Nucleic Acid Polymers Research Articles

State-of-the-art and emerging antivirals for chronic hepatitis B infection

ABSTRACT Introduction Current treatment options for chronic hepatitis B virus (HBV) infection cannot achieve functional cure [hepatitis B surface antigen (HBsAg) loss]; therefore, new approaches are under investigation. This review summarizes the most promising approaches in emerging antivirals against HBV, after search in Medline (2016–2022) and European and American liver meetings (2019–2022). Areas covered Classes of antivirals include entry inhibitors (bulevirtide), capsid assembly modulators (CAMs), translation inhibitors [small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs)], and HBsAg secretion inhibitors [nucleic acid polymers (NAPs)]. Bulevirtide has good efficacy in hepatitis B and D coinfection, but there is limited data in HBV monoinfection. CAMs profoundly reduce serum HBV DNA/RNA levels, but have minimal effects on antigen levels. siRNAs and ASOs mostly reduce HBsAg levels, but small proportions of patients reach HBsAg seroclearance. NAPs reduce serum HBV DNA and especially HBsAg levels offering substantial HBsAg seroconversion rates, but having limited data over a long period. Combinations of agents of different classes are starting to be evaluated. Expert opinion Continued efforts are required in order to address many unanswered questions about the optimal combined regimens of finite duration which will be safe and well tolerated achieving functional cure in a substantial proportion of chronic HBV patients.

The hepatitis delta virus and chronic hepatitis D.

The hepatitis delta virus and chronic hepatitis D.

FUS Microphase Separation: Regulation by Nucleic Acid Polymers and DNA Repair Proteins.

Fused in sarcoma (FUS) is involved in the regulation of RNA and DNA metabolism. FUS participates in the formation of biomolecular condensates driven by phase transition. FUS is prone to self-aggregation and tends to undergo phase transition both with or without nucleic acid polymers. Using dynamic light scattering and fluorescence microscopy, we examined the formation of FUS high-order structures or FUS-rich microphases induced by the presence of RNA, poly(ADP-ribose), ssDNA, or dsDNA and evaluated effects of some nucleic-acid-binding proteins on the phase behavior of FUS-nucleic acid systems. Formation and stability of FUS-rich microphases only partially correlated with FUS's affinity for a nucleic acid polymer. Some proteins-which directly interact with PAR, RNA, ssDNA, and dsDNA and are possible components of FUS-enriched cellular condensates-disrupted the nucleic-acid-induced assembly of FUS-rich microphases. We found that XRCC1, a DNA repair factor, underwent a microphase separation and formed own microdroplets and coassemblies with FUS in the presence of poly(ADP-ribose). These results probably indicated an important role of nucleic-acid-binding proteins in the regulation of FUS-dependent formation of condensates and imply the possibility of the formation of XRCC1-dependent phase-separated condensates in the cell.

Length-Controlled Nanofiber Micelleplexes as Efficient Nucleic Acid Delivery Vehicles.

Micelleplexes show great promise as effective polymeric delivery systems for nucleic acids. Although studies have shown that spherical micelleplexes can exhibit superior cellular transfection to polyplexes, to date there has been no report on the effects of micelleplex morphology on cellular transfection. In this work, we prepared precision, length-tunable poly(fluorenetrimethylenecarbonate)-b-poly(2-(dimethylamino)ethyl methacrylate) (PFTMC16-b-PDMAEMA131) nanofiber micelleplexes and compared their properties and transfection activity to those of the equivalent nanosphere micelleplexes and polyplexes. We studied the DNA complexation process in detail via a range of techniques including cryo-transmission electron microscopy, atomic force microscopy, dynamic light scattering, and ζ-potential measurements, thereby examining how nanofiber micelleplexes form, as well the key differences that exist compared to nanosphere micelleplexes and polyplexes in terms of DNA loading and colloidal stability. The effects of particle morphology and nanofiber length on the transfection and cell viability of U-87 MG glioblastoma cells with a luciferase plasmid were explored, revealing that short nanofiber micelleplexes (length < ca. 100 nm) were the most effective delivery vehicle examined, outperforming nanosphere micelleplexes, polyplexes, and longer nanofiber micelleplexes as well as the Lipofectamine 2000 control. This study highlights the potential importance of 1D micelleplex morphologies for achieving optimal transfection activity and provides a fundamental platform for the future development of more effective polymeric nucleic acid delivery vehicles.

