Aptamers are synthetic single-strand DNA or RNA oligonucleotides that adopt secondary and tertiary conformations, which allow them to bind and inhibit the target molecules to which they have been selected with high affinity and specificity . The laboratory technique of aptamer selection, Selective Evolution of Ligands by EXponential enrichment (SELEX), was first described in 1990 [2,3] and the ongoing evolution of aptamer selection has been reviewed by multiple authors [1,4]. Most aptamer selections have targeted protein molecules, many of which are enzymes. Aptamers selected against enzymes cleaved from zymogens, a characteristic of coagulation proteases, typically bind both an enzyme exosite [5–7,8▪] and the corresponding zymogen proexosite [9–12,13▪] if the latter is exposed. Enzyme-inhibiting aptamers generally function by inhibiting one or more kinetically important macromolecular interactions between the enzyme and either a cofactor or a substrate of that enzyme [9–11].
In 1992, Bock et al.  identified a 15-nt DNA aptamer that bound thrombin, the first nonnuclear protein SELEX target. That aptamer inhibited thrombin's procoagulant activity, and in 1994, DeAnda et al.  found that it significantly prolonged the activated clotting times of dogs, on which they performed cardiopulmonary bypass (CPB) surgery. Those findings and the drawbacks of unfractionated heparin (UFH) prompted the selection of additional thrombin aptamers [6,11,16,17] as well as aptamers against FVIIa [7,18], FIXa , and factor X (FX)a  by investigators seeking to identify rapid onset heparinless anticoagulation strategies. In addition, the subsequent discovery that the anticoagulant activity of such aptamers could be rapidly neutralized by using sequence-specific, complementary antidote oligonucleotides further fueled efforts to develop aptamers as a novel class of antidote-controllable anticoagulants [20,21]. One of those aptamers, the 35-nt FIXa RNA aptamer 9.3t (Table 1) was found to inhibit over 99% of FIXa's catalytic activity  and to achieve potent in-vivo anticoagulant and antithrombotic effects, which could be promptly reversed using a specific 17-mer antidote oligonucleotides . A high molecular weight polyethylene glycol (PEG) conjugate of 9.3t, subsequently named pegnivacogin , was found to have a significantly longer in-vivo half-life than 9.3t, and an anticoagulation strategy constituting of pegnivacogin and its antidote oligonucleotides (later named anivamersen) was tested in human volunteers  and later in over 2000 patients who underwent percutaneous coronary intervention (PCI) procedures during several clinical trials [24,25,26▪▪].
In this review, we first discuss recent developments in the anticoagulant field including important lessons learned from preclinical and clinical studies of pegnivacogin. Some of these have inspired newer attempts to develop alternative strategies for potent antidote-controllable CPB anticoagulation that utilize the FXa aptamer 11F7t. In addition, we examine recent efforts to identify aptamers that inhibit contact pathway factors such as FXIa and kallikrein, which may prove to be valuable as antithrombotic therapeutics that minimize the associated risk of bleeding in certain clinical settings. Finally, we also discuss early studies of pro-hemostatic aptamers that inhibit activated protein C (APC) and tissue plasminogen activator (tPA), which may help to control bleeding in patients with hemophilia or other coagulopathies.
LESSONS LEARNED FROM THE FIXa APTAMER PEGNIVACOGIN
Several authors [1,27▪▪,28] have previously reviewed in detail the clinical trial experience of pegnivacogin and anivamersen (together referred to as the REG1 anticoagulation system). Briefly, following successful demonstration of both efficacy and safety in Phase I and Phase IIa trials [23,29,30], REG1 was compared with UFH in patients undergoing PCI in the Phase IIb RADAR trial. Administration of REG1 not only facilitated safe and effective PCI with minimal bleeding observed after femoral sheath removal but also reduced the incidence of ischemic events to 3.0% compared with 5.7% in the UFH arm . Those encouraging results prompted commencement of REGULATE-PCI, a large Phase III trial that aimed to assess the efficacy of REG1 versus bivalirudin for PCI in more than 13 000 patients . However, this study was halted in 2014 following enrollment of 3232 participants after 10 serious allergic reactions to pegnivacogin, including one fatality, occurred . In fact, even during the previous RADAR trial, 3 of the 640 study participants who received pegnivacogin had similar reactions .
