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Vascular thiol isomerases (VTIs) encompass proteins such as protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERp5), ERp46, ERp57, ERp72, thioredoxin-related transmembrane protein 1 (TMX1), and TMX4, and play pivotal functions in platelet aggregation and formation of thrombosis. Investigating vascular thiol isomerases, their substrates implicated in thrombosis, the underlying regulatory mechanisms, and the development of inhibitors targeting these enzymes represents a rapidly advancing frontier within vascular biology. In this review, we summarize the structural characteristics and functional attributes of VTIs, describe the associations between these enzymes and thrombosis, and outline the progress in developing inhibitors of VTIs for potential antithrombotic therapeutic applications., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Both protein disulfide isomerase (PDI) and SARS-CoV-2 main protease (M pro ) are reliant on active site cysteines stabilized by adjacent amino acids. We reasoned that redox active compounds might interfere with both enzymes by acting in the vicinity of these reactive sites thus interfering with viral replication and thrombus formation. Our previous screen of 1019 flavonoids identified several compounds that inhibit SARS-CoV-2 M pro ., Aims: Our goal was to identify phytochemical inhibitors of SARS-CoV-2 M pro that block thiol isomerases and are antithrombotic., Methods: PDI, ERp57, ERp5, ERp46, isolated domains of PDI, and PDI mutants were used to evaluate effects of galloylated polyphenols and their analogs on thiol isomerase reductase activity. Laser-injury and FeCl 3 models of thrombus formation and a tail snip assay were used to assess effects on thrombosis and hemostasis., Results: Pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG) inhibited both PDI and SARS-CoV-2 M pro . Evaluation of isolated PDI fragments and active site cysteine mutants showed that PGHG acts at the catalytic domains. Structure-function studies showed that PGHG interacts with histidines within the CGHC motifs of PDI. PGHG was equally active against other thiol isomerases, including ERp57, ERp5, ERp72, and ERp46. Screening numerous galloylated polyphenols demonstrated a class effect on thiol isomerase inhibition. Structure-activity relationships indicated that the galloyl moieties within large galloylated polyphenols were important for their inhibitory activity. PGHG and punicalagin were antithrombotic in murine models of thrombus formation., Conclusions: Galloylated polyphenols represent a large class of antithrombotic compounds with broad activity against thiol isomerases. Many of these compounds also inhibit SARS-CoV-2 M pro and viral replication., (Copyright © 2025. Published by Elsevier Inc.)

Background: Protein disulfide isomerase (PDI) is a promising target for combating thrombosis. Extensive research over the past decade has identified numerous PDI-targeting compounds. However, limited information exists regarding how these compounds control PDI activity, which complicates further development., Objectives: To define the mechanism of action of 2 allosteric antithrombotic compounds of therapeutic interest, quercetin-3-O-rutinoside and bepristat-2a., Methods: A multipronged approach that integrates single-molecule spectroscopy, steady-state kinetics, single-turnover kinetics, and site-specific mutagenesis., Results: PDI is a thiol isomerase consisting of 2 catalytic a domains and 2 inactive b domains arranged in the order a-b-b'-a'. The active sites CGHC are located in the a and a' domains. The binding site of quercetin-3-O-rutinoside and bepristat-2a is in the b' domain. Using a library of 9 Förster resonance energy transfer sensors, we showed that quercetin-3-O-rutinoside and bepristat-2a globally alter PDI structure and dynamics, leading to ligand-specific modifications of its shape and reorientation of the active sites. Combined with enzyme kinetics and mutagenesis of the active sites, Förster resonance energy transfer data reveal that binding of quercetin-3-O-rutinoside results in a twisted enzyme with reduced affinity for the substrate. In contrast, bepristat-2a promotes a more compact conformation of PDI, in which a greater enzymatic activity is achieved by accelerating the nucleophilic step of the a domain, leading to faster formation of the covalent enzyme-substrate complex., Conclusion: This work reveals the mechanistic basis underlying PDI regulation by antithrombotic compounds quercetin-3-O-rutinoside and bepristat-2a and points to novel strategies for furthering the development of PDI-targeting compounds into drugs., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Coordinated initiation of DNA replication is essential to ensure efficient and timely DNA synthesis. Yet, molecular mechanism describing how replication initiation is coordinated in eukar-yotic cells is not completely understood. Herein, we present data demonstrating a novel feature of RNAs transcribed in the proximity of actively replicating gene loci. We show that RNAs aN-Choring ORC1 (ANCORs) to the histone variant H2A.Z are licensors of the DNA replication process. This ANCOR-H2A.Z interaction is essential for cells to initiate duplication of their ge-netic material. Widespread and locus-specific perturbations of these transcripts correlate with anomalous replication patterns and a notable loss of the H2A.Z replicative marker at the origin site. Collectively, we present a previously undescribed RNA-mediated mechanism that is associ-ated with the generation of active replication origins in eukaryotic cells. Our findings delineate a strategy to modulate the origins of replication in human cells at a local and global level, with potentially broad biomedical implications.

The angiopoietin (Ang)-Tie axis, critical for endothelial cell function and vascular development, is a promising therapeutic target for treating vascular disorders and inflammatory conditions like sepsis. This study aimed to enhance the binding affinity of recombinant Ang1 variants to the Tie2 and explore their therapeutic potential. Structural insights from the Ang1-Tie2 complex enabled the identification of key residues within the Ang1 receptor binding domain (RBD) critical for Tie2 interaction. Molecular dynamics simulations revealed that Met 436 Arg (M436R) and Ala 451 Asp (A451D) could improve Ang1's Tie2 binding affinity. One variant, Ang1-RBD A451D , demonstrated a 100-fold increase compared to the wild type. Cellular assays revealed that Ang1 A451D enhanced Tie2 phosphorylation, promoting endothelial cell migration and tube formation. In vivo, this variant effectively reduced inflammatory cytokines and attenuated organ damage in septic mice. These findings highlight Ang1 A451D as a promising therapeutic candidate for vascular diseases, offering notable clinical potential for mitigating sepsis-related vascular dysfunction.

Competing Interests: Declaration of competing interests R.F. is a founder and consultant for Platelet Diagnostics. Beth Israel Deaconess Medical Center manages the interests of R.F.

The actin cytoskeleton serves an important, but poorly characterized, role in controlling granule exocytosis. The dynamic nature of actin remodeling allows it to act both as a barrier to prevent indiscriminate granule release and as a facilitator of membrane fusion. In its capacity to promote exocytosis, filamentous actin binds to components of the exocytotic machinery through actin binding proteins, but also through direct interactions with SNAREs. The platelet is an excellent cellular model to evaluate SNARE-actin interactions because of the marked reorganization of its actin cytoskeleton that occurs with activation and because of its abundance of secretory granules. This chapter will describe methods to evaluate SNARE-actin interactions in platelets using isolated platelet actin cytoskeleton, granule-enriched membrane fractions in a cell-free secretory system, and purified actin and recombinant SNAREs., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Platelets are essential for hemostasis and thrombosis and play vital roles during metastatic cancer progression and infection. Hallmarks of platelet function are activation, cytoskeletal rearrangements, and the degranulation of their cellular contents upon stimulation. While α-granules and dense granules are the most studied platelet secretory granules, the dense tubular system (DTS) also functions as a secretory system for vascular thiol isomerases. However, how DTS cargo is packaged and transported from megakaryocytes (MKs) to platelets is poorly understood., Objectives: To underpin the mechanisms responsible for DTS cargo transport and leverage those for therapeutic protein packaging into platelets., Methods: A retroviral expression system combined with immunofluorescence confocal microscopy was employed to track protein DTS cargo protein disulfide isomerase fused to enhanced green fluorescent protein (eGFP-PDI) during platelet production. Murine bone marrow transplantation models were used to determine the release of therapeutic proteins from platelets., Results: We demonstrated that the endoplasmic reticulum retrieval motif Lys-Asp-Glu-Leu (KDEL) located at the C-terminus of protein disulfide isomerase was essential for the regular transport of eGFP-PDI-containing granules. eGFP-PDI ΔKDEL , in which the retrieval signal was deleted, was aberrantly packaged, and its expression was upregulated within clathrin-coated endosomes. Finally, we found that ectopic transgenic proteins, such as tissue factor pathway inhibitor and interleukin 2, can be packaged into MKs and proplatelets by adding a KDEL retrieval sequence., Conclusion: Our data corroborate the DTS as a noncanonical secretory system in platelets and demonstrate that in vitro-generated MKs and platelets may be used as a delivery system for transgenic proteins during cellular therapy., Competing Interests: Declaration of competing interests J.E.I. has a financial interest in and is a founder of Stellular Bio, a biotechnology company focused on making donor-independent platelet-like cells at scale, and Spry Bio. Boston Children’s Hospital manages the interests of J.E.I.. R.F. is a founder of and consultant for Platelet Diagnostics. Beth Israel Deaconess Medical Center manages the interests of R.F.. All other authors declare no competing financial interests., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Flavonoids are naturally occurring metabolites of plants that can inhibit the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), which is required for viral replication. Here, we present a protocol to identify flavonoid antagonists of the SARS-CoV-2 Mpro. We describe steps for the expression and purification of Mpro and a kinetic enzymatic assay for Mpro activity using a dequenching fluorescence resonance energy transfer peptide substrate. We then detail procedures for using this enzymatic assay to test flavonoid antagonism and reversible inhibition. For complete details on the use and execution of this protocol, please refer to Lin et al. 1 ., Competing Interests: Declaration of interests R.F. and L.L. have a patent pending entitled, “Compounds and Methods for Treating Viral Infections”. R.F. is on the Scientific Advisory Board of NytroPonix and is a founder and consultant at PlateletDiagnostics, LLC., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract: Antiprothrombin antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered type I and type II antibodies and proposed that type I recognizes the open form. In this study, we report the discovery and structural and functional characterization in human plasma of a type I antibody, POmAb (prothrombin open monoclonal antibody). Using surface plasmon resonance and single-molecule spectroscopy, we show that POmAb interacts with kringle-1 of prothrombin, shifting the equilibrium toward the open form. Using single-particle cryogenic electron microscopy (cryo-EM), we establish that the epitope targeted by POmAb is in kringle-1, comprising an extended binding interface centered at residues R90-Y93. The 3.2-Å cryo-EM structure of the complex reveals that the epitope overlaps with the position occupied by the protease domain of prothrombin in the closed state, explaining the exclusive binding of POmAb to the open form. In human plasma, POmAb prolongs phospholipid-initiated and diluted Russell's viper venom clotting time, which could be partly rescued by excess phospholipids, indicating POmAb is an anticoagulant but exerts a weak lupus anticoagulant effect. These studies reveal the structural basis of prothrombin recognition by a type I antiphospholipid antibody and uncover an exciting new strategy to achieve anticoagulation in human plasma., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Background: Respiratory infection is associated with microvascular thrombus formation and marked elevation in cytokine levels. The role of cytokines elaborated by the pulmonary epithelium in thrombotic responses is poorly understood., Objectives: Our goal was to identify cytokines of pulmonary epithelial cell origin that enhance thrombin generation in the endothelium at concentrations equal to or less than those found in the circulation during infection., Methods: We screened multiple cytokines produced by the pulmonary epithelium for the ability to enhance toll-like receptor (TLR)-mediated endothelial thrombin generation. Effects of cytokines on tissue factor and thrombomodulin expression, cytokine selectivity for different TLRs, and prothrombotic activity of endogenous cytokines in conditioned medium from pulmonary human epithelial cells were evaluated., Results: MIP-1β, MCP-1, IL-10, IL-6, IL-1β, TNFα, IFNα, IFNβ, and IFNγ were tested for their ability to enhance TLR3-mediated thrombin generation on endothelial cells. Only interferons (IFNs) and TNFα promoted TLR3-mediated thrombin generation at levels that circulate during infection. IFNs robustly enhanced tissue factor expression when used in conjunction with TLR agonists and reduced thrombomodulin expression in the endothelium independently of TLRs. IFNα, which is typically elevated with viral infection, only synergized with TLR3 agonists mimicking viral pathogen-associated molecular patterns. In contrast, IFNγ, which is typically observed in bacterial infection, synergized more effectively with TLR4 agonists released by bacteria. Conditioned media from inflamed pulmonary epithelial cells primed the endothelium for TLR-mediated thrombin generation. Anti-IFN type I antibodies blocked this effect, indicating that endogenous IFNs prime the endothelium for TLR-mediated thrombin generation., Conclusion: IFNs elaborated by the pulmonary epithelium are necessary and sufficient to enhance TLR-mediated thrombin generation., Competing Interests: Declaration of competing interests R.F. is a founder and consultant for Platelet Diagnostics. The other authors declare no competing financial interests., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.

Thrombosis is a common complication of advanced cancer, yet the cellular mechanisms linking malignancy to thrombosis are poorly understood. The unfolded protein response (UPR) is an ER stress response associated with advanced cancers. A proteomic evaluation of plasma from patients with gastric and non-small cell lung cancer who were monitored prospectively for venous thromboembolism demonstrated increased levels of UPR-related markers in plasma of patients who developed clots compared with those who did not. Release of procoagulant activity into supernatants of gastric, lung, and pancreatic cancer cells was enhanced by UPR induction and blocked by antagonists of the UPR receptors inositol-requiring enzyme 1α (IRE1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK). Release of extracellular vesicles bearing tissue factor (EVTFs) from pancreatic cancer cells was inhibited by siRNA-mediated knockdown of IRE1α/XBP1 or PERK pathways. Induction of UPR did not increase tissue factor (TF) synthesis, but rather stimulated localization of TF to the cell surface. UPR-induced TF delivery to EVTFs was inhibited by ADP-ribosylation factor 1 knockdown or GBF1 antagonism, verifying the role of vesicular trafficking. Our findings show that UPR activation resulted in increased vesicular trafficking leading to release of prothrombotic EVTFs, thus providing a mechanistic link between ER stress and cancer-associated thrombosis.