Oligonucleotide-Based Therapies for Chronic HBV Infection: A Primer on Biochemistry, Mechanisms and Antiviral Effects.

Three types of oligonucleotide-based medicines are under clinical development for the treatment of chronic HBV infection. Antisense oligonucleotides (ASOs) and synthetic interfering RNA (siRNA) are designed to degrade HBV mRNA, and nucleic acid polymers (NAPs) stop the assembly and secretion of HBV subviral particles. Extensive clinical development of ASOs and siRNA for a variety of liver diseases has established a solid understanding of their pharmacodynamics, accumulation in different tissue types in the liver, pharmacological effects, off-target effects and how chemical modifications and delivery approaches affect these parameters. These effects are highly conserved for all ASO and siRNA used in human studies to date. The clinical assessment of several ASO and siRNA compounds in chronic HBV infection in recent years is complicated by the different delivery approaches used. Moreover, these assessments have not considered the large clinical database of ASO/siRNA function in other liver diseases and known off target effects in other viral infections. The goal of this review is to summarize the current understanding of ASO/siRNA/NAP pharmacology and integrate these concepts into current clinical results for these compounds in the treatment of chronic HBV infection.

Medical Advances in Hepatitis D Therapy: Molecular Targets.

An approximate number of 250 million people worldwide are chronically infected with hepatitis B virus, making them susceptible to a coinfection with hepatitis D virus. The superinfection causes the most severe form of a viral hepatitis and thus drastically worsens the course of the disease. Until recently, the only available therapy consisted of interferon-α, only eligible for a minority of patients. In July 2020, the EMA granted Hepcludex conditional marketing authorization throughout the European Union. This first-in-class entry inhibitor offers the promise to prevent the spread in order to gain control and eventually participate in curing hepatitis B and D. Hepcludex is an example of how understanding the viral lifecycle can give rise to new therapy options. Sodium taurocholate co-transporting polypeptide, the virus receptor and the target of Hepcludex, and other targets of hepatitis D therapy currently researched are reviewed in this work. Farnesyltransferase inhibitors such as Lonafarnib, targeting another essential molecule in the HDV life cycle, represent a promising target for hepatitis D therapy. Farnesyltransferase attaches a farnesyl (isoprenyl) group to proteins carrying a C-terminal Ca1a2X (C: cysteine, a: aliphatic amino acid, X: C-terminal amino acid) motif like the large hepatitis D virus antigen. This modification enables the interaction of the HBV/HDV particle and the virus envelope proteins. Lonafarnib, which prevents this envelopment, has been tested in clinical trials. Targeting the lifecycle of the hepatitis B virus needs to be considered in hepatitis D therapy in order to cure a patient from both coexisting infections. Nucleic acid polymers target the hepatitis B lifecycle in a manner that is not yet understood. Understanding the possible targets of the hepatitis D virus therapy is inevitable for the improvement and development of a sufficient therapy that HDV patients are desperately in need of.

A single-molecule approach to unravel the molecular mechanism of the action of Deinococcus radiodurans RecD2 and its interaction with SSB and RecA in DNA repair