To examine the cause of these adverse reactions, recent post hoc studies by Ganson et al. [27▪▪] and Povsic et al. [32▪▪] in 2016 conducted using plasma from patients in both the RADAR and REGULATE-PCI studies revealed a strong correlation between the incidence of allergic reactions and the presence of preexisting circulating anti-PEG antibodies . Moreover, patients from REGULATE-PCI who experienced the most severe reactions were those with the highest levels of preexisting anti-PEG antibodies [27▪▪,31,32▪▪]. Thus, investigators concluded that reactions to the PEG moiety of pegnivacogin – and not its aptamer component – were responsible for the adverse responses [27▪▪,31,32▪▪]. This important finding highlights the potential of aptamer therapeutics for use in similar clinical applications, provided individuals with anti-PEG antibodies be screened for and excluded from such therapies or that alternative chemical formulations that avoid PEG can be identified to facilitate suitable pharmacokinetics. In this light, certain groups are exploring the design of aptamer–albumin conjugates and aptamer amphiphiles that self-assemble into aptamer-displaying micelles [33▪–35▪].
Although further clinical development of pegnivacogin has been discontinued, the overall results of the RADAR and REGULATE-PCI trials demonstrate that aptamer-based inhibition of FIXa can provide effective anticoagulation during PCI. However, a 2016 study in baboons exploring the use of REG1 for CPB suggests that targeting FIXa alone does not achieve satisfactory CPB anticoagulation [36▪]. Baboons that received pegnivacogin, in contrast to those that received UFH, displayed evidence of microthrombi in the bypass cannulae and kidney cortical infarcts [36▪]. The authors concluded that the inability of the FIXa-targeting aptamer to effectively inhibit tissue factor-mediated coagulation – a critical component of the hemostatic activation that occurs during CPB – was responsible for the inadequate anticoagulation observed in this highly thrombogenic setting [36▪]. These findings have motivated newer efforts (described in the following section) to develop more potent anticoagulant strategies using aptamer inhibitors of common pathway factors FXa and thrombin, in order to provide effective anticoagulation for CPB that overcomes hemostatic activation triggered by both the intrinsic and extrinsic pathways.
ANTICOAGULANT APTAMER STRATEGIES INVOLVING FXa INHIBITION
Three heparinless anticoagulation strategies that utilize 11F7t, a 36-nt RNA FXa aptamer originally identified in 2010  (Table 2), have now been found to mimic UFH's potent anticoagulant effects. 11F7t binds a FXa exosite (K d = 1.26 nmol/l) and the corresponding FX proexosite (K d = 0.79 nmol/l) in a manner that inhibits FXa from binding FVa to form prothrombinase, inhibits FXa-catalyzed cleavage of FVIII, and inhibits FIXa-catalyzed activation of FX . Without binding or inhibiting FXa's catalytic site, 11F7t achieves a significant anticoagulant effect, although one that is less potent than that of UFH .
In 2014, Bompiani et al.  tested the ability of R9D-14t (Table 2), a 58-nt RNA thrombin/prothrombin aptamer, to augment 11F7t's anticoagulant intensity . R9D-14t binds both exosite I of thrombin (K d = 1.0 nmol/l), thereby inhibiting thrombin from binding fibrinogen, FV, FVIII, among others, and the corresponding proexosite of prothrombin (K d = 10.0 nmol/l), thereby inhibiting FXa-catalyzed cleavage of prothrombin, but nevertheless also achieves an anticoagulant effect less potent than UFH's [11,37]. The authors showed that the combination of 0.5 μmol/l 11F7t with 5.0 μmol/l R9D-14t could prevent clot formation in human whole blood that was recirculated continuously for 2 h within a functioning miniature ex-vivo CPB oxygenator circuit as effectively as 5 U/ml UFH, the standard UFH concentration used for CPB . Moreover, administration of complementary antidote oligonucleotides to either 11F7t and R9d14t could rapidly reverse their individual and combined anticoagulant effects . Expanding upon this discovery, Soule et al. [38▪] described in 2016 the design of an 81-nt bivalent RNA aptamer RNABA4 constituting covalently linked truncated sequences of 11F7t and R9D-14t (Table 2). RNABA4 retains the binding and anticoagulant activity of both parent aptamers, yet can be effectively neutralized using a single complementary antidote oligonucleotides [38▪]. These results underscore novel approaches for developing highly potent, antidote-controllable anticoagulant alternatives to UFH for CPB anticoagulation and exemplify the chemical versatility of constructing bivalent aptamer conjugates to engineer a desired functionality.
In 2018, Gunaratne et al. [39▪▪] tested the ability of rivaroxaban, apixaban, edoxaban, or fondaparinux to augment the anticoagulant effect of 11F7t in blood subjected to the same experimental protocols used by Bompiani et al.  and Soule et al. [38▪]. The first three of the aforementioned anticoagulants directly, specifically, and reversibly bind and inhibit FXa's catalytic site [40–42], whereas the fourth accelerates antithrombin-catalyzed irreversible inhibition of that site. None of those FXa catalytic site inhibitors by itself can achieve the anticoagulant effect of UFH. However, the combination of 2.0 μmol/l 11F7t with a 2.0 μmol/l concentration of any of those FXa catalytic site inhibitors replicated the anticoagulant potency of 5 U/ml UFH in blood recirculated within the ex-vivo CPB oxygenator circuit. Importantly, postcirculation levels of thrombin generation, which are associated with thrombotic, coagulopathic, and inflammatory complications after CPB, were significantly lower in blood anticoagulated with 11F7t with each FXa catalytic site inhibitor than in blood anticoagulated with UFH. Moreover, an inactive FXa decoy protein, GD-FXaS195A, which closely resembles an antidote for FXa inhibitors (Andexanet Alfa) that received Food and Drug Administration (FDA) approval in 2018, could be used to concomitantly neutralize the anticoagulant effects of 11F7t and each FXa catalytic site inhibitor [39▪▪]. In addition, purified IgG from patients with heparin-induced thrombocytopenia (HIT) induced pathologic platelet aggregation in the presence of UFH, but not 11F7t, alone or in combination with each FXa catalytic site inhibitor. These findings not only suggest that an anticoagulation strategy constituting of 11F7t with a FXa catalytic site inhibitor may prove to be a safer and more effective alternative to UFH for CPB especially in patients with HIT antibodies, but also highlight the unique ability of exosite-targeting aptamers to complement catalytic site-directed small molecule drugs.
ANTITHROMBOTIC APTAMERS TARGETING INTRINSIC PATHWAY FACTORS
Several lines of evidence [43–46] have prompted recent identification of aptamers that inhibit intrinsic pathway factors such as FXIa, FXIIa, or kallikrein (Table 1) [8▪,12,13▪,47▪]. Patients with deficiencies in those factors demonstrate evidence of impaired thrombin generation and fibrin formation in coagulation tests such as the activated partial thromboplastin time (aPTT) as well as reduced incidence of stroke and deep vein thrombosis, yet display no or only mild bleeding tendencies [48–51]. Moreover, inhibition of these factors in preclinical and clinical studies appears to be protective against thrombosis, and unlike inhibition of FXa or thrombin, is not associated with any significant bleeding risk [12,43,52,53]. For example, in patients undergoing total knee arthroplasty (TKA), serial subcutaneous injections of a FXI antisense oligonucleotide (FXI-ASO) carried out during the 36 days preceding the TKAs significantly reduced both circulating FXI levels and the incidence of postoperative DVT . Likewise, the FXIIa inhibitory antibody 3F7 has been shown to prevent clot formation during extracorporeal membrane oxygenation (ECMO) in rabbits in which thrombin generation is triggered primarily by contact activation . Thus, aptamer inhibitors of FXIIa, FXIa, or kallikrein, might also reduce thrombosis in such settings without a concomitant elevation in bleeding risk. The following paragraphs describe recent efforts to identify aptamers targeting FXIa and kallikrein. An RNA aptamer, R4cXII-1t, which binds a FXIIa exosite and the corresponding FXII proexosite in a manner that inhibits FXIIa-catalyzed activation of FXI and FXII autoactivation has been reviewed recently [12,56] (Table 1).
In 2017, Donkor et al. [8▪] reported the first FXIa aptamer, FELIAP (Factor ELeven Inhibitory APtamer), a 36-nt DNA aptamer that binds FXIa with high specificity and affinity (K d = 1.8 nmol/l) (Table 1). FELIAP inhibits FXIa-catalyzed cleavage of its tripeptide chromogenic substrate, FXIa-catalyzed cleavage of FIX, and AT-catalyzed inhibition of FXIa [8▪]. Thus, in contrast to aptamers selected to date against procoagulant proteases thrombin, FXa, FIXa, FVIIa, and FXIIa, FELIAP appears to directly inhibit access to the catalytic site of its target protease. FELIAP does not inhibit thrombin-catalyzed cleavage of FXI, suggesting that FELIAP, unlike the other aforementioned anticoagulant aptamers, does not bind the zymogen of its target enzyme with high affinity. Indeed, the authors do not report FELIAP's K d for FXI or whether FELIAP inhibits FXIIa-catalyzed cleavage of FXI. In modified CAT assays in which the activator is silica instead of TF, 30 μmol/l FELIAP prolonged the lag time of thrombin generation and reduced ETP.
Subsequently in 2017, Woodruff et al. reported the selection of two FXIa RNA aptamers, the 29-nt 11.16 (K d = 122.4 nmol/l for FXIa) and the 40-nt 12.7 (K d = 84.9 nmol/l for FXIa; K d = 189.2 nmol/l for FXI) (Table 1). Like FELIAP, each of those aptamers also directly inhibits FXIa-catalyzed cleavage of FIX. Alanine scanning experiments suggest that the inhibitory effect each RNA FXIa aptamer depends to varying degrees on its binding to FXIa's anion-binding exosite 2 and FXIa's autolysis loop. The proximity of each of those positively charged exosites to FXIa's catalytic site may partly explain the inhibitory effect of 11.16 and 12.7 on FXIa-catalyzed substrate cleavage. The fact that FXIa-ASO injections significantly reduced the risk of post-TKA DVT  suggests that aptamer-induced inhibition of FXIa might achieve a similar result.
In 2017, Steen Burrell et al. [47▪] identified a 54-nt RNA aptamer, Kall1-T4, that binds kallikrein and prekallikrein with high specificity and subnanomolar affinity (Table 1). As expected, the aptamer does not prolong the prothrombin time, but it does extend the aPTT in a dose-dependent manner. Kall1-T4 does not inhibit kallikrein-catalyzed cleavage of chromogenic tripeptide substrates, suggesting that the aptamer neither binds kallikrein's catalytic site nor alters that site through an allosteric mechanism, but instead binds a kallikrein exosite. In plasma, FXIIa catalyzes the cleavage of prekallikrein to kallikrein, which in turn catalyzes the cleavage of FXII to FXIIa in a reciprocal activation feed-back loop. Kinetic studies performed in systems of purified proteins suggest that Kall1-T4 inhibits that feedback loop mainly by inhibiting FXIIa-catalyzed prekallikrein activation [47▪]. The aptamer has a significantly smaller inhibitory effect on kallikrein-mediated FXII activation. At concentrations over 250 nmol/l, Kall1-T4 also inhibits kallikrein-catalyzed release of bradykinin from high molecular weight kininogen by about 65%.
PROCOAGULANT APTAMERS TARGETING ANTICOAGULANT FACTORS
Although initial efforts to develop aptamers against anticoagulant factors to control bleeding in clinical settings such as hemophilia, trauma, or other coagulopathies focused on tissue factor pathway inhibitor (TFPI)-targeting aptamers (reviewed previously in , Table 3), more recent work has also explored the development of aptamers that inhibit APC and tissue plasminogen activator (tPA). APC catalyzes the inactivation of FVa and FVIIIa, and its overactivation has been shown to play a critical role in the development of trauma-induced coagulopathy (TIC), leading to interest in identifying small molecule and other therapeutics that can neutralize its activity . In 2009, Muller et al.  identified a 52 nt DNA aptamer, HS02-52G, that selectively binds a basic exosite of human APC with nanomolar affinity in a manner that significantly reduces APC-mediated cleavage of FVIIIa and FVa (Table 3). In doing so, HS02-52G dose-dependently shortens the aPTT clotting time reflecting its procoagulant activity. To enable rapid control of aptamer activity, Hamedani et al. [59▪] in 2016 designed a 22nt ssDNA oligonucleotide antisense antidote, AD 22, which completely reverses the procoagulant function of HS02-52G in plasma and whole blood clotting as well as thrombin generation assays. Thus, such an aptamer–antidote pair targeting APC has the potential for clinical application as a controllable procoagulant therapeutic for TIC or hemophilia.
Although recombinant tissue plasminogen activator (r-tPA) is routinely used as a life-saving fibrinolytic agent for treatment of acute thrombotic stroke, its administration is associated with significant risk of cerebral hemorrhage, leading to cautious use by clinicians [60▪]. Antifibrinolytic agents such as tranexamic acid have been used to counteract r-tPA-induced bleeding, but adverse effects linked to these products have motivated investigators to identify aptamer-based antidotes against r-tPA to better control its activity and allow for safer clinical use [60▪]. In 2017, Bjerragaard et al. [60▪] designed a bivalent 82 nt 2′-fluoropyrimidine-modified RNA aptamer termed 3218 that targets human tPA (Table 3). 3218 was designed as a conjugate of two previously identified tPA-targeting aptamers : K18v2 (32 nt), which binds tPA's catalytic domain and K32v2 (32 nt), which binds the A domain, joined by an 18 nt linker. Although K18v2 and K32v2 individually have minimal effects on the fibrinolytic properties of rTPA, the bivalent aptamer not only inhibited activation of plasminogen by rTPA 40-fold better than each aptamer alone but also inhibited fibrinolysis for up to 600 min in tPA-treated human plasma, underscoring its clinical potential as an alternative therapy to control tPA-induced hemorrhage [60▪]. Moreover, this study provides another example of leveraging the biochemical properties of coagulation factor-targeting aptamers to design complementary conjugates that optimize therapeutic utility.
In summary, this review highlights recent progress in the development of aptamers for anticoagulant, antithrombotic, and pro-hemostatic indications. Several of these advances exemplify important features of aptamers that arise from their inherent biochemical malleability and unique ability to modulate exosite-dependent macromolecular interactions. These include the capacity to utilize aptamers in conjugation with one another or in complementation with other drug types (e.g. small molecules or antibodies) in order to rationally engineer therapeutic strategies with enhanced functionality. Such properties of aptamers may prove to be valuable for designing novel, improved therapies in a variety of clinical contexts even beyond anticoagulation or hemostasis.
Financial support and sponsorship
This work was supported by NIH grants HL-065222 (B.A.S), F30 HL-127977 (R.G.), and T32 GM-007171 (R.G.).
Conflicts of interest
Duke University (B.A.S.) have applied for patents on aptamer-based anticoagulant strategies. There are no conflicts of interests for the remaining authors.
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