoagulation is associated with increased bleeding following surgery, the ability to rapidly reverse such robust anticoagulation is also important. Previously, we observed that no single aptamer was as potent as heparin for anticoagulating blood. However, we discovered that combinations of two aptamers were as potent as heparin. Herein, we sought to combine two individual anticoagulant aptamers into a single bivalent RNA molecule in an effort to generate a single molecule that retained the potent anticoagulant activity of the combination of individual aptamers. We created four bivalent aptamers that can inhibit Factor X/Xa and prothrombin/thrombin and anticoagulate plasma, as well as the combination of individual aptamers. Detailed characterization of the shortest bivalent aptamer indicates that each aptamer retains full binding and functional activity when presented in the bivalent context. Finally, reversal of this bivalent aptamer with a single antidote was explored, and anticoagulant activity could be rapidly turned off in a dose-dependent manner. These studies demonstrate that bivalent anticoagulant aptamers represent a novel and potent approach to actively and reversibly control coagulation.

Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation of the aptamers and thereby to maximize anticoagulant activity in functional assays. By judicious engineering of the DNA nanostructures, we have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than free aptamer. Reversal of the thrombin inhibition was also achieved by the use of single-stranded DNA antidotes, thus enabling significant control over blood coagulation., From the Clinical Editor: Thrombin inhibition via DNA aptamers has recently become a possibility. In this study, thrombin-binding DNA aptamers were further optimized by nanoscale organization on DNA nanostructures. The authors have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than that of free aptamer. Reversal of thrombin inhibition was also achieved by single-stranded DNA antidotes, enabling significant control over the coagulation pathway., (Copyright © 2012. Published by Elsevier Inc.)

The interaction of factor Xa with factor Va on membranes to form prothrombinase profoundly increases the rate of the proteolytic conversion of prothrombin to thrombin. We present the characterization of an RNA aptamer (RNA(11F7t)) selected from a combinatorial library based on its ability to bind factor Xa. We show that RNA(11F7t) inhibits thrombin formation catalyzed by prothrombinase without obscuring the active site of Xa within the enzyme complex. Selective inhibition of protein substrate cleavage arises from the ability of the aptamer to bind to factor Xa and exclude interactions between the proteinase and cofactor within prothrombinase. Competition for enzyme complex assembly results from the binding of RNA(11F7t) to factor Xa with nanomolar affinity in a Ca(2+)-dependent interaction. RNA(11F7t) binds equivalently to the zymogen factor X as well as derivatives lacking gamma-carboxyglutamic acid residues. We suggest that the ability of RNA(11F7t) to compete for the Xa-Va interaction with surprisingly high affinity likely reflects a significant contribution from its ability to indirectly impact regions of Xa that participate in the proteinase-cofactor interaction. Thus, despite the complexity of the macromolecular interactions that underlie the assembly of prothrombinase, efficient inhibition of enzyme complex assembly and thrombin formation can be achieved by tight binding ligands that target factor Xa in a discrete manner.

With an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs, but, unfortunately, it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Here we describe the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents based upon aptamers. These universal antidotes exploit the fact that, when systemically administered, aptamers are the only free extracellular oligonucleotides found in circulation. We show that protein- and polymer-based molecules that capture oligonucleotides can reverse the activity of several aptamers in vitro and counteract aptamer activity in vivo. The availability of universal antidotes to control the activity of any aptamer suggests that aptamers may be a particularly safe class of therapeutics.

Heparin and protamine are the standard anticoagulant-antidote regimen used in almost every cardiopulmonary bypass (CPB) procedure even though both are associated with an array of complications and toxicities. Here we demonstrate that an anticoagulant aptamer-antidote pair targeting factor IXa can replace heparin and protamine in a porcine CPB model and also limit the adverse effects on thrombin generation, inflammation, and cardiac physiology associated with heparin and protamine use. These results demonstrate that targeting clotting factors upstream of thrombin in the coagulation cascade can potentially reduce the perioperative pathologies associated with CPB and suggest that the aptamer-antidote pair to FIXa may improve the outcome of patients undergoing CPB. In particular, this novel anticoagulant-antidote pair may prove to be useful in patients diagnosed with heparin-induced thrombocytopenia or those who have been sensitized to protamine, particularly patients who have insulin-dependent diabetes.

Useful additional options for anticoagulant therapy have been introduced over the last 15 years, including low-molecular-weight heparins and direct thrombin inhibitors. Despite these impressive advances, a need for safer effective anticoagulants remains. Aptamers represent a therapeutic modality that has the potential to address this unmet need. Aptamers are small nucleic acid molecules that function as direct protein inhibitors, much like monoclonal antibodies. Aptamers are delivered by parenteral administration, can be formulated to possess a very short or sustained half-life, and are purported to be nonimmunogenic. Perhaps most relevant to the development of safer anticoagulant therapies, recent studies have shown that antidotes can be rationally designed to control the pharmacologic effects of aptamers in vivo, paving the way for a new class of antidote-controlled therapeutics. This review discusses the limitations of current anticoagulant therapies, the properties of aptamers and how these properties can be exploited to address the unmet needs within this therapeutic class, and the progress to date in developing new aptamer-based anticoagulant therapies.

Patient safety and treatment outcome could be improved if physicians could rapidly control the activity of therapeutic agents in their patients. Antidote control is the safest way to regulate drug activity, because unlike rapidly clearing drugs, control of the drug activity is independent of underlying patient physiology and co-morbidities. Until recently, however, there was no general method to discover antidote-controlled drugs. Here we demonstrate that the activity and side effects of a specific class of drugs, called aptamers, can be controlled by matched antidotes in vivo. The drug, an anticoagulant aptamer, systemically induces anticoagulation in pigs and inhibits thrombosis in murine models. The antidote rapidly reverses anticoagulation engendered by the drug, and prevents drug-induced bleeding in surgically challenged animals. These results demonstrate that rationally designed drug-antidote pairs can be generated to provide control over drug activities in animals.

Proteins generally execute the key physiological activities required for normal growth and homeostasis. As such, many different classes of proteins, including proteases, kinases, cellular receptors and signalling proteins, represent attractive targets for diagnosis or therapy. Aptamers are small nucleic acid molecules that function as direct protein inhibitors, much like monoclonal antibodies. After a decade of intensive research, technology development and initial clinical evaluation, aptamers have now demonstrated broad potential as direct protein ligands and inhibitors, and thus represent an exciting class of compounds for the development of new therapeutic and diagnostic agents. This review will discuss the basic properties and isolation of aptamers, their use in animals and the clinic, and describe an exciting recent advance in the development of antidotes to certain aptamers, which will add a repertoire of new agents with regulatable activity for clinical use.

Many therapeutic agents are associated with adverse effects in patients. Anticoagulants can engender acute complications such as significant bleeding that increases patient morbidity and mortality. Antidote control provides the safest means to regulate drug action. For this reason, despite its known limitations and toxicities, heparin use remains high because it is the only anticoagulant that can be controlled by an antidote, the polypeptide protamine. To date, no generalizable strategy for developing drug-antidote pairs has been described. We investigated whether drug-antidote pairs could be rationally designed by taking advantage of properties inherent to nucleic acids to make antidote-controlled anticoagulant agents. Here we show that protein-binding oligonucleotides (aptamers) against coagulation factor IXa are potent anticoagulants. We also show that oligonucleotides complementary to these aptamers can act as antidotes capable of efficiently reversing the activity of these new anticoagulants in plasma from healthy volunteers and from patients who cannot tolerate heparin. This generalizable strategy for rationally designing a drug-antidote pair thus opens up the way for developing safer regulatable therapeutics.

Abstract: Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation of the aptamers and thereby to maximize anticoagulant activity in functional assays. By judicious engineering of the DNA nanostructures, we have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than free aptamer. Reversal of the thrombin inhibition was also achieved by the use of single-stranded DNA antidotes, thus enabling significant control over blood coagulation. From the Clinical Editor: Thrombin inhibition via DNA aptamers has recently become a possibility. In this study, thrombin-binding DNA aptamers were further optimized by nanoscale organization on DNA nanostructures. The authors have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than that of free aptamer. Reversal of thrombin inhibition was also achieved by single-stranded DNA antidotes, enabling significant control over the coagulation pathway. [Copyright &y& Elsevier]

An aptamer targeting factor IXa has been evaluated in animal models and several clinical studies as a potential antithombotic therapy. We elucidate the molecular mechanism by which this aptamer acts as an anticoagulant. The aptamer binds tightly to factor IXa and prolongs the clotting time of human plasma. The aptamer completely blocks factor IXa activation of factor X regardless of the presence of factor VIIIa. However, the aptamer does not completely block small synthetic substrate cleavage, although it does slow the rate of cleavage. These data are consistent with the aptamer binding to the catalytic domain of factor IXa in such a way as to block an extended substrate-binding site. Therefore, unlike small molecule inhibitors, aptamers appear to be able to bind surfaces surrounding an active site and thereby sterically interfere with enzyme activity. Thus, aptamers may be useful agents to probe and block substrate-binding sites outside of the active site of an enzyme. [ABSTRACT FROM AUTHOR]

Thrombotic disorders and their common clinical phenotypes of acute myocardial infarction, ischemic stroke, and venous thromboembolism are the proximate cause of substantial morbidity, mortality, and health care expenditures worldwide. Accordingly, therapies designed to attenuate thrombus initiation and propagation, reflecting integrated platelet-mediated and coagulation protease-mediated events, respectively, represent a standard of care. Unfortunately, there are numerous inherent limitations of existing therapies that include target nonselectivity, variable onset and offset of pharmacodynamic effects, a narrow efficacy–safety profile, and the absence of a safe and reliable platform for either accurate titration, based on existing patient-specific, disease-specific, and clinical conditions, or active reversibility. Herein, we summarize our experience with oligonucleotide antithrombotic agents and their complementary antidotes, targeting the platelet adhesive protein von Willebrand factor and the pivotal coagulation protease factor IXa. [ABSTRACT FROM AUTHOR]

Coordinated enzymatic reactions regulate blood clot generation. To explore the contributions of various coagulation enzymes in this process, we utilized a panel of aptamers against factors VIIa, IXa, Xa, and prothrombin. Each aptamer dose-dependently inhibited clot formation, yet none was able to completely impede this process in highly procoagulant settings. However, several combinations of two aptamers synergistically impaired clot formation. One extremely potent aptamer combination was able to maintain human blood fluidity even during extracorporeal circulation, a highly procoagulant setting encountered during cardiopulmonary bypass surgery. Moreover, this aptamer cocktail could be rapidly reversed with antidotes to restore normal hemostasis, indicating that even highly potent aptamer combinations can be rapidly controlled. These studies highlight the potential utility of using sets of aptamers to probe the functions of proteins in molecular pathways for research and therapeutic ends. [ABSTRACT FROM AUTHOR]

Millions of individuals are prescribed platelet inhibitors, such as aspirin and clopidogrel, to reduce their risk of thrombosis-related clinical events. Unfortunately many platelet inhibitors are contraindicated in surgical settings because of their inherent bleeding risk complicating the treatment of patients who require surgery. We describe the development of a potent antiplatelet agent, an RNA aptamer-termed Ch-9.14-T10 that binds von Willebrand factor (VWF) with high affinity and inhibits thrombosis in a murine carotid artery damage model. As expected, when this potent antiplatelet agent is administered, it greatly increases bleeding from animals that are surgically challenged. To improve this antiplatelet agent's safety profile, we describe the generation of antidotes that can rapidly reverse the activity of Ch-9.14-T10 and limit blood loss from surgically challenged animals. Our work represents the first antidote controllable antiplatelet agent, which could conceivably lead to improved medical management of patients requiring antiplatelet medication who also need surgery. [ABSTRACT FROM AUTHOR]