Helicases are ATP-driven molecular machines that directionally remodel nucleic acid polymers in all three domains of life. They are responsible for resolving double-stranded DNA (dsDNA) into single-strands, which is essential for DNA replication, nucleotide excision repair, and homologous recombination. RecD2 from Deinococcus radiodurans (DrRecD2) has important contributions to the organism's unusually high tolerance to gamma radiation and hydrogen peroxide. Although the results from X-ray Crystallography studies have revealed the structural characteristics of the protein, direct experimental evidence regarding the dynamics of the DNA unwinding process by DrRecD2 in the context of other accessory proteins is yet to be found. In this study, we have probed the exact binding event and processivity of DrRecD2 at single-molecule resolution using Protein-induced fluorescence enhancement (smPIFE) and Forster resonance energy transfer (smFRET). We have found that the protein prefers to bind at the 5′ terminal end of the single-stranded DNA (ssDNA) by Drift and has helicase activity even in absence of ATP. However, a faster and iterative mode of DNA unwinding was evident in presence of ATP. The rate of translocation of the protein was found to be slower on dsDNA compared to ssDNA. We also showed that DrRecD2 is recruited at the binding site by the single-strand binding protein (SSB) and during the unwinding, it can displace RecA from ssDNA.

Generating experimentally unrelated target molecule-binding highly functionalized nucleic-acid polymers using machine learning

In vitro selection queries large combinatorial libraries for sequence-defined polymers with target binding and reaction catalysis activity. While the total sequence space of these libraries can extend beyond 1022 sequences, practical considerations limit starting sequences to ≤~1015 distinct molecules. Selection-induced sequence convergence and limited sequencing depth further constrain experimentally observable sequence space. To address these limitations, we integrate experimental and machine learning approaches to explore regions of sequence space unrelated to experimentally derived variants. We perform in vitro selections to discover highly side-chain-functionalized nucleic acid polymers (HFNAPs) with potent affinities for a target small molecule (daunomycin KD = 5–65 nM). We then use the selection data to train a conditional variational autoencoder (CVAE) machine learning model to generate diverse and unique HFNAP sequences with high daunomycin affinities (KD = 9–26 nM), even though they are unrelated in sequence to experimental polymers. Coupling in vitro selection with a machine learning model thus enables direct generation of active variants, demonstrating a new approach to the discovery of functional biopolymers.

SAT398 - Characterizing of hepatitis B virus serum RNA kinetics during TDF plus pegylated interferon alfa-2a with and without nucleic acid polymers

SAT398 - Characterizing of hepatitis B virus serum RNA kinetics during TDF plus pegylated interferon alfa-2a with and without nucleic acid polymers

Recent breakthroughs in the treatment of chronic hepatitis Delta.

Hepatitis Delta virus (HDV) is responsible for the most aggressive form of chronic hepatitis, which may evolve towards cirrhosis, hepatocellular carcinoma and death within few years. During the last 30 years the only available therapy was interferon or peg-IFN, which was characterized by poor tolerability and modest results. The detailed knowledge of the HDV replication cycle and its interaction with HBV allowed the introduction of new drugs which are currently in phase II or III of experimentation. Basically, bulevirtide, to date the only one approved by EMA, inhibits the entry of the virus into the hepatocytes and hence its intrahepatic spread; lonafarnib inhibits the pharnesylation process of the L-HDAg, which is critical for the assembly of the HDV virion; the nucleic acid polymers (NAPs) mainly block the production/release of HBsAg. The available clinical trials with these compounds showed an excellent anti-viral activity against HDV.

Self-Consistent Parameterization of DNA Residues for the Non-Polarizable AMBER Force Fields.

Fixed-charge (non-polarizable) forcefields are accurate and computationally efficient tools for modeling the molecular dynamics of nucleic acid polymers, particularly DNA, well into the µs timescale. The continued utility of these forcefields depends in part on expanding the residue set in step with advancing nucleic acid chemistry and biology. A key step in parameterizing new residues is charge derivation which is self-consistent with the existing residues. As atomic charges are derived by fitting against molecular electrostatic potentials, appropriate structural models are critical. Benchmarking against the existing charge set used in current AMBER nucleic acid forcefields, we report that quantum mechanical models of deoxynucleosides, even at a high level of theory, are not optimal structures for charge derivation. Instead, structures from molecular mechanics minimization yield charges with up to 6-fold lower RMS deviation from the published values, due to the choice of such an approach in the derivation of the original charge set. We present a contemporary protocol for rendering self-consistent charges as well as optimized charges for a panel of nine non-canonical residues that will permit comparison with literature as well as studying the dynamics of novel DNA polymers.

Multiple Regions Drive Hepatitis Delta Virus Proliferation and Are Therapeutic Targets.

Hepatitis Delta Virus (HDV) is the smallest mammalian single-stranded RNA virus. It requires host cells and hepatitis B virus (HBV) to complete its unique life cycle. The present review summarizes the specific regions on hepatitis D antigen (HDAg) and hepatitis B surface antigen (HBsAg) that drive HDV to utilize host cell machinery system to produce three types of RNA and two forms of HDAg, and hijack HBsAg for its secretion and de novo entry. Previously, interferon-α was the only recommended therapy for HDV infection. In recent years, some new therapies targeting these regions, such as Bulevirtide, Lonafarnib, Nucleic acid polymers have appeared, with better curative effects and fewer adverse reactions.

HBsAg isoform dynamics during NAP-based therapy of HBeAg-negative chronic HBV and HBV/HDV infection.

Nucleic acid polymers block the assembly of hepatitis B virus (HBV) subviral particles, effectively preventing hepatitis B surface antigen (HBsAg) replenishment in the circulation. Nucleic acid polymer (NAP)–based combination therapy of HBV infection or HBV/hepatitis D virus (HDV) co‐infection is accompanied by HBsAg clearance and seroconversion, HDV‐RNA clearance in co‐infection, and persistent functional cure of HBV (HBsAg < 0.05 IU/ml, HBV‐DNA target not dected, normal alanine aminotransferase) and persistent clearance of HDV RNA. An analysis of HBsAg isoform changes during quantitative HBsAg declines (qHBsAg), and subsequent treatment‐free follow‐up in the REP 301/REP 301‐LTF (HBV/HDV) and REP 401 (HBV) studies was conducted. HBsAg isoforms were analyzed from frozen serum samples using Abbott Research Use Only assays for HBsAg isoforms (large [L], medium [M], and total [T]). The relative change over time in small HBsAg relative to the other isoforms was inferred by the change in the ratio over time of T‐HBsAg to M‐HBsAg. HBsAg isoform declines followed qHBsAg declines in all participants. No HBsAg isoforms were detectable in any participants with functional cure. HBsAg declines > 2 log10 IU/ml from baseline were correlated with selective clearance of S‐HBsAg in 39 of 42 participants. Selective S‐HBsAg decline was absent in 9 of 10 participants with HBsAg decline < 2 log10 IU/ml from baseline. Mild qHBsAg rebound during follow‐up <10 IU/ml consisted mostly of S‐HBsAg and M‐HBsAg and not accompanied by significant covalently closed circular DNA activity. Conclusion: The faster observed declines in S‐HBsAg indicate the selective clearance of subviral particles from the circulation, consistent with previous mechanistic studies on NAPs. Trace HBsAg rebound in the absence of HBV DNA may reflect HBsAg derived from integrated HBV DNA and not rebound of viral infection.

HBsAg Loss as a Treatment Endpoint for Chronic HBV Infection: HBV Cure.

Despite the availability of effective vaccines and antiviral therapy over the past two to three decades, chronic hepatitis B virus (HBV) infection remains a major global health threat as a leading cause of cirrhosis and liver cancer. Functional HBV cure defined as hepatitis B surface antigen (HBsAg) loss and undetectable serum HBV DNA is associated with improved clinical outcomes in patients with chronic HBV infection. However, spontaneous loss of HBsAg is rare and occurs in only 1% of all HBsAg-positive individuals annually. Furthermore, the rate of functional cure with currently available antiviral therapy is even lower, <1% patients on treatment per year. Nonetheless, HBsAg loss has become the new target or therapeutic endpoint for antiviral treatment. Recently, there has been much excitement surrounding the development of novel antiviral agents such as small interfering RNA (siRNA), core assembly modulators (CAMs), nucleic acid polymers (NAPs) among others, which may be used in combination with nucleos(t)ide analogs and possibly immunomodulatory therapies to achieve functional cure in a significant proportion of patients with chronic hepatitis B. Novel assays with improved sensitivity for detection of very low levels of HBsAg and to determine the source of HBsAg production will also be required to measure efficacy of newer antiviral treatments for HBV cure. In this narrative review, we will define HBV cure, discuss various sources of HBsAg production, evaluate rates of HBsAg loss with current and future antiviral agents, review clinical factors associated with spontaneous HBsAg loss, and explore clinical implications of functional cure.

Fluorophore-mediated Photooxidation of the Guanine Heterocycle.

Fluorescent dyes are routinely used to visualize DNA or RNA in various experiments, and some dyes also act as photosensitizers capable of catalyzing oxidation reactions. The present studies explored whether the common labeling dyes fluorescein, rhodamine, BODIPY, or cyanine3 (Cy3) can function as photosensitizers to oxidize nucleic acid polymers. Photoirradiation of each dye in the presence of the guanine (G) heterocycle, which is the most sensitive toward oxidation, identified slow rates of nucleobase oxidation in the nucleoside and DNA contexts. For all four fluorophores studied, the only product detected was spiroiminodihydantoin (Sp) suggesting the dyes functioned as Type II photosensitizers and generate singlet oxygen (1O2). The nucleoside reactions were then conducted in D2O solutions, known to increase the lifetime of 1O2, which resulted in a ~6-fold increase in the Sp yield, further supporting the classification of these dyes as Type II photosensitizers. Lastly, we inspected the pattern of G reactivity with the dyes upon photoirradiation in the context of a parallel-stranded G-quadruplex. The G nucleotides in the two exterior G-tetrads were found to be oxidation prone, providing the third line of evidence that the dyes are Type II photooxidants. The present work found that the common dyes fluorescein, rhodamine, BODIPY, or Cy3 can drive G oxidation but with a slow rate and low overall yield. This will likely not impact many experiments using dyes to study nucleic acids except for those that have long exposures with high-intensity lights, such as sequencing-by-synthesis experiments using fluorescence as the readout.

Making Sense of Antisense Oligonucleotide Therapeutics Targeting Bcl-2.

The B-cell lymphoma 2 (Bcl-2) family, comprised of pro- and anti-apoptotic proteins, regulates the delicate balance between programmed cell death and cell survival. The Bcl-2 family is essential in the maintenance of tissue homeostasis, but also a key culprit in tumorigenesis. Anti-apoptotic Bcl-2, the founding member of this family, was discovered due to its dysregulated expression in non-Hodgkin’s lymphoma. Bcl-2 is a central protagonist in a wide range of human cancers, promoting cell survival, angiogenesis and chemotherapy resistance; this has prompted the development of Bcl-2-targeting drugs. Antisense oligonucleotides (ASO) are highly specific nucleic acid polymers used to modulate target gene expression. Over the past 25 years several Bcl-2 ASO have been developed in preclinical studies and explored in clinical trials. This review will describe the history and development of Bcl-2-targeted ASO; from initial attempts, optimizations, clinical trials undertaken and the promising candidates at hand.

Systematic Investigation of Biocompatible Cationic Polymeric Nucleic Acid Carriers for Immunotherapy of Hepatocellular Carcinoma.

