Journal of Thrombosis and Haemostasis

Understanding how specific VWF variants disrupt endothelial processing and function is central to elucidating von Willebrand disease (VWD) pathophysiology. However, current in vitro systems lack either the endothelial specificity or the genetic flexibility required for systematic variant characterization. Here, we present a genetically defined VWF-knockout cord-blood-derived endothelial colony-forming cell (VWF-KO cbECFC) model that enables controlled reintroduction of VWF variants in a physiologically relevant endothelial context. Using a patient with type 3 VWD carrying the homozygous pathogenic variant p.M771V and a second homozygous variant of uncertain significance p.R2663P as a reference, we demonstrate that expression of p.M771V in VWF-KO cbECFCs reproduces the patients intracellular processing defect and loss of high-molecular-weight multimers, whereas p.R2663P behaves as a benign allele. These findings establish the models ability to accurately distinguish pathogenic from non-pathogenic variants. Comparative analyses with HEK293 cells show that VWF-KO cbECFCs provide superior subcellular resolution, reliably forming authentic Weibel-Palade bodies (WPBs) and faithfully revealing ER retention phenotypes that remain ambiguous in non-endothelial systems. The proliferative capacity of cbECFCs further enables scalable and reproducible experimentation, overcoming major limitations associated with patient-derived ECFCs. Looking ahead, the VWF-KO cbECFC platform offers broad potential for VWF and VWD research. Its endothelial identity and genetic flexibility make it suitable for investigating VWF biosynthesis and trafficking, secretion dynamics, WPB biology, angiogenic processes, and shear-dependent VWF function. This system therefore provides a versatile foundation for mechanistic studies, systematic variant assessment, and future translational applications.

BackgroundLong COVID is characterised by persistent systemic inflammation and endothelial dysfunction, with increasing evidence implicating thromboinflammatory mechanisms. Platelet-monocyte aggregates (PMA) represent a sensitive marker of platelet activation and immune-vascular interactions, but their role in Long COVID remains incompletely defined. MethodsThis study quantified circulating PMA in 20 Long COVID patients and 20 healthy controls using a two-colour imaging flow cytometry assay targeting CD14 (a monocyte receptor for pathogen-associated molecular patterns, PAMPs) and CD62P (P-selectin). PMA were expressed as a percentage of total monocytes, and platelet attachment patterns were classified into single versus multiple platelet binding. Statistical analyses included Shapiro-Wilk normality testing, unpaired t-tests, Mann-Whitney U tests or two-way ANOVA as appropriate, and linear regression for correlation analysis. ResultsCirculating PMA were significantly elevated in Long COVID patients compared with controls (29.19 [20.02-37.26] vs 4.59 [2.67-7.16], p < 0.0001). Long COVID samples showed a reduced proportion of monocytes with single platelet attachment and a corresponding increase in multiple platelet binding (p < 0.0001). In controls, %PMA increased with age (p < 0.01), whereas no age association was observed in Long COVID, indicating an elevated baseline independent of age. ConclusionsLong COVID is associated with markedly increased platelet-monocyte aggregation and altered platelet attachment dynamics, consistent with sustained thromboinflammatory activity. PMA represent a sensitive cellular marker of platelet-driven immune activation and may have utility as an accessible biomarker for stratifying thromboinflammatory burden in Long COVID.

