T-cell acute lymphoblastic leukemia (T-ALL) represents a group of aggressive hematological malignancies characterized by unfavorable prognosis, urging the need for innovative therapeutic strategies. LEF1 is a member of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of DNA-binding transcription factors, known for their interaction with nuclear β-catenin in the context of the Wnt signaling pathway. Although LEF1's implication in colon cancer is well-documented, its clinical relevance and functional consequences remain elusive in T-ALL. In this study, we provide valuable insights into the prevalence and significance of LEF1 alterations in a comprehensive cohort of 474 pediatric and adult T-ALL patients enrolled in the FRALLE 2000 and GRAALL-2003-2005 trials (NCT00222027; NCT00327678) respectively. LEF1 alterations were detected in 63 cases (13%), including 9 point-mutations (14.3%), 18 large deletions (28.6%), and - strikingly - 36 focal deletions (57.1%), which emerge as the most recurrent subtype. LEF1-altered cases were associated with increased central nervous system involvement and improved initial treatment response. Importantly, we unveil the existence of a previously undescribed dominant-negative LEF1 isoform resulting from focal deletions of the exons 2-3. This novel truncated protein, previously unreported in the literature, is associated with the disruption of the Wnt pathway and TCR signaling, which can be exploited as a therapeutic strategy to enhance chemosensitivity in LEF1-deleted T-ALL cases.

Fetal hemoglobin (HbF) reactivation is a promising therapy for β-hemoglobinopathies. We developed a prime-editing strategy that introduces multiple mutations in the fetal γ-globin promoters that are expected to increase their activity. We tested multiple targets and optimized a variety of parameters to achieve ~50% of precise edits in a hematopoietic cell line, with minimal off-targets effects. This work improved our understanding of the complex DNA repair mechanisms involved in prime editing. We tested this strategy in patients' hematopoietic stem/progenitor cells (HSPCs). Although the editing efficiency was variable amongst donors, erythroid clones carrying multiple mutations express a significantly higher γ-globin level compared to cells carrying individual mutations, confirming the potential therapeutic benefit of our combined strategy for patients with β-hemoglobinopathies.

Adoptive transfer of NK cells can induce complete remissions in 30-50% of patients with refractory acute myeloid leukemia and lymphoma. While blood chimerism occurs, attaining functional homing to the site of tumor without exhaustion has been elusive. During chronic infections and tumorigenesis, exposure to activating stimuli weakens the effector activity of NK cells. Despite this knowledge, there is little known about the mechanisms that govern this dysregulation and whether these disparate activating stimuli use distinct pathways to downregulate effector immunity. In this study, we reveal that chronic NK cell activating receptor (NKAR) stimulation and chronic IL-15 exposure impart distinct modes of dysregulation, with NKAR stimulation inducing a tissue resident-like state that resembles that of tumor-infiltrating NK cells in cancer patients. Using loss- and gain-of-function studies, we identify the transcription factor KLF2 as a master regulator of the NK cell response to chronic activation and provide evidence that KLF2 overexpression promotes NK cell cytotoxicity, cytokine production, and chemotaxis, and inhibits the development of dysfunctional, tissue resident-like features. Using KLF2 reporter mice, we show that in certain tissues, tissue resident NK cells are predominantly KLF2-negative while circulating NK cells in these tissues are overwhelmingly KLF2+. Lastly, using mixed bone marrow chimeras, we demonstrate that conditional KLF2 deficiency in NK cells leads to altered homing and the acquisition of tissue resident-like features in vivo. Together, these findings highlight the profound changes NK cells undergo during prolonged activation and advance our understanding of how some NK cell therapies fail during malignant relapse.

Platelets are dynamic cells that perform vital roles both in thrombosis and in immune regulation. Platelets are activated through well described signaling pathways following injury to the vascular wall to prevent excess bleeding and initiate vascular repair. However, platelet functions extend beyond this initial physical phase of platelet activation, through their expression and secretion of angiogenic factors and immune mediators that initiate wound healing and recruit leukocytes to prevent infection. Not only do activated platelets induce inflammation and vascular repair but circulating resting platelets maintain vascular integrity and immune homeostasis. Furthermore, many studies have shown that platelets are not homogenous, demonstrating transcriptomic and proteomic differences that associate with platelet phenotypes and functions in multiple disease states. Platelets are produced by megakaryocytes that are found not only in the bone marrow but also in the lungs and spleen. Megakaryocytes within the bone marrow are heterogeneous, which may contribute to platelet heterogeneity. Additionally, lung megakaryocytes are immune-differentiated compared to those in other tissues and proportionately produce more platelets at times of increased demand, perhaps adding to platelet heterogeneity in disease contexts. In this review, we will discuss underlying mechanisms that may lead to platelet heterogeneity impacting both health maintenance and disease.

Artificial intelligence (AI) and its subdiscipline, machine learning (ML), have the potential to revolutionize health care, including hematology. The diagnosis and treatment of hematologic disorders depend on the integration of diverse data sources, such as imaging, pathology, omics, and laboratory parameters. The increasing volume and complexity of patient data have made clinical decision-making more challenging. AI/ML hold significant potential for enhancing diagnostic accuracy, risk stratification, and treatment response prediction through advanced modeling techniques. Generative AI, a recent advancement within the broader field of AI, is poised to have a profound impact on health care and hematology. Generative AI can enhance the development of novel therapeutic strategies, improve diagnostic workflows by generating high-fidelity images or pathology reports, and facilitate more personalized approaches to patient management. Its ability to augment clinical decision-making and streamline research represents a significant leap forward in the field. However, despite this potential, few AI/ML tools have been fully implemented in clinical practice due to challenges related to data quality, equity, advanced infrastructure, and the establishment of robust evaluation metrics. Despite its promise, AI implementation in hematology faces critical challenges, including bias, data quality issues, and a lack of regulatory frameworks and safety standards that keep pace with rapid technological advancements. In this review, we provide an overview of the current state of AI/ML in hematology as of 2025, identify existing gaps, and offer insights into future developments.

Desmopressin (DDAVP) is often administered to correct factor VIII (FVIII) levels in female hemophilia A carriers (HACs). However, the post-DDAVP FVIII pharmacokinetic profiles have been reported only in small series in HACs. Therefore, this study analyzed the post-DDAVP FVIII and von Willebrand factor (VWF) response in 361 HACs. A population pharmacokinetic/pharmacodynamic model was developed to analyze the VWF and FVIII levels by taking into account the F8 gene variants (n = 143 [39.6%] with null; and n = 218 [60.4%] with non-null variants), demographic and laboratory covariates. The before/after DDAVP mean basal, peak and recovery FVIII activity (FVIII:C) levels were 0.34 IU/mL (0.08-0.65), 1.13 IU/mL (0.19-2.69), and 2.85 IU/mL (1.06-7.13), respectively. Peak FVIII:C was ≥0.5 IU/mL in 95.6% (345/361) and ≥0.8 IU/mL in 78.7% (284/361) of patients. The covariate analysis showed a poorer DDAVP FVIII:C response for null than non-null F8 variants: lower mean FVIII:C peak (1.04 vs 1.23 IU/mL; P < 5 × 10-5) and patients percentage with normalized FVIII:C (91.6% vs 98.1%; P = .0068), higher mean FVIII:C clearance (5898.83 vs 2704.06 IU/h; P = 1.58 × 10-15), lower mean FVIII:C area under the curve during the first 12 hours (AUC0-12h =7.73 vs 9.06 IU/mL per hour; P < 5 × 10-6), and shorter mean time with FVIII:C ≥0.8 IU/mL (1.9 vs 4.1 hours; P < 6 × 10-6). HAC with body weight <35 kg had lower peak FVIII:C, higher FVIII:C clearance, lower AUC0-12h, and shorter time with FVIII:C ≥0.8 IU/mL than HAC with body weight 35 to 70 kg. In HACs, the post-DDAVP FVIII response is strongly influenced by the F8 genotype and body weight.

Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-related mortality. Though the precise mechanism is not fully understood, a 2-hit model is widely accepted, involving both a predisposing patient condition and the transfusion itself. Mitochondrial damage-associated molecular patterns (mtDAMPs), such as mitochondrial DNA (mtDNA) and N-formylated peptides (NFPs), are elevated in patients who have experienced trauma, and stored blood products, and have been implicated in adverse transfusion outcomes, prompting us to investigate whether mtDAMPs could serve as a priming "first hit" in TRALI. Using a murine model, we found that injection of purified mitochondria followed by a monoclonal anti-major histocompatibility complex class I antibody (34-1-2s) induced significantly greater lung injury compared with the isotype control. This was evidenced by increased pulmonary edema, elevated plasma macrophage inflammatory protein 2, enhanced neutrophil lung infiltration, hypothermia, and respiratory distress. Similar effects were observed using a Toll-like receptor 9 (TLR9) agonist (oligonucleotide 2395), purified mtDNA, and a synthetic NFP (WKYMVm), agonist of formyl peptide receptor (FPR). Notably, a TLR9 antagonist blocked the mtDAMP-induced TRALI response, whereas 2 FPR antagonists did not, underscoring a key role for mtDNA and TLR9 signaling in disease priming. These findings suggest that mtDAMPs, particularly mtDNA, present in both transfusion products and recipient plasma, may predispose patients to antibody-mediated TRALI. Targeting mtDAMPs or their receptors may offer a novel therapeutic strategy to mitigate TRALI risk.

Until recently, the JAK1/2 inhibitor ruxolitinib (Jakafi) was the only therapy for steroid-refractory acute graft-versus-host disease approved by the US Food and Drug Administration (FDA) for use in patients aged >12 years. The FDA has now approved a potent mesenchymal stromal cell product, remestemcel-L-rknd (Ryoncil), for children aged ≤18 years, showing 70% response rates and ∼70% 6-month survival. In this spotlight, we highlight this important advance in the field.

Functional high-risk (FHR) multiple myeloma (MM) is defined as an unexpected, early relapse (ER) of disease in the absence of baseline molecular or clinical risk factors (RF), making FHR MM inherently dependent on which RFs were assessed at diagnosis, but also on what treatment patients received. To establish the true incidence of FHR, we analysed uniformly treated, transplant-eligible (TE) patients from the UK NCRI Myeloma-XI trial that had been profiled for IMS/IMWG defined high-risk cytogenetic aberrations (HRCA) and the SKY92 gene expression HR signature (GEP-HR). 135 TE MyXI patients meeting these criteria were studied, with a median follow-up of 88 months. 25 patients (18.5%) experienced ER, defined as relapse <18 months from maintenance randomization post-autologous stem-cell transplantation. Hereof, 15 (60%) were classified as IMS/IMWG-HR at diagnosis, of whom 8 were also GEP-HR. Another 6 patients were GEP-HR only and would have been missed by IMS/IMWG-HR. Among 4 patients with both IMS/IMWG- & GEP-standard risk (SR), 2 had isolated HR markers at diagnosis, leaving only 2 patients (8% of ER; 1.5% of all) truly meeting all FHR criteria. The combination of IMS/IMWG-HR and GEP-HR profiling identified 84% of ER, and differentiated long-term outcome across all 135 patients: co-occurring IMS/IMWG-HR and GEP-HR was associated with very short overall survival compared to the absence of both (HR=13.1, 95%-CI: 6.5-26.1, P<0.0001), followed by GEP-HR only (HR=5.1, 95%-CI: 2.4-11.1, P<0.0001) and IMS/IMWG-HR only (HR=3.2, 95%-CI: 1.6-6.2, P=0.0007). Our results support more comprehensive baseline diagnostic profiling to identify those at risk of ER upfront. ISRCTN49407852, NCT01554852.

Two gene therapy products have been approved by the US Food and Drug Administration for sickle cell disease. Nearly all patients in the clinical trials that led to approval either were sickle hemoglobin (HbS) gene homozygotes (sickle cell anemia) or had HbS-β0 thalassemia. HbSC disease, caused by compound heterozygosity for HbS and hemoglobin C genes, is the second most common genotype of sickle cell disease. Gene therapy has not been tested in patients with HbSC disease who are severely symptomatic. We discuss the pathophysiology and clinical features of HbSC disease and how gene therapy is likely to provide a curative option for some individuals. We also discuss the mechanism through which fetal hemoglobin (HbF) and HbF-like HbA (HbAT87Q) might mitigate adverse clinical outcomes and end-organ damage in patients with HbSC disease and other compound heterozygous sickle hemoglobinopathies.

Repeated bleeding into joints in hemophilia leads to chronic inflammation that plays a central role in the pathogenesis of hemophilic arthropathy (HA). Our recent studies revealed that factor VIIa (FVIIa) treatment releases extracellular vesicles from the endothelium (eEVs) and FVIIa-released eEVs exhibit anti-inflammatory and barrier protective functions. The present study was undertaken to investigate the effect of FVIIa-released eEVs on HA and the mechanism of their protective effect. Joint bleeding in hemophilia (F8-/- ) mice was induced by a needle puncture injury. Injured mice were treated with saline, control eEVs, or FVIIa-released eEVs, and the changes in the knee joints were analyzed by gross examination of knees as well as histological and immunohistochemical analysis. Joint tissues were examined for evidence of synovial hyperplasia, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. The data showed that treatment of mice with control eEVs had no significant effect on the development of HA whereas treatment with FVIIa-released eEVs markedly reduced all pathological features of joint bleed-induced HA. Incorporation of microRNA10a (miR10a) inhibitor into FVIIa-released eEVs abrogated the protective effect of FVIIa-released eEVs on HA. More importantly, loading miR10a mimic into control eEVs conferred a protective effect. Administration of miR10a containing FVIIa-released eEVs or control eEVs loaded with miR10a mimic were found to abrogate joint bleed-induced IL-6 production in the synovium. miR10a in eEVs had no effect on hemostasis. Cumulatively, our data indicates that EVs containing miR10a that effectively suppress synovial inflammation would have immense therapeutic value in treating HA.

Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy that increasingly affects the elderly population, with its post-transcriptional landscape remaining largely elusive. Establishing a stable proteomics-based classification system and systematically screening age-related proteins and regulatory networks are crucial for understanding the pathogenesis and outcomes of AML. In this study, we leveraged a multi-omics cohort of 374 newly diagnosed AML patients, integrating proteome, phosphoproteome, genome, transcriptome, and drug screening data. Through similarity network fusion clustering, we established eight proteomic subtypes with distinct clinical and molecular properties, including S1 (CEBPA mutations), S3 (myelodysplasia-related AML), S4 (PML::RARA), S5 (NPM1 mutations), S6 (PML::RARA and RUNX1::RUNX1T1), S8 (CBFB::MYH11), S2 and S7 (mixed), aligning well with and adding actionable value to the latest World Health Organization nomenclature of AML. Hematopoietic lineage profiling of proteins indicated that megakaryocyte/platelet- and immune-related networks characterized distinct aging patterns in AML, which were consistent with our recent findings at the RNA level. Phosphosites also demonstrated distinct age-related features. The high protein abundance of megakaryocytic signatures was observed in S2, S3, and S7 subtypes, which were associated with advanced age and dismal prognosis of patients. A hematopoietic aging score with an independent prognostic value was established based on proteomic data, where higher scores correlated with myelodysplasia-related AML, NPM1 mutations, and clonal hematopoiesis-related gene mutations. Collectively, this study provides an overview of the molecular circuits and regulatory networks of AML during the aging process, advancing current classification systems and offering a comprehensive perspective on the disease.

Immunotherapy has transformed the treatment landscape of multiple myeloma (MM), a hematological cancer predominantly affecting older individuals. Yet, whether immune aging, shaped by intrinsic aging processes, genetics, and external factors, affects treatment efficacy remains unclear. To address this, we investigated the influence of age on the immune system in patients with MM and explored whether immune aging associates with clinical outcomes in older patients. Using flow cytometry, we conducted high-dimensional profiling of T cells and natural killer cells in peripheral blood and bone marrow samples of 124 older (>65 years) and 145 younger (≤65 years) patients with newly diagnosed MM (ages 34-92 years) enrolled in the HOVON-143 and CASSIOPEIA/HOVON-131 trials. On average, older patients exhibited a more activated, differentiated, and senescent T-cell compartment than younger patients. Nonetheless, substantial interindividual variation in T-cell subset frequencies within both age groups indicated that calendar age inadequately reflects an individual's immune status. We therefore developed an immune clock on high-dimensional phenotypic T-cell data to quantify each patient's "immune age," revealing substantial variation in immune ages among patients of similar calendar age. Importantly, immune age appeared a stronger predictor of clinical outcomes than calendar age in older, nonfit patients with newly diagnosed MM receiving daratumumab-ixazomib-dexamethasone, even after adjusting for frailty and other established risk factors. Overall, these findings highlight immune age as a clinically relevant composite metric that better reflects a patient's immune status than their calendar age. Validating this methodology in other immunotherapy settings may improve our ability to predict immunotherapy efficacy in older patients with MM or other hematological cancers.

Platelets are specialized cells for hemostasis that circulate in close contact to the glycocalyx, an extracellular layer of interwoven glycoproteins, proteoglycans, and glycosaminoglycans that maintain vascular homeostasis. Platelets survey their circulating environment, balancing inhibitory signals that prevent inappropriate activation with activating signals that initiate thrombus formation. Disease can disrupt this delicate balance of endogenous inhibitory signaling, leading to an increased risk of thrombosis as in patients with inflammatory bowel disease (IBD). In this study, we demonstrate that physiological concentrations of hyaluronan (HA), an essential component of the glycocalyx, acts as an inhibitor of activation and aggregation in human platelets. Using a combination of affinity chromatography, and functional assays of platelets from humans and genetically modified mice, we identify layilin as the receptor for HA and an endogenous inhibitor of platelet activation. LAYN KO platelets display agonist-induced hyperactivation of αiiBβ3 and increased adhesion to fibrinogen under venous shear. Loss of layilin results in dysregulation of Rho GTPase family members-RAC1, Cdc42, RhoA, and Ras-like Rap1,- via layilin's binding partner, merlin, and downstream PAK1. Furthermore, IBD patient platelets contain reduced layilin protein levels correlating with heightened basal Rac1-GTP levels and increased reactivity. Finally, although IBD platelets show enhanced sensitivity to activation, pharmacological inhibition of RAC1 effectively reduces platelet hyperactivity in IBD patient platelets. These findings highlight a novel role for layilin and HA in the maintenance of platelet homeostasis that becomes disrupted in patients with IBD.

Older patients with diffuse large B-cell lymphoma (DLBCL) present unfavorable genetic and microenvironmental alterations. In this phase 2 trial, we assessed the efficacy and safety of zanubrutinib in combination with rituximab and lenalidomide (ZR2) in patients with de novo DLBCL aged ≥75 years. Forty patients were enrolled, and the primary end point was the complete response rate, which was 65.0% (95% confidence interval [CI], 48.3-78.9) at the end of induction treatment. The 2-year progression-free and overall survival rates were 67.1% (95% CI, 50.1-79.4) and 82.4% (95% CI, 66.5-91.2). The most common grades 3 and 4 hematologic adverse event (AE) was neutropenia (n = 14 [35.0%]). The most common grades 3 and 4 nonhematologic AEs were increased alanine transaminase (n = 5 [12.5%]) and aspartate transaminase levels (n = 5; 12.5%), and pulmonary infection (n = 5 [12.5%]). No events of atrial fibrillation were observed. Importantly, the efficacy of ZR2 was more dependent on tumor microenvironmental than genetic alterations, and was associated with upregulation of class I and II human leukocyte antigen and increased number and function of conventional type 1 dendritic cells. Preexisting expansion of intratumoral CD8+ T cells and treatment-induced clonal T-cell receptor (TCR) repertoire contributed to better clinical outcome. TCR sequencing of the peripheral blood mononuclear cell samples from patients with durable remission detected the expanded T-cell clones 3 years after treatment. These findings thus improve the understanding of the effect of T-cell immunological memory on ZR2-based immunotherapy, and support a paradigm shift toward mechanism-based targeted therapy of aggressive lymphoma. This trial was registered at www.clinicaltrials.gov as #NCT04460248.

The enhancer of zeste homolog 2 (EZH2) histone methyltransferase inhibitors tazemetostat and valemetostat recently have received approval for clinical use in follicular lymphoma and adult T-cell leukemia/lymphoma, respectively. In follicular lymphoma, the expression of activation-induced cytidine deaminase (AID) is responsible for increased mutational signatures and genomic instability. Because EZH2 inhibitors induce epigenetic and transcriptional changes in normal and lymphoma B cells, we studied whether these inhibitors could alter the pattern of AID-mediated chromosomal translocations. In this study, we showed that treatment with EZH2 inhibitors did not significantly change AID expression or AID-dependent chromosomal translocation frequency when used as monotherapy in either CH12F3 mouse B cells or MEC-1 human B cells. In contrast, when combined with phosphoinositide 3-kinase δ (PI3Kδ) inhibition, which enhances AID expression, EZH2 inhibition significantly increased the frequency of chromosomal translocations when compared with either EZH2 or PI3Kδ inhibition alone both in mouse CH12F3 cells and human MEC-1 cells. EZH2 inhibition also further enhanced translocation formation in mouse B cells that were DNA ligase IV (Ligase4) deficient. Mechanistically, EZH2 inhibition in B cells depletes the repressive histone modification H3 trimethylation at lysine 27 (H3K27me3) while concurrently enhancing the active histone modification H3 acetylation at lysine 27 (H3K27ac), thereby selectively increasing transcriptional activity and facilitating chromosomal translocation formation in the presence of high AID activity or Ligase4 deficiency. These findings highlight the impact of drugs that induce epigenetic changes to influence chromosomal translocations, and demonstrate the genetic safety of EZH2 inhibitors as monotherapy while highlighting the increased risk of genomic instability when used in cells prone to translocations, such as B cells with high AID levels or DNA repair deficiency.

Detection of KIT p.D816V is a cornerstone in the diagnosis and classification of mast cell activation syndromes (MCAS) and mastocytosis. However, KIT p.D816V may be undetected due to a low mutated cell burden in blood and bone marrow (BM) of many patients, particularly among those without skin lesions. These findings underscore the need for ultra-sensitive molecular techniques for the detection of KIT p.D816V in these clinical settings. Here, we evaluated for the first time the sensitivity and specificity of a novel Flow-SuperRCA® KIT p.D816V assay, compared to conventional ASOqPCR, through the analysis of 548 blood and BM samples from 337 adults MCAS and mastocytosis patients. Our results demonstrated greater sensitivity of the new technique vs ASOqPCR (limit-of-detection: 0.001% vs 0.01% VAF), with higher rates of positivity for KIT p.D816V in whole-blood and/or BM of 64% of monoclonal-MCAS (MMAS) and 55% of BM mastocytosis patients (p<.0001). Notably, the sensitivity of the new assay went even beyond that of ASOqPCR in purified BM mast cells (p<.0001). Additionally, clonality was newly identified in 18% of patients previously diagnosed with non-clonal-MCAS, who presented with a unique cutaneous MCA-related symptomatic profile. These results confirm the high-specificity and ultra-sensitivity of the Flow-SuperRCA® assay for the detection of KIT p.D816V, emerging as a well-suited whole-blood and whole-BM test for the diagnostic screening and classification of MCAS and mastocytosis patients. These findings further highlight the clonal nature of an unprecedently high fraction of patients who presented with anaphylaxis and MCAS, with important pathogenic, diagnostic and clinical implications.

Recent advances have transformed the treatment landscape for relapsed and refractory follicular lymphoma. Although chemotherapy has long served as the backbone of treatment, the availability of novel targeted, immunomodulatory, and immunotherapeutic approaches is challenging its relevance. These approaches have focused on targeting epigenetic regulators, components of the B-cell receptor or its downstream intracellular pathways and the follicular lymphoma tumor microenvironment. The recent development of bispecific antibodies and chimeric antigen receptor T-cell therapies, which target both tumor-associated and host-specific antigens, has enabled a redirection of the immune system, enhancing the innate antitumor immune response. Rational combinations of these strategies are actively being evaluated in the relapsed and refractory setting and will inevitably move forward into earlier lines of treatment. The success of these approaches has led to numerous and parallel options for patients and clinicians. The emerging challenge now lies in how best to approach each individual patient with relapsed or refractory follicular lymphoma, addressing complex decision-making that considers a patient's previous treatment history, goals of care, clinical and biological characteristics of recurrence, and personal preferences. Understanding the implications of refractory and transformed disease, as well as the timing and biology of relapse will be critical to support a more personalized treatment approach in the modern era.

Blood clotting is triggered in hemostasis and thrombosis when the membrane-bound tissue factor (TF)/factor VIIa (FVIIa) complex activates factor X (FX). There are no structures of TF/FVIIa on membranes, with or without FX. Using cryo-EM to address this gap, we assembled TF/FVIIa complexes on nanoscale membrane bilayers (nanodiscs), bound to XK1 and an antibody fragment. XK1 is a FX mimetic whose protease domain is replaced by the first Kunitz-type (K1) domain of tissue factor pathway inhibitor, while 10H10 is a non-inhibitory, anti-TF antibody. We determined a cryo-EM structure of a TF/FVIIa/XK1/10H10/nanodisc complex with a resolution of 3.7 Å, allowing us to model all the protein backbones. TF/FVIIa extends perpendicularly from the membrane, interacting with a "handle shaped" XK1 at two locations: the K1 domain docks into FVIIa's active site, while the γ-carboxyglutamate-rich (GLA) domain binds to TF's substrate-binding exosite. The FX and FVIIa GLA domains also contact each other and the membrane surface. Except for a minor contact between the first epidermal growth factor (EGF)-like domain of XK1 and TF, the rest of the FX light chain does not interact with TF/FVIIa. The structure reveals a previously unrecognized, membrane-dependent allosteric activation mechanism between FVIIa and TF where a serine-rich loop in TF that partially obscures the TF exosite must undergo a shift to allow access of the FX GLA domain to its final binding location on the membrane-bound TF/FVIIa complex. This mechanism also provides a novel explanation for the otherwise puzzling phenomenon of TF encryption/decryption on cell surfaces.

Venous thromboembolism (VTE) is a frequent (annual incidence of 1-2 per 1000 individuals) and potentially life-threatening (case-fatality rate up to 10%) disease. VTE is associated with serious short-term and long-term complications, including a recurrence rate approaching 20% within 5 years. Anticoagulant therapy, the mainstay of VTE treatment, drastically reduces the risk of early VTE recurrence, but it exposes patients to a substantial risk of bleeding. We analyzed the genomic architecture of VTE recurrence using data from 6355 patients across 8 cohorts (including 1775 recurrences), enriched by subgroup analyses, according to sex and clinical manifestation of first VTE, which led to the identification of 28 molecular markers. Through genome-wide association studies, we identified 1 locus associated with VTE recurrence, GPR149/MME. Among all variants known to be associated with first VTE, KNG1, and FGG were associated with recurrence. Additionally, Mendelian randomization analyses identified 7 proteins as risk factors for recurrence: elevated plasma levels of coagulation factor XI, coagulation factor VIII, von Willebrand factor, histo-blood group ABO system transferase, and Golgi membrane protein 2; and decreased levels of proprotein convertase subtilisin/kexin 9 and pro-interleukin-16. Subgroup analyses revealed 18 molecular determinants associated with VTE recurrence, with notable differences between subgroups. For example, the exonic variant SLC4A1 p.Glu40Lys was significantly associated in patients who experienced pulmonary embolism but showed no effect in those with deep vein thrombosis. These findings emphasize the role of specific genetic loci and protein pathways in influencing VTE recurrence and provide valuable insights into potential therapeutic targets. Further research is needed to clarify the biological mechanisms driving these associations.