A man in his early 60s presented with dyspnoea superimposed on a 2-month to 3-month history of spontaneous mucocutaneous bleeding. Laboratory testing revealed unmeasurably elevated international normalised ratio (INR) and activated partial thromboplastin time. CT of the neck demonstrated supraglottic soft tissue swelling with severe airway narrowing, requiring intubation for airway protection. Further history revealed occupational exposure to rodenticides during pest control work without consistent use of personal protective equipment. Markedly reduced activity of vitamin K-dependent clotting factors (II, VII, IX and X) supported a presumptive diagnosis of superwarfarin toxicity. Treatment with 4-factor prothrombin complex concentrate and vitamin K1 rapidly corrected the coagulopathy, although prolonged high-dose oral vitamin K1 was required because of recurrent INR elevation. He was discharged after 9 days and completed a 3-week vitamin K1 taper. This case highlights a rare presentation of presumed transdermal superwarfarin toxicity causing life-threatening coagulopathy and airway compromise.

Coagulation factor IX (FIX) is a serine protease that is crucial for normal hemostasis in vertebrates. Unlike other soluble vitamin K-dependent procoagulant enzymes, there is an "extravascular reservoir" of FIX in various tissues. The extravasation of FIX and its subsequent maintenance outside of circulation have long been attributed to its ability to bind collagen IV. This property is conferred by a short, specialized region of its phospholipid-binding gamma-carboxyglutamic acid (Gla) domain. Collagen IV is a nonfibrillar extracellular protein found primarily in basement membranes that assembles into sheet-like supportive structures around blood vessels and epithelia, providing a convenient scaffold for this "hemostatic barrier." Early seminal investigations from 1980 to 2000 suggested the existence of a large extravascular reservoir containing the majority of a vertebrate organism's FIX. The subsequent development of a FIX-deficient mouse model, identification of the amino acid residues responsible for FIX's collagen binding (K5 and V10), and development of enzyme mutants with high and low affinities for collagen IV afforded new insights into the hemostatic importance of this property from 2000 through 2020. Recent interest on the topic has been catalyzed by therapeutic implications for hemophilia treatment. For example, studies have examined the hemostatic and pharmacokinetic effects of enhanced collagen binding on the hyperactive FIX variant (Padua) currently in trials for hemophilia B gene therapy. In this review, we highlight research updates on extravascular FIX since 2020 and present remaining questions to help guide future investigations.

The emergence of seekRNA- and bridgeRNA-guided recombination has introduced a distinct paradigm in genome engineering by coupling programmable RNA-directed DNA recognition with recombinase-mediated insertion, excision, inversion, and genomic rearrangement without canonical double-strand breaks. Since their discovery in 2024, these systems have progressed rapidly from bacterial mobile genetic elements and mechanistic characterization to structural elucidation and programmable genome engineering in human cells. However, these advances remain distributed across foundational and rapidly emerging studies, creating a need for an integrated molecular perspective on their mechanisms, technological development, and position within contemporary genome engineering. This review synthesizes the molecular architecture, RNA-guided recognition, strand-exchange mechanisms, programmability, and engineering of seekRNA and bridgeRNA systems, with particular emphasis on complementary human-cell advances involving ISCro4 and engineered IS621. Whereas ISCro4 systems have enabled multikilobase DNA insertion, genomic excision, and near-megabase inversion, the enIS621-tebRNA platform has enabled scarless kilobase-scale integration across multiple human cell types, including proof-of-concept functional CD19 chimeric antigen receptor and factor IX gene insertion. The review further integrates recent genome-scale bacterial rewriting and Targetable Recombinase Assisted DNA Exchange (TRADE)-mediated DNA replacement, while benchmarking RNA-guided recombination against conventional site-specific recombination, clustered regularly interspaced short palindromic repeats (CRISPR)-based editing, Programmable Addition via Site-specific Targeting Elements (PASTE), CRISPR-associated transposases, and emerging large-payload genome-writing strategies, including kilobase-scale nickase-targeting (KNIT) editing, Prime Assembly, engineered R2 retrotransposons, and TransCRISTI. This comparative framework highlights a broader transition from programmable sequence modification toward direct engineering of genomic architecture, while identifying recognition-site constraints, mismatch-tolerant recombination, unintended recombination products, delivery, and genome-wide specificity as key translational barriers. By integrating foundational mechanisms with recent mammalian engineering, genome-scale bacterial rewriting, large-payload technologies, and emerging computational design strategies, this review provides a contemporary framework for defining the distinctive capabilities, current limitations, and future development of programmable RNA-guided DNA recombination.

Hemophilia is an X-linked hereditary bleeding disorder caused by loss-of-function mutations in the genes encoding coagulation factors, leading to excessive bleeding and potentially being life-threatening. Currently, regular treatment for hemophilia is the infusion of recombinant blood coagulation factors. This approach is not only costly but can also give rise to complications such as the development of neutralizing antibodies (Nabs), which impede the therapeutic outcome. Hemophilia is a monogenic disease, making gene therapy a promising curative measure that includes gene addition and gene editing approaches. Adeno-associated virus (AAV) vectors have gained massive attention as the premier delivery vehicle for clinical gene therapy due to their diverse tissue tropisms dictated by the natural and engineered AAV capsids, their excellent safety profile since AAVs do not cause any known human diseases, low immunogenicity, and long-lasting gene expression. Several AAV-based gene addition therapies have been approved for hemophilia B and hemophilia A, and dozens of similar AAV-based clinical trials are underway. Gene editing technologies, like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), have shown immense potential in the treatment of genetic diseases. CRISPR/Cas9-mediated transgene integration provides critical advantages over regular gene insertion therapy by enabling site-specific, targeted genomic integration rather than random integration. Clinical trials focusing on AAV-CRISPR/Cas9-mediated knock-in of human genes represent the frontier of in vivo genetic medicine, and one such prominent ongoing clinical trial for hemophilia B is designed to insert the human Factor IX gene into hepatocytes. This review outlines recent advances and feasible strategies of CRISPR/Cas9-based genome editing for hemophilia therapy, highlights the potential applications of next-generation gene editing and vector delivery technologies, and offers new insights for the advantages, limitations, and future directions for these novel treatment modalities.

Accurate measurement of therapeutic protein concentration is critical for ensuring manufacturing consistency. Synonymous gene recoding is often used to improve protein yield, including for coagulation Factor IX (FIX), used to treat Hemophilia B. Although synonymous recoding does not alter primary amino acid sequence, it can affect protein conformation, potentially influencing the accuracy of protein quantification depending on the method used. In this study, we investigated whether synonymous changes in F9 gene, which encodes FIX, impacted reliability of commonly used protein quantitation methods. HEK293- FlpIn cells were used to generate FIX proteins expressed from seven different synonymous F9 sequences. Purified proteins were initially measured using absorbance at 280 nm, followed by quantification using various colorimetric, and fluorometric protein assays. Relative differences among protein quantification methods were inconsistent between wild-type and synonymous F9 variants. Compared with A280 measurements, mean differences across variants ranged from - 33% to - 8% for Bradford and from 14 to 54% for BCA, whereas wider ranges were observed for OPA (- 23% to 133%), CBQCA (14% to 282%), and Lowry (1% to 185%). Bradford and BCA also exhibited lower intra-assay variability, with standard deviations of 6% and 10%, respectively, compared with 12%, 18%, and 33% for OPA, CBQCA, and Lowry assays. These findings indicate that synonymous codon substitutions influence protein quantification outcomes, with Bradford and BCA producing more consistent results and lowest variability, whereas amine-reactive fluorescence-based assays (OPA and CBQCA) showed greater inconsistencies. In conclusion, protein quantification methods may not produce consistent accuracy among proteins translated from synonymously recoded sequences.

Only a handful of anticoagulants have reached the clinic to treat and prevent thrombotic episodes in the millions of patients suffering from cardiovascular, cancer, viral (COVID-19) and metabolic diseases. In this work, we report the discovery, chemical synthesis, mechanistic biochemistry, pharmacology, and toxicology of SB-01, a novel, allosteric, human factor XIa (FXIa) inhibitor that displays potent antithrombotic activity in preclinical models. SB-01 is a synthetic, highly sulfated, aromatic agent that binds in both anion-binding exosites of hFXIa to induce inhibition of factor IX activation. SB-01 inhibited rodent arterial and venous thrombosis at doses of 6 and 3 mg/kg, respectively. Additionally, a twice-a-day regimen of 6 mg/kg SB-01 was significantly more effective (p<0.001) in preventing deep vein thrombosis in animals with lung cancer. Michaelis-Menten kinetics and competition with unfractionated heparin and polyphosphate revealed SB-01 to be an allosteric inhibitor. Critically, at the effective dose of 6 mg/kg, SB-01 did not significantly increase mouse tail bleeding time or blood loss. Further, SB-01's anticoagulation could be readily reversed with protamine sulfate, the FDA-approved antidote for heparin. Finally, SB-01 showed no major adverse effects across multiple toxicological assays including gross, cardiac, metabolic, hematological, and clinical chemistry. Overall, this work presents a structurally and functionally unique, allosteric small molecule as a highly promising candidate for safer anticoagulation.

BACKGROUND Cholecystectomy alters enterohepatic bile circulation, impairing fat-soluble vitamin absorption and reducing bioavailability of vitamin D and vitamin K, required for hepatic synthesis of coagulation factors II, VII, IX, and X. Despite the high global frequency of this procedure, evidence regarding clinically significant postoperative changes in fat-soluble vitamin status remains inconsistent. This retrospective single-center study compared preoperative and postoperative serum 25-hydroxyvitamin D (25[OH]D) levels and vitamin K-dependent coagulation profiles in patients undergoing cholecystectomy. MATERIAL AND METHODS All data were retrieved from electronic medical records of patients who underwent cholecystectomy between January 2020 and January 2025. Two distinct cohorts were formed: the vitamin K cohort (n=101), in which prothrombin time (PT), activated partial thromboplastin time (aPTT), and international normalized ratio (INR) were evaluated at ≥7 days postoperatively; and the 25(OH)D cohort (n=78), in which serum 25(OH)D levels were evaluated at ≥3 months postoperatively. Patients with comorbidities or medications affecting these parameters were excluded. RESULTS In the 25(OH)D cohort, no significant difference was found between preoperative and postoperative median levels (19.76 vs 19.13 µg/L, P=0.248). In the vitamin K cohort, no significant changes were detected in PT (11.80 vs 11.67 s, P=0.301), aPTT (29.83 vs 30.40 s, P=0.131), or INR (1.03 vs 1.03, P=0.859). Sex-stratified analyses revealed no significant differences. CONCLUSIONS Cholecystectomy was not associated with a clinically significant decline in serum 25(OH)D or alterations in vitamin K-dependent coagulation profiles. Postoperative insufficiencies appear driven by pre-existing metabolic risk profiles. Clinical management should prioritize preoperative metabolic optimization over routine post-surgical supplementation.

The 2026 Lasker~DeBakey Clinical Medical Research Award has been awarded to Kunihiro Hattori, Takehisa Kitazawa, and Tomoyuki Igawa (Chugai Pharmaceuticals, Tokyo, Japan) for invention of a bispecific antibody that joins blood clotting Factors IXa (FIXa) and X (FX), restoring deficient Factor VIII (FVIII) activity in patients with hemophilia A and preventing severe bleeding in this hereditary disorder. Starting from the 1960s, types of FVIII-replacement therapy remarkably improved on-demand hemostatic treatment for ongoing bleeds and enabled prophylactic reduction of bleeding episodes. However, these agents required burdensome intravenous infusions multiple times per week, and ~30% of patients developed neutralizing anti-FVIII antibodies (FVIII inhibitors). To address these unmet medical needs, in 2000, Hattori-a molecular and hematology biologist, Kitazawa-a hematology biologist and translational researcher, and Igawa-a biochemist and protein engineer, conceived, designed, and created over the span of 10 y a novel FVIII-mimetic. Instead of a native protein construct, they engineered a bispecific IgG antibody against FIXa and FX, circumventing the need for FVIII and enabling subcutaneous administration up to once per month. Importantly, they identified the biophysical requirements for the bispecific to mimic FVIII function effectively and safely, and their breakthrough protein engineering design made possible favorable pharmacokinetics and physicochemical properties with efficient manufacturability. Their collective research efforts demonstrate how imagination, determination, and multidisciplinary scientific research come together to improve patients' lives.

Arteriovenous fistula (AVF) is the preferred vascular access for patients with end-stage renal disease; however, its failure is primarily due to neointimal hyperplasia. Five patients who underwent initial AVF surgery served as the control group, and another five patients with failed AVF surgery served as the experimental group. Herein, we employed mass spectrometry (MS)-based quantitative proteomics coupled with tandem mass tag labeling to screen differentially expressed proteins (DEPs) in the anastomotic cephalic vein, followed by bioinformatics analyses and verification experiments. A total of 121 DEPs were identified in the failed AVF group. GO analysis was primarily enriched in protein binding, nucleic acid binding, enzyme binding, mRNA binding, cadherin binding, catalytic activity, and cell adhesion molecule binding. KEGG pathways were mainly enriched in cell aggregation and adhesion, actin cytoskeleton, extracellular matrix-receptor interaction, PI3K-Akt signaling pathway, complement and coagulation cascades, and cholesterol metabolism. Protein-protein interaction network consisted of 86 (71.07%) DEPs, including complement VII (C7), factor IX (F9), SERPINC1, microfibril-associated glycoprotein 4 (MFAP4), complement C1s subcomponent, complement C1q subcomponent subunit A, complement C1q subcomponent subunit B, tissue factor, and von Willebrand factor, which interacting with numerous other proteins. In the expanded validation for different patients, C7, F9, SERPINC1, and MFAP4, were verified by immunohistochemical staining and Western blotting, which were consistent with the proteomics results. Collectively, this study identifies a series of potential diagnostic biomarkers, and explores the underlying mechanisms associated with AVF dysfunction.

Anthracycline-induced cardiotoxicity remains a major limitation of cancer therapy, and effective preventive strategies are lacking. Topoisomerase IIb has been implicated as a central driver of this toxicity, suggesting that epigenetic regulators may interfere with the pathological cardiac response. Here, we show that doxorubicin promotes topoisomerase IIb accumulation at cardiomyocyte-specific gene promoters (e.g., Actc1, Myl2, and Myh7) overlapping myocyte enhancer factor 2 binding sites and enhances myocyte enhancer factor 2 -dependent transcription. This response is attenuated by the pan-histone deacetylase inhibitor suberoylanilide hydroxamic acid. Suberoylanilide hydroxamic acid -mediated cardioprotection requires class IIa histone deacetylases, as genetic loss of HDAC4 abolishes its effect. Mechanistically, suberoylanilide hydroxamic acid induces acetylation of the chaperone 14-3-3, disrupting its interaction with HDAC4/5, promoting their nuclear accumulation, and repressing myocyte enhancer factor 2 - driven transcription. In vivo, suberoylanilide hydroxamic acid mitigates doxorubicin-induced cardiotoxicity. These findings identify histone deacetylase inhibition as a cardioprotective repurposing strategy and reveal a mechanistic link between epigenetic regulation and anthracycline-associated cardiotoxicity.

Photodynamic therapy (PDT) is a clinically established approach that relies on the localized generation of reactive oxygen species (ROS) mediated by a photosensitizer (PS), light, and molecular oxygen. Protoporphyrin IX (PpIX) is an approved PS; however, its intracellular retention is limited, factor that can alter its overall photodynamic efficiency. In this study, we report how the design of PpIX derivatives bearing piperazine (PpIX-Pip) or morpholine (PpIX-Morp) substituents may modulate lipophilicity, cellular uptake, oxidative stress, and phototoxicity. The photodynamic activity of these derivatives was evaluated in the aggressive triple-negative human breast cancer cell line MDA-MB-231. Both derivatives showed greater time-dependent cellular retention and enhanced photodynamic activity compared to PpIX, promoting oxidative stress, and triggering loss of mitochondrial membrane potential and caspase-3 activation. These results highlight the impact of rational structural modification of PpIX on photodynamic efficiency and provide valuable structure-activity relationship insights for the development of improved porphyrin-based PS for PDT.