Background and PurposeNeurointerventional outcomes depend on clot composition and may be influenced by clot contraction. Thus, a priori identification of clot composition and contraction could inform procedural strategies and improve outcomes. The goal of our work is to conduct an in vitro test to determine whether MRI can reliably predict both clot composition and contractile state. Materials and MethodsTo this end, we prepared blood clots spanning clinically observed compositions (0-80% red blood cells (RBCs)) in both contracted and uncontracted states. Contraction was controlled by coagulating blood with or without thrombin. We imaged these clots using quantitative, clinical, and investigational MRI sequences. Using these data, we then determined whether MRI signal intensities, quantitative parameters, and radiomic features capturing intensity and texture patterns can (i) predict clot hematocrit and (ii) classify clots by composition (RBC-rich vs. fibrin-rich) and contraction state. ResultsQuantitative MRI parameters (T1, T2, ADC) decreased with increasing hematocrit (R2 = 0.56-0.85, p < 0.001), while signal intensities from clinical sequences showed weaker correlations (R2 = 0.46-0.62, p < 0.001). Radiomic models predicted hematocrit with performance comparable to MRI parameters. When applied to classification, radiomic features accurately discriminated RBC-versus fibrin-rich clots, with AUCs exceeding 0.90 across nearly all sequences. In contrast, classification of contraction state showed greater variability in AUCs across sequences but remained high for quantitative T1 and T2 values (AUCs up to 0.88). Trends were consistent across clots coagulated with and without thrombin. Pooling features across sequences did not outperform the best individual sequence for either regression or classification. ConclusionsWe demonstrate that MRI-based radiomic analysis quantitatively characterizes clot composition and contraction in vitro. These findings support the feasibility of using MRI for pre-interventional clot phenotyping, with potential to inform thrombolytic and mechanical thrombectomy strategies. Thus, in vivo studies validating these results are warranted.

Background: SARS-CoV-2 infection is an established prothrombotic trigger, yet the population-level temporal relationship between circulating viral activity and pulmonary embolism (PE) remains poorly characterized. We aimed to evaluate the short-term association between respiratory viral activity and PE hospitalizations, accounting for specific temporal lags. Methods: We conducted a population-level time-series analysis of incident PE hospitalizations in Ontario, Canada, from 2011 to 2024. Using distributed lag non-linear models, we assessed the association between standardized weekly activity levels of SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) and PE risk over a 5-week lag period. Relative risks (RR) per standard deviation (SD) increase in viral activity were estimated via negative binomial regression using cross-basis terms to account for both exposure-response and lag-response non-linearities. Models were adjusted for Fourier seasonal terms and secular trends. Findings: Among 70,670 incident PE cases identified between 2011 and 2024, SARS-CoV-2 activity demonstrated a significant temporal association with PE. A cumulative RR increase of 20% per SD in SARS-CoV-2 activity was observed over the five weeks following exposure (RR 1.20; 95% CI 1.05-1.37). The risk followed a distinct delay trajectory: weekly cumulative RRs peaked at week 3 (RR 1.21; 95% CI 1.01-1.45). For the 2020-2024 period, influenza A also showed an association peaking at week 3 without statistical significance (RR 1.17; 95% CI 0.95-1.45). Interpretation: Increased population-level SARS-CoV-2 activity is associated with a heightened risk of PE, peaking at approximately the third week. This delayed peak suggests a protracted thrombo-inflammatory window, likely driven by sustained endothelial injury. These findings highlight the vascular burden of COVID-19 and suggest that infection prevention measures, including vaccination, may provide significant downstream protection against thromboembolic disease.

BackgroundCerebral ischemia-reperfusion injury (CIRI) is a major determinant of poor outcome after recanalization therapy in acute ischemic stroke. Microglial functional heterogeneity underpins neuroinflammation, yet the molecular mechanisms governing microglial phenotypic transitions remain incompletely understood. Metabolite-driven post-translational modifications (PTMs) have emerged as key regulators of microglial metabolism and inflammation, but whether PTM regulatory enzymes form co-expression modules that define microglial states is unknown. MethodsWe analyzed single-cell RNA-seq datasets from five GEO studies (GSE174574, GSE227651, GSE245386, GSE267240, GSE319237) covering tMCAO reperfusion and permanent ischemia models. Microglia were purified using double filtration (P2ry12/Tmem119/Cx3cr1+, Cd68/Adgre1/Ly6c-). PTM enzyme co-expression modules were identified by non-negative matrix factorization (NMF). Spatiotemporal dynamics were assessed by module projection across timepoints (Sham, 1d, 3d, 7d) and pseudotime analysis. Independent validation was performed in an additional tMCAO dataset (GSE245386). Sex differences were explored in a mixed-sex permanent ischemia dataset (GSE267240). ResultsThree robust PTM enzyme co-expression modules were identified: Metabolic stress-associated (M1), Pro-inflammatory-associated (M2), and Reparative-associated (M3). M1 was enriched in TCA cycle enzymes, M2 in inflammatory pathways (leukocyte activation, chemotaxis), and M3 in vascular development and translation. Module proportions and scores showed dynamic transitions: M1 decreased after reperfusion, M2 peaked at day 1-3, and M3 slightly increased at day 7. Independent validation in GSE245386 yielded high module conservation (cosine similarity = 0.874). Sex-specific differences in module distribution were observed in permanent ischemia ({chi}2 = 14.98, p = 0.00056). ConclusionsPTM enzyme co-expression modules delineate metabolic, pro-inflammatory, and reparative microglial states in CIRI with distinct spatiotemporal dynamics. This transcriptional framework supports the "PTM enzyme code" hypothesis and provides stage-specific targets for stroke therapy.