Simple SummaryImmunotherapy, including adaptive and innate immunotherapy, exhibits promising future for the treatment of hepatocellular carcinoma. As a common tool for immunotherapy, the safe and efficient gene delivery turns to be especially important. Biocompatible polymers are a category of promising materials used in gene delivery, while there still lacks a comprehensive review article to discuss the updates on multiple disciplines covering biocompatible polymers, gene therapy, tumor immune microenvironment, and immunotherapy. This review is well-integrated with biocompatible polymers, nonviral gene therapy, and cancer immunotherapy. Our investigation will provide different perspective for the scientists focusing on the domains of biomaterials, gene therapy, and oncologists to move their research work forward.Hepatocellular carcinoma (HCC) is the third-largest cause of cancer death worldwide, while immunotherapy is rapidly being developed to fight HCC with great potential. Nucleic acid drugs are the most important modulators in HCC immunotherapy. To boost the efficacy of therapeutics and amplify the efficiency of genetic materials, biocompatible polymers are commonly used. However, under the strong need of a summary for current developments of biocompatible polymeric nucleic acid carriers for immunotherapy of HCC, there is rare review article specific to this topic to our best knowledge. In this article, we will discuss the current progress of immunotherapy for HCC, biocompatible cationic polymers (BCPs) as nucleic acid carriers used (or potential) to fight HCC, the roles of biocompatible polymeric carriers for nucleic acid delivery, and nucleic acid delivery by biocompatible polymers for immunotherapy. At the end, we will conclude the review and discuss future perspectives. This article discusses biocompatible polymeric nucleic acid carriers for immunotherapy of HCC from multidiscipline perspectives and provides a new insight in this domain. We believe this review will be interesting to polymer chemists, pharmacists, clinic doctors, and PhD students in related disciplines.

Current Designs of Polymeric Platforms Towards the Delivery of Nucleic Acids Inside the Cells with Focus on Polyethylenimine.

Gene delivery is a promising technology for treating diseases linked to abnormal gene expression. Since nucleic acids are the therapeutic entities in such approach, a transfecting vector is required because the macromolecules are not able to efficiently enter the cells by themselves. Viral vectors have been evidenced to be highly effective in this context; however, they suffer from fundamental drawbacks, such as the ability to stimulate immune responses. The development of synthetic vectors has accordingly emerged as an alternative. Gene delivery by using non-viral vectors is a multi-step process that poses many challenges, either regarding the extracellular or intracellular media. We explore the delivery pathway and afterwards, we review the main classes of non-viral gene delivery vectors. We further focus on the progresses concerning polyethylenimine-based polymer-nucleic acid polyplexes, which have emerged as one of the most efficient systems for delivering genetic material inside the cells. The complexity of the whole transfection pathway, along with a lack of fundamental understanding, particularly regarding the intracellular trafficking of nucleic acids complexed to non-viral vectors, probably justifies the current (beginning of 2021) limited number of formulations that have progressed to clinical trials. Truly, successful medical developments still require a lot of basic research. Advances in macromolecular chemistry and high-resolution imaging techniques will be useful to understand fundamental aspects towards further optimizations and future applications. More investigations concerning the dynamics, thermodynamics and structural parameters of polyplexes would be valuable since they can be connected to the different levels of transfection efficiency hitherto evidenced.

Novel Therapies of Hepatitis B and D.

Hepatitis B virus (HBV) infection is a global public health issue and is a major cause of cirrhosis and hepatocellular carcinoma (HCC). Hepatitis D virus (HDV) requires the hepatitis B surface antigen (HBsAg) to replicate. The eradication of HBV, therefore, can also cure HDV. The current therapies for chronic hepatitis B and D are suboptimal and cannot definitely cure the viruses. In order to achieve functional or complete cure of these infections, novel therapeutic agents that target the various sites of the viral replicative cycle are necessary. Furthermore, novel immunomodulatory agents are also essential to achieve viral clearance. Many of these new promising compounds such as entry inhibitors, covalently closed circular DNA (cccDNA) inhibitors, small interfering RNAs (siRNAs), capsid assembly modulators and nucleic acid polymers are in various stages of clinical developments. In this review article, we provided a comprehensive overview of the structure and lifecycle of HBV, the limitations of the current therapies and a summary of the novel therapeutic agents for both HDV and HBV infection.

Skeletal Muscle Myosin Is Procoagulant By Binding Factor XI Via Its A3 Domain and Enhancing Factor XI Activation By Thrombin

Blood coagulation mechanisms play key roles in health and disease. Pilot studies using selected human plasmas showed the potential associations of plasma skeletal muscle myosin (SkM) isoforms and phenotypes with pulmonary embolism and thrombin generation, suggesting SkM may contribute to blood coagulation reactions in plasma. Here we report that ex vivo studies of the coagulability of fresh flowing human blood over SkM-coated surfaces showed that an anti-factor XI (FXI) mAb, but not an anti-tissue factor mAb, inhibited clot formation, indicating that FXI is an essential contributor for the normal observed procoagulant response of blood during its exposure to immobilized SkM. This raised the question of whether procoagulant SkM's requirement for FXI involves direct or indirect effects on FXI.To assess direct interactions between SkM and FXI, Bio-Layer Interferometry (BLI) (Octet Red system) was used to record kinetics for binding of soluble FXI to immobilized SkM. BLI data showed that FXI bound to SkM with a Kd of 0.2 nM (k on= 2.92x10 6 M -1s -1 and k off=9.25x10 -3 s -1) (Fig. 1A). In contrast, prekallikrein (PK) did not bind to the SkM (Fig.1A), indicating the specificity of SkM for binding FXI. The anti-FXI mAb1A6, which recognizes the Apple (A)3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying SkM binds the FXI A3 domain.Studies using purified clotting factors were made to identify which FXI-related activities might be affected by SkM. When FXI activation by thrombin was evaluated under conditions where polyphosphate (PolyP) 100-mer and 700-mer enhance FXI activation, SkM concentration-dependently enhanced FXI activation by thrombin (Fig. 1B). Whereas alkaline phosphatase destroyed PolyP's ability to stimulate FXI activation by thrombin, it did not cause a reduction of SkM's ability to enhance FXI activation, indicating SkM's activity is independent of PolyP-like sequences in SkM. Small unilamellar phospholipid vesicles (20% phosphatidylserine (PS) / 80% phosphatidylcholine) did not affect FXI activation by thrombin; furthermore, reagents that neutralize procoagulant PS, i.e., lactadherin, annexin V, and phospholipase A2, did not affect SkM's enhancement of FXI activation by thrombin, indicating that this activity is not due to anionic phospholipids linked to SkM.The effects of SkM on FXI autoactivation and FXI activation by FXIIa were evaluated. As is well known, PolyP and some other anionic reagents, e.g., nucleic acid polymers, enhance not only FXI activation by thrombin but also FXI autoactivation and FXI activation by FXIIa. However, SkM did not significantly affect FXI autoactivation or FXI activation by factor XIIa, further emphasizing that SkM's enhancement of FXI activation by thrombin is not due to any PolyP-like compounds and that it is a unique property of procoagulant SkM. This also suggests that SkM has a unique mechanism for its procoagulant activity on FXI activation which is limited to the thrombin positive feedback loop.To evaluate further the basis for interactions between FXI and SkM, we employed FXI- PK chimeras because BLI binding studies showed that, in contrast to FXI, PK did not bind to SkM. Recombinant FXI proteins in which each of the four A domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding A domain from PK and were used to identify the site of factor XI to interact with SkM for FXI activation by thrombin. The FXI chimera with the substitution of the PKA1 domain was not activated by thrombin, which is consistent with the fact that the FXI A1 domain is an interactive site for thrombin. Thrombin activation of the two FXI chimeras (FXI/PKA3 and FXI/PKA4) with substitutions of either the A3 or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain (FXI/PKA2) did not reduce the enhancement of activation by thrombin compared to wild type FXI. Furthermore, mAb1A6, which recognizes the A3 domain and which inhibited the prothrombotic activity of fresh blood flowing over a SkM-coated surface, potently inhibited FXI binding to SkM in BLI studies. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains of FXI.In summary, we found that SkM's ex vivo procoagulant activity requires FXI, that SkM enhances FXI activation by thrombin and this requires FXI's A3 and A4 domains, and that SkM's high affinity binding of FXI requires the FXI A3 domain (Fig. 1C). [Display omitted] DisclosuresGruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment. Gailani: Anthos Therapeutics: Consultancy; Aronora: Membership on an entity's Board of Directors or advisory committees; Bayer Pharma: Consultancy; Bristol Myer Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees.