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Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain. [ABSTRACT FROM AUTHOR]

Background: Andexanet alfa (andexanet) is the only FDA-approved antidote for direct factor Xa (FXa) inhibitors but has been reported to cause resistance to unfractionated heparin (UFH). This has delayed anticoagulation for procedures requiring cardiopulmonary bypass (CPB). The mechanism, andexanet and UFH dose dependence, and thrombotic risk of andexanet-associated heparin resistance are unknown., Methods: The effect of andexanet in vitro was determined using activated clotting times (ACT) and thromboelastography (TEG). Ex vivo CPB circuits were used to determine whether andexanet impaired anticoagulation for extracorporeal circulation. Kinetics of antithrombin (AT) inhibition of FXa and thrombin were measured in the presence of andexanet. Equilibrium modeling and thrombin generation assay (TGA) validation were used to predict the role of andexanet, AT, and UFH concentrations in andexanet-associated heparin resistance., Results: Andexanet prevented UFH-mediated prolongation of ACT and TEG times. At lower concentrations of andexanet, heparin resistance could be overcome with suprapharmacologic doses of UFH, but not at higher andexanet concentrations. Andexanet rendered standard doses of UFH inadequate to prevent circuit thrombosis, and suprapharmacologic UFH doses were only partially able to overcome this. Scanning electron microscopy demonstrated coagulation activation in circuits. Andexanet prevented UFH enhancement of AT-mediated inhibition of FXa and thrombin. Equilibrium modeling and TGA validation demonstrated that andexanet creates a triphasic equilibrium with UFH and AT: initial UFH unresponsiveness, normal UFH responsiveness when andexanet is depleted, and finally AT depletion. Sufficient CPB heparinization can only occur at low therapeutic andexanet doses and normal AT levels. Higher andexanet doses or AT deficiency may require both AT supplementation and very high UFH doses., Conclusions: Andexanet causes heparin resistance due to redistribution of UFH-bound AT. If andexanet cannot be avoided prior to heparinization and direct thrombin inhibitors are undesirable, our in vitro study suggests excess UFH should be considered as a potential strategy prior to AT supplementation., Highlights: Andexanet alfa causes heparin resistance not by depleting antithrombin, but rather by sequestering heparin-bound antithrombin such that it cannot act as an anticoagulant.Heparin responsiveness in the presence of Andexanet alfa is triphasic such that the effect of a dose of heparin can now be predicted in vitro based on the relative concentrations of andexanet, heparin, and antithrombin.The in vitro insights provided by this work provide a rational starting point for further clinical elucidation of the problem and management of andexanet-associated heparin resistance.

Abstract: Secreted microRNAs (miRNAs) have been detected in various body fluids including the cerebrospinal fluid, yet their direct role in regulating synaptic transmission remains uncertain. We found that intrathecal injection of low dose of let-7b (1 μg) induced short-term (<24 hours) mechanical allodynia and heat hyperalgesia, a response that is compromised in Tlr7-/- or Trpa1-/- mice. Ex vivo and in vivo calcium imaging in GCaMP6-report mice revealed increased calcium signal in spinal cord afferent terminals and doral root ganglion/dorsal root ganglia neurons following spinal perfusion and intraplantar injection of let-7b. Patch-clamp recordings also demonstrated enhanced excitatory synaptic transmission (miniature excitatory postsynaptic currents [EPSCs]) in spinal nociceptive neurons following let-7b perfusion or optogenetic activation of axonal terminals. The elevation in spinal calcium signaling and EPSCs was dependent on the presence of toll-like receptor-7 (TLR7) and transient receptor potential ion channel subtype A1 (TRPA1). In addition, endogenous let-7b is enriched in spinal cord synaptosome, and peripheral inflammation increased let-7b in doral root ganglion/dorsal root ganglia neurons, spinal cord tissue, and the cerebrospinal fluid. Notably, let-7b antagomir inhibited inflammatory pain and inflammation-induced synaptic plasticity (EPSC increase), suggesting an endogenous role of let-7b in regulating pain and synaptic transmission. Furthermore, intrathecal injection of let-7b, at a higher dose (10 μg), induced persistent mechanical allodynia for >2 weeks, which was abolished in Tlr7-/- mice. The high dose of let-7b also induced microgliosis in the spinal cord. Of interest, intrathecal minocycline only inhibited let-7b-induced mechanical allodynia in male but not female mice. Our findings indicate that the secreted microRNA let-7b has the capacity to provoke pain through both neuronal and glial signaling, thereby establishing miRNA as an emerging neuromodulator., (Copyright © 2024 International Association for the Study of Pain.)

Background: Severe COVID-19 carries a high morbidity and mortality. Previous studies have shown an association between COVID-19 severity and SARS-CoV-2 viral load (VL). We sought to measure VL in multiple compartments (urine, plasma, lower respiratory tract) in patients admitted to the intensive care unit (ICU) with severe COVID-19 pneumonia and correlate with clinical outcomes., Methods: Plasma, urine, and endotracheal aspirate (ETA) samples were obtained on days 1, 3, 7, 14, and 21 from subjects admitted to the ICU with severe COVID-19. VL was measured via reverse transcriptase polymerase chain reaction. Clinical data was collected from the electronic health record. Grouped comparisons were performed using Student's t-test or 1-way ANOVA. Linear regression was used to correlate VL from different compartments collected at the same time. Logistic regression was performed to model ventilator-freedom at 28 days as a function of peak plasma VL., Results: We enrolled 57 subjects with severe COVID-19 and measured VL in plasma (n = 57), urine (n = 25), and ETA (n = 34). Ventilator-associated pneumonia developed in 63% of subjects. 49% of subjects were viremic on study day 1. VL in plasma and ETA both significantly decreased by day 14 (P < 0.05), and the two were weakly correlated on study day 1 (P = 0.0037, r 2  = 0.2343) and on all study days (P < 0.001, r 2  = 0.2211). VL were not detected in urine. While no associations were observed with peak ETA VL, subjects with higher peak plasma VL experienced a greater number of respiratory complications, including ventilator-associated pneumonia and fewer ventilator-free and hospital-free days. There was no association between VL in either plasma or ETA and mortality. In viremic patients, plasma VL was significantly lower in subjects that were ICU-free and ventilator-free (P < 0.05), with trends noted for hospital-freedom, ventilator-associated pneumonia, and survival to discharge (P < 0.1). By logistic regression, plasma VL was inversely associated with ventilator-freedom at 28 days (odds ratio 0.14, 95% confidence interval 0.02-0.50)., Conclusions: Elevated SARS-CoV-2 VL in the plasma but not in the lower respiratory tract is a novel biomarker in severe COVID-19 for respiratory complications., (© 2024. The Author(s).)

The ability to reverse the binding of aptamers to their target proteins has received considerable attention for developing controllable therapeutic agents. Recently, use of aptamers as reversible cell-sorting ligands has also sparked interest. Antibodies are currently utilized for isolating cells expressing a particular cell surface receptor. The inability to remove antibodies from isolated cells following sorting greatly limits their utility for many applications. Previously, we described how a particular aptamer-antidote oligonucleotide pair can isolate cells and clean them. Here, we demonstrate that this approach is generalizable; aptamers can simultaneously recognize more than one cell type during fluorescent activated cell sorting (FACS). Moreover, we describe a novel approach to reverse aptamer binding following cell sorting using a nuclease. This alternative strategy represents a cleaning approach that does not require the generation of antidote oligonucleotides for each aptamer and will greatly reduce the cost and expand the utility of Clean FACS.