BackgroundThe success of hematopoietic stem cell transplantation (HSCT) depends critically on human leukocyte antigen (HLA) matching between donor and recipient. While traditional matching focuses on five classical HLA loci (A, B, C, DRB1, DQB1), clinical practice increasingly considers extended typing at nine loci, including DPA1, DQA1, DPB1, and DRB3/4/5. Furthermore, emerging evidence supports transplantation with up to three HLA mismatches under post-transplant cyclophosphamide (PTCy) regimens. However, current donor search algorithms cannot efficiently identify donors with multiple mismatches across extended HLA loci in real-time. MethodsWe developed GRIMM-II (GRaph IMputation and Matching, version II), which comprises two novel algorithms: ML-GRIM (Multi-Locus GRIM) for HLA imputation across multiple loci, and ML-GRMA (Multi-Locus GRMA) for real-time donor-patient matching with up to three mismatches. Both algorithms employ a two-stage approach that combines efficient candidate reduction through graph-theoretic frameworks with detailed genotype comparison. ML-GRIM partitions genotypes into class I (HLA-A, B, C) and class II (remaining loci) components, enabling memory-efficient storage and rapid candidate identification. ML-GRMA searches a pre-imputed donor graph composed of donor genotypes and their sub-components, then computes asymmetric graft-versus-host (GvH) and host-versus-graft (HvG) mismatch probabilities to provide clinically relevant compatibility assessments. Both imputation and matching tools are available as a web application at https://grimmard.math.biu.ac.il/ and through GitHub repositories at https://github.com/nmdp-bioinformatics/py-graph-imputation (imputation) and https://github.com/nmdp-bioinformatics/py-graph-match (matching). ResultsWe validated ML-GRMA and ML-GRIM using the WMDA3 (World Marrow Donor Association) validation dataset, successfully reproducing all previously reported matches while identifying numerous additional candidate donors not detected by previous algorithms. Further validation of ML-GRMA using 3,000 patients with artificially introduced mismatches (0-3 allele substitutions) demonstrated 100% sensitivity and specificity in identifying matching donors at expected mismatch levels. We validated ML-GRIM using simulated nine-locus typings derived from 8,078,224 US donors in the NMDP registry. The algorithm successfully imputed genotypes across variable numbers of typed loci while incorporating multiethnic haplotype frequencies. The algorithm achieved real-time performance with typical imputation times under one second and matching times of 1-13 seconds per patient for up to three mismatches, even when searching databases exceeding 8 million donors. Notably, ML-GRMA identified substantially more potentially suitable donors than traditional algorithms by accounting for the biological reality that GvH and HvG mismatches often differ, particularly for donors homozygous at specific loci. To evaluate ML-GRIM performance with low-resolution typing, we tested it on simulated 3-locus typings from the same population. The resulting imputation accuracy correlated with the mutual information between typed loci and complete genotypes. ConclusionsGRIMM-II provides a scalable, memory-efficient solution for nine-locus HLA imputation and real-time identification of donors with up to three mismatches. The graph-based framework supports dynamic registry updates and can readily accommodate additional HLA loci and matching criteria as clinical knowledge evolves. By expanding the pool of acceptable donors while maintaining computational efficiency, GRIMM-II addresses a critical need in contemporary transplantation practice, particularly for patients from underrepresented ethnic minorities who face lower probabilities of finding perfectly matched donors.

Circulating stem and progenitor cells (SPCs), including mesenchymal stromal cells (MSCs) and hematopoietic stem/progenitor cells (HSPCs), are mobilised after tissue injury but their temporal behaviour after hemorrhagic shock (HS) and relationship to cytokine milieus and outcome remain unclear. In a prospective observational cohort at JPN Apex Trauma Centre, AIIMS, New Delhi we studied 100 participants: 50 trauma patients with hemorrhagic shock and traumatic brain injury (HS index group), 25 trauma patients without HS, and 25 minor-injury controls. Peripheral blood was collected at admission (day 0) for all groups and additionally at days 3, 7 and 14 for the HS group. PBMCs were phenotyped by flow cytometry (HSPC markers: CD45, CD123, CD38, CD34; MSC markers: CD105, CD73, CD90) and serum SDF-1, VEGF-A, EGF, GRO- and GRO-{beta}, GM-CSF and G-CSF were measured by ELISA; group and time effects were evaluated with mixed-effects models and correlations by Spearman tests (two-tailed p<0.05). At admission, trauma patients without HS had significantly higher MSC and HSPC-like populations versus controls (p<0.0001). In the HS cohort SPC percentages rose modestly at day 0-3 then declined sharply by days 7-14 (time effect p<0.0001); non-survivors exhibited significantly higher early SPC and cytokine levels that persisted until death while survivors showed an early rise followed by decline (outcome and time interaction p<0.0001). All cytokines were up-regulated in trauma groups, peaked at day 0-3 in HS patients, and correlated positively with SPC counts (notably SDF-1, VEGF-A, G-CSF, Gro- and GM-CSF; Spearman p<0.05); higher early SPC and cytokine signatures associated with greater organ dysfunction (higher SOFA) and with timing of sepsis. These findings indicate that trauma provokes an early SPC and cytokine response that in HS is followed by later decline, and that persistent early elevation predicts worse outcomes, suggesting serial SPC and cytokine profiling may have prognostic value and identify an early therapeutic window for regenerative or immunomodulatory interventions.

Ischemic stroke triggers a cascade of molecular and cellular processes leading to fibrotic scar formation, entailing activation of brain platelet-derived growth factor receptor (PDGFR){beta}+ cells. Kruppel-like factor (KLF)4 plays an important role in regulating the activation of peripheral PDGFR{beta}+ perivascular cells in response to hypoxia/ischemia. Herein, we aimed to characterize the spatiotemporal responses of brain PDGFR{beta}+ cells while assessing the contribution of KLF4. This was achieved using transgenic mice that enable tracking or conditionally depleting KLF4 in PDGFR{beta}+ cells, which were subjected to experimental ischemic stroke. Next, we employed point pattern analysis (PPA) and topological data analysis (TDA) to quantitatively characterize cell phenotypic changes and spatial distribution over injury progression after ischemic stroke. We show that brain PDGFR{beta}+ cells rapidly become reactive and early localize to regions prone to irreversible damage. We report the emergence of parenchymal PDGFR{beta}+ cells, which cannot be causally linked to proliferation or vascular detachment. Moreover, our analysis reveals that KLF4 is barely expressed in brain PDGFR{beta}+ cells under normal conditions, and that its expression is slightly induced in reactive cells in the injured brain. Notably, specific attenuation of KLF4 induced expression in PDGFR{beta}+ cells does not affect cell reactivity and spatiotemporal distribution, nor scar formation and injury severity. These observations suggest that in contrast with the periphery, KLF4 is not implicated in regulating the responses of brain PDGFR{beta}+ cells. Our results indicate that the reactivity of brain PDGFR{beta}+ cells after stroke is spatiotemporally diverse, evolve over injury progression, and is distinct from peripheral perivascular cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/712632v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1149c62org.highwire.dtl.DTLVardef@26edaaorg.highwire.dtl.DTLVardef@1bd3d35org.highwire.dtl.DTLVardef@fd8030_HPS_FORMAT_FIGEXP M_FