Recent advances in the design and immobilization of heparin for biomedical application: A review.

Heparin, a member of the glycosaminoglycan family, is renowned as the most negatively charged biomolecule discovered within the realm of human biology. This polysaccharide serves a vital role as a regulator for various proteins, cells, and tissues within the human body, positioning itself as a pivotal macromolecule of significance. The domain of biology has witnessed substantial interest in the intricate design of heparin and its derivatives, particularly focusing on heparin-based polymers and hydrogels. This intrigue spans a wide spectrum of applications, encompassing diverse areas such as protein adsorption, anticoagulant properties, controlled drug release, development of implants, stent innovation, enhancement of blood compatibility, acceleration of wound healing, and pioneering strides in tissue engineering. This comprehensive overview delves into a multitude of developed heparin conjugates, employing various methods, and explores their functions in both the biomedicine and electronics fields. The efficacy of materials derived from heparin is also thoroughly investigated, encompassing considerations such as thrombogenicity, drug release kinetics, affinity for growth factors (GFs), biocompatibility, and electrochemical analyses. We firmly believe that by redirecting focus towards research and advancements in heparin-related polymers/hydrogels, this study will ignite further research and accelerate potential breakthroughs in this promising and evolving field of discovery.

Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin.

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N-sulfoheparosan with N-sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products.

Nutrition and Cardiovascular Disease: The Potential Role of Marine Bioactive Proteins and Peptides in Thrombosis Prevention.

Thrombus and cardiovascular diseases pose a significant health threat, and dietary interventions have shown promising potential in reducing the incidence of these diseases. Marine bioactive proteins and peptides have been extensively studied for their antithrombotic properties. They can inhibit platelet activation and aggregation by binding to key receptors on the platelet surface. Additionally, they can competitively anchor to critical enzyme sites, leading to the inhibition of coagulation factors. Marine microorganisms also offer alternative sources for the development of novel fibrinolytic proteins, which can help dissolve blood clots. The advancements in technologies, such as targeted hydrolysis, specific purification, and encapsulation, have provided a solid foundation for the industrialization of bioactive peptides. These techniques enable precise control over the production and delivery of bioactive peptides, enhancing their efficacy and safety. However, it is important to note that further research and clinical studies are needed to fully understand the mechanisms of action and therapeutic potential of marine bioactive proteins and peptides in mitigating thrombotic events. The challenges and future application perspectives of these bioactive peptides also need to be explored.

Structure and Binding Properties to Blood Co-Factors of the Least Sulfated Galactan Found in the Cell Wall of the Red Alga Botryocladia occidentalis.

Three different populations of sulfated polysaccharides can be found in the cell wall of the red alga Botryocladia occidentalis. In a previous work, the structures of the two more sulfated polysaccharides were revised. In this work, NMR-based structural analysis was performed on the least sulfated polysaccharide and its chemically modified derivatives. Results have revealed the presence of both 4-linked α- and 3-linked ß-galactose units having the following chemical features: more than half of the total galactose units are not sulfated, the α-units occur primarily as 3,6-anhydrogalactose units either 2-O-methylated or 2-O-sulfated, and the ß-galactose units can be 4-O-sulfated or 2,4-O-disulfated. SPR-based results indicated weaker binding of the least sulfated galactan to thrombin, factor Xa, and antithrombin, but stronger binding to heparin cofactor II than unfractionated heparin. This report together with our previous publication completes the structural characterization of the three polysaccharides found in the cell wall of the red alga B. occidentalis and correlates the impact of their composing chemical groups with the levels of interaction with the blood co-factors.

Crystal structure of tick tyrosylprotein sulfotransferase reveals the activation mechanism of the tick anticoagulant protein madanin.

Ticks pose a substantial public health risk as they transmit various pathogens. This concern is related to the adept blood-sucking strategy of ticks, underscored by the action of the anticoagulant, madanin, which is known to exhibit an approximately 1000-fold increase in anticoagulant activity following sulfation of its two tyrosine residues, Tyr51 and Tyr54. Despite this knowledge, the molecular mechanism underlying sulfation by tick tyrosylprotein sulfotransferase (TPST) remains unclear. In this study, we successfully prepared tick TPST as a soluble recombinant enzyme. We clarified the method by which this enzyme proficiently sulfates tyrosine residues in madanin. Biochemical analysis using a substrate peptide based on madanin and tick TPST, along with the analysis of the crystal structure of the complex and docking simulations, revealed a sequential sulfation process. Initial sulfation at the Tyr51 site augments binding, thereby facilitating efficient sulfation at Tyr54. Beyond direct biochemical implications, these findings considerably improve our understanding of tick blood-sucking strategies. Furthermore, combined with the utility of modified tick TPST, our findings may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention and advancements in the field of public health.

Structural and biofunctional diversity of sulfated polysaccharides from the genus Codium (Bryopsidales, Chlorophyta): A review.

It is believed that polysaccharides will become a focal point for future production of food, pharmaceuticals, and materials due to their ubiquitous and renewable nature, as well as their exceptional properties that have been extensively validated in the fields of nutrition, healthcare, and materials. Sulfated polysaccharides derived from seaweed sources have attracted considerable attention owing to their distinctive structures and properties. The genus Codium, represented by the species C. fragile, holds significance as a vital economic green seaweed and serves as a traditional Chinese medicinal herb. To date, the cell walls of the genus Codium have been found to contain at least four types of sulfated polysaccharides, specifically pyruvylated ß-d-galactan sulfates, sulfated arabinogalactans, sulfated ß-l-arabinans, and sulfated ß-d-mannans. These sulfated polysaccharides exhibit diverse biofunctions, including anticoagulant, immune-enhancing, anticancer, antioxidant activities, and drug-carrying capacity. This review explores the structural and biofunctional diversity of sulfated polysaccharides derived from the genus Codium. Additionally, in addressing the impending challenges within the industrialization of these polysaccharides, encompassing concerns regarding scale-up production and quality control, we outline potential strategies to address these challenges from the perspectives of raw materials, extraction processes, purification technologies, and methods for quality control.

Daboialipase, a phospholipase A2 from Vipera russelli russelli venom posesses anti-platelet, anti-thrombin and anti-cancer properties.

Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A2 with molecular mass of 14.3 kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules.

Structural characterization of a distinct fucan sulfate from Pattalus mollis through an oligosaccharide mapping approach.

The elucidation of the precise structure of fucan sulfate is essential for understanding the structure-activity relationship and promoting potential biomedical applications. In this work, the structure of a distinct fucan sulfate fraction V (PmFS in Ref 15 and FSV in Ref 16 → PFV) from Pattalus mollis was investigated using an oligosaccharide mapping approach. Six size-homogeneous fractions were purified from the mild acid hydrolyzed PFV and identified as fucitols, disaccharides and trisaccharides by 1D/2D NMR and MS analysis. Significantly, the sulfation pattern, glycosidic linkages, and sequences of all the oligosaccharides were unambiguously identified. The common 2-desulfation of the reducing end residue of the oligosaccharides was observed. Overall, the backbone of PFV was composed of L-Fuc2S (major) and L-Fuc3S (minor) linked by α1,4 glycosidic bonds. Importantly, the branches contain both monosaccharide and disaccharide linked to the backbone by α1,3 glycosidic linkages. Thus, the tentative structure of natural PFV was shown to be {-(R-α1,3)-L-Fuc2S-α1,4-(L-Fuc2S/3S-α1,4)x-}n, where R is L-Fuc(2S)4S-α1,3/4-L-Fuc4S(0S)- or L-Fuc(2S)4S-. Our results provide insight into the heterogeneous structure of the fucan sulfate found in sea cucumbers. Additionally, PFV and its fractions showed strong anticoagulant and anti-iXase activities, which may be related to the distinct structure of PFV.

TFPIα anticoagulant function is highly dependent on protein S in vivo.

Tissue factor pathway inhibitor α (TFPIα) is the major physiological regulator of the initiation of blood coagulation. In vitro, TFPIα anticoagulant function is enhanced by its cofactor, protein S. To define the role of protein S enhancement in TFPIα anticoagulant function in vivo, we blocked endogenous TFPI in mice using a monoclonal antibody (14D1). This caused a profound increase in fibrin deposition using the laser injury thrombosis model. To explore the role of plasma TFPIα in regulating thrombus formation, increasing concentrations of human TFPIα were coinjected with 14D1, which dose-dependently reduced fibrin deposition. Inhibition of protein S cofactor function using recombinant C4b-binding protein ß chain significantly reduced the anticoagulant function of human TFPIα in controlling fibrin deposition. We report an in vivo model that is sensitive to the anticoagulant properties of the TFPIα-protein S pathway and show the importance of protein S as a cofactor in the anticoagulant function of TFPIα in vivo.

Characterization of complexes of PF4 and heparins by size-exclusion chromatography coupled with multi-angle light scattering detector.

Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy. Antibodies binding to complexes of platelet factor 4 (PF4) and heparin is the trigger of HIT. A method using size exclusion chromatography with multi-angle laser light scattering detector (SEC-MALS) was developed in this work. The soluble ultra-large complex (ULC) was separated from the small complex (SC) and their molecular weights (MWs) were firstly measured. The complexes of PF4 and three heparins with different MW, including unfractionated heparin (UFH), dalteparin (Daltep) and enoxaparin (Eno) were characterized using this method. The contents and the sizes of ULC increased gradually when heparins were added to PF4 to certain amounts. While, they reduced after more heparins were added. It is the first time to measure the MWs of the biggest ULC of PF4-heparins as millions of Dalton. at the proper ratios of PF4 to heparin (PHR). Meanwhile, those mixtures at those certain PHRs induced the higher expression of CD83 and CD14 markers on dendritic cells (DCs) suggesting that they had stronger immunogenicity and is critical for HIT.

Structure, anticoagulant and cytotoxic activity of a sulfated polysaccharide from green seaweed Chaetomorpha linum.

In this article, chemical structure and conformation in an aqueous solution of a new sulfated polysaccharide, PCL, extracted from green seaweed Chaetomorpha linum were elucidated by SEC-MALL, IR, NMR and SAXS. The results indicated that the obtained polysaccharide is a sulfated arabinogalactan with a molecular weight of 223 kDa, and is mainly composed of →3,6)-α-D-Galp4S→ and →2)-α-L-Araf→ connecting together through 1→3 glycoside linkages. It has a broken rod-like conformation in solution with Rgc estimated as 0.43 nm from SAXS measurements. The polysaccharide exhibited a notable anticoagulant activity measured by the assays of activated partial thromboplastintime, thrombintime and prothrombine time as well as a significant cytotoxic activity against hepatocellular, human breast cancer, and cervical cancer cell lines.

Surface modification of polyvinyl chloride with sodium alginate/carboxymethyl chitosan and heparin for realizing the anticoagulation.

Thrombosis of extracorporeal circuits causes significant morbidity and mortality worldwide. In this study, plasma treatment technology and chemical grafting method were used to construct heparinized surfaces on the PVC substrate, which could not only reduce thrombosis but also decrease the side effects of the direct injection of anticoagulants. The PVC substrate was modified by plasma treatment technology firstly to obtain the active surface with the hydroxyl groups used for grafting. Then, heparin was grafted onto the modified PVC surface using different grafting strategies to prepare different heparinized surfaces. The experimental results indicated that the sodium alginate (SA) and carboxymethyl chitosan (CCS) used as interlayers could significantly increase the graft density of heparin to improve the anticoagulant effects and hemocompatibility of heparinized surfaces. In addition, the modification of heparin can further improve the anticoagulant effects. The CCS/low-molecular-weight heparin (LWMH) surface has the best anticoagulant properties, which can prolong the activated partial thromboplastin time (APTT) values of human plasma for about 35 s, reduce the hemolysis rates to <0.3 %, and perform well in the in-vitro blood circulation test. The heparinized surfaces prepared in this work have great application potential in anticoagulant treatment for medical devices.

Purification, Characterization and Evaluation of the Anticoagulant Effect of an Uncompetitive Trypsin Inhibitor obtained from Bauhinia pulchella (Benth) Seeds.

INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.

Potential antithrombotic effect of two new phenylpropanoid sucrose esters and other secondary metabolites of Canna indica L. rhizome.

Canna indica L. has been traditionally used to treat various diseases. Based on previously reported antithrombotic effect for this plant, two new phenylpropanoid sucrose esters (canindicoside A (1) and canindicoside B (2)) and seven known compounds: nepetoidin B (3), caffeic acid (4), ferulic acid (5), (R)-(+)-rosmarinic acid (6), isorinic acid (7), (S)-(-)-rosmarinic acid (8) and (S)-(-)-rosmarinic acid methyl ester (9) were isolated from the ethyl acetate extract. Compounds were elucidated by NMR and MS spectroscopic methods. The antiplatelet effect was evaluated using turbidimetric method. Anticoagulant activity was examined by measuring activated partial thromboplastine time (APTT), prothrombin time, and thrombine time (TT). It was shown for the first time that both new phenylpropanoid sucrose esters 1 and 2, 7 and 9 displayed dose-dependent antiplatelet effects. 2 and 9 had the highest inhibitory activity on both adenosine diphosphate (ADP)- and collagen-induced platelet aggregation. Moreover, 1, 7 and 9 also exhibited anticoagulant activity. At 0.4 mg/mL, both 1 and 7 prolonged APTT compared to the negative control (p < 0.05), suggesting the possible inhibitory impact on the intrinsic coagulation pathway. Moreover, 9 at 0.4 mg/mL exerted higher TT values than the negative control (p < 0.05). C. indica and its bioactive phytochemicals are potential candidates for development of anti-thrombosis therapy.

Preanalytical Quality Evaluation of Citrate Evacuated Blood Collection Tubes-Ultraviolet Molecular Absorption Spectrometry Confronted with Ion Chromatography.

We previously enabled a direct insight into the quality of citrate anticoagulant tubes before their intended use for specimen collection by introducing an easy-to-perform UV spectrometric method for citrate determination on a purified water model. The results revealed differences between the tubes of three producers, Greiner BIO-ONE (A), LT Burnik (B), and BD (C). It became apparent that tubes C contain an additive, which absorbs light in the ultraviolet range and prevents reliable evaluation of citrate anticoagulant concentration with the suggested method. In this research, we re-evaluate the quality of citrate-evacuated blood collection tubes by complementing UV spectrometry with ion chromatography. (1) Comparable results were obtained for tubes B at 220 nm. (2) Citrate concentrations determined with ion chromatography were lower for tubes A and C. Chromatograms reveal additional peaks for both. (3) Influences of heparin on absorption spectra and chromatograms of citrate were studied. Some similarities with the shape of the anticoagulant spectra of tubes A and C were observed, and the lithium heparin peak in chromatograms is close to them, but a confident judgment was not possible. (4) Contamination of anticoagulant solution with potassium, magnesium, and calcium was confirmed for all the brands, and contamination with lithium for B and C.

Structural Characterization and Anticoagulant Activities of a Keratan Sulfate-like Polysaccharide from the Sea Cucumber Holothuria fuscopunctata.

A sulfated polysaccharide (AG) was extracted and isolated from the sea cucumber H. fuscopunctata, consisting of GlcNAc, GalNAc, Gal, Fuc and lacking any uronic acid residues. Importantly, several chemical depolymerization methods were used to elucidate the structure of the AG through a bottom-up strategy. A highly sulfated galactose (oAG-1) and two disaccharides labeled with 2,5-anhydro-D-mannose (oAG-2, oAG-3) were obtained from the deaminative depolymerized product along with the structures of the disaccharide derivatives (oAG-4~oAG-6) identified from the free radical depolymerized product, suggesting that the repeating building blocks in a natural AG should comprise the disaccharide ß-D-GalS-1,4-D-GlcNAc6S. The possible disaccharide side chains (bAG-1) were obtained with mild acid hydrolysis. Thus, a natural AG may consist of a keratan sulfate-like (KS-like) glycosaminoglycan with diverse modifications, including the sulfation types of the Gal residue and the possible disaccharide branches α-D-GalNAc4S6S-1,2-α/ß-L-Fuc3S linked to the KS-like chain. Additionally, the anticoagulant activities of the AG and its depolymerized products (dAG1-9) were evaluated in vitro using normal human plasma. The AG could prolong activated partial thromboplastin time (APTT) in a dose-dependent manner, and the activity potency was positively related to the chain length. The AG and dAG1-dAG3 could prolong thrombin time (TT), while they had little effect on prothrombin time (PT). The results indicate that the AG could inhibit the intrinsic and common coagulation pathways.

Sulfated Galactans from Agarophytes: Review of Extraction Methods, Structural Features, and Biological Activities.

Agarophytes are important seaweeds of the Rhodophyta type, which have been highly exploited for industrial use as sources of a widely consumed polysaccharide of agar. In addition to that, sulfated galactans (SGs) from agarophytes, which consist of various functional sulfate groups, have attracted the attention of scientists in current studies. SGs possess various biological activities, such as anti-tumor, anticoagulant, anti-inflammatory, antioxidant, anti-obesity, anti-diabetic, anti-microbial, anti-diarrhea, and gut microbiota regulation properties. Meanwhile, the taxonomy, ecological factors, i.e., environmental factors, and harvest period, as well as preparation methods, i.e., the pretreatment, extraction, and purification conditions, have been found to influence the chemical compositions and fine structures of SGs, which have, further, been shown to have an impact on their biological activities. However, the gaps in the knowledge of the properties of SGs due to the above complex factors have hindered their industrial application. The aim of this paper is to collect and systematically review the scientific evidence about SGs and, thus, to pave the way for broader and otherwise valuable industrial applications of agarophytes for human enterprise. In the future, this harvested biomass could be sustainably used not only as a source of agar production but also as natural materials in functional food and pharmaceutical industries.

Sulfated polysaccharides in sea cucumbers and their biological properties: A review.

Sea cucumbers contain a wide range of biomolecules, including sulfated polysaccharides (SPs), with immense therapeutic and nutraceutical potential. SPs in sea cucumbers are mainly fucosylated chondroitin sulfate (FCS) and fucan sulfate (FS) which exhibit a series of pharmacological effects, including anticoagulant activity, in several biological systems. FCS is a structurally distinct glycosaminoglycan in the sea cucumber body wall, and its biological properties mainly depend on the degree of sulfation, position of sulfate group, molecular weight, and distribution of branches along the backbone. So far, FCS and FS have been recognized for their antithrombotic, anti-inflammatory, anticancer, antidiabetic, anti-hyperlipidemic, anti-obesity, and antioxidant potential. However, the functions of these SPs are mainly dependent on the species, origins, harvesting season, and extraction methods applied. This review focuses on the SPs of sea cucumbers and how their structural diversities affect various biological activities. In addition, the mechanism of actions of SPs, chemical structures, factors affecting their bioactivities, and their extraction methods are also discussed.

Probing naphthalene diimide and 3-hydroxypropylphosphate as end-conjugating moieties for improved thrombin binding aptamers: Structural and biological effects.

The limitations associated with the in vivo use of the thrombin binding aptamer (TBA or TBA15) have dramatically stimulated the search of suitable chemically modified analogues in order to discover effective and reversible inhibitors of thrombin activity. In this context, we previously proposed cyclic and pseudo-cyclic TBA analogues with improved stability that proved to be more active than the parent aptamer. Herein, we have investigated a novel library of TBA derivatives carrying naphthalene diimide (NDI) moieties at the 3'- or 5'-end. In a subset of the investigated oligonucleotides, additional 3-hydroxypropylphosphate (HPP) groups were introduced at one or both ends of the TBA sequence. Evaluation of the G-quadruplex thermal stability, serum nuclease resistance and in vitro anticoagulant activity of the new TBA analogues allowed rationalizing the effect of these appendages on the activity of the aptamer on the basis of their relative position. Notably, most of the different TBA analogues tested were more potent thrombin inhibitors than unmodified TBA. Particularly, the analogue carrying an NDI group at the 5'-end and an HPP group at the 3'-end, named N-TBA-p, exhibited enhanced G-quadruplex thermal stability (ΔTm + 14° C) and ca. 10-fold improved nuclease resistance in serum compared to the native aptamer. N-TBA-p also induced prolonged and dose-dependent clotting times, showing a ca. 11-fold higher anticoagulant activity compared to unmodified TBA, as determined by spectroscopic methods. Overall, N-TBA-p proved to be in vitro a more efficient thrombin inhibitor than all the best ones previously investigated in our group. Its interesting features, associated with its easy preparation, make it a very promising candidate for future in vivo studies.

Aptamer Conjugated RNA/DNA Hybrid Nanostructures Designed for Efficient Regulation of Blood Coagulation.

Disruptions to the hemostatic pathway can cause a variety of serious or even life-threatening complications. Situations in which the coagulation of blood has become disturbed necessitate immediate care. Thrombin-binding aptamers are single-stranded nucleic acids that bind to thrombin with high specificity and affinity. While they can effectively inhibit thrombin, they suffer from rapid degradation and clearance in vivo. These issues are resolved, however, by attaching the therapeutic aptamer to a nucleic acid nanostructure. The increased size of the nanostructure-aptamer complex elongates the post-infusion half-life of the aptamer. These complexes are also immunoquiescent. A significant benefit of using nucleic acids as anticoagulants is their rapid deactivation by the introduction of a nanostructure made fully from the reverse complement of the therapeutically active nanostructure. These advantages make nanoparticle conjugated antithrombin aptamers a promising candidate for a rapidly reversible anticoagulant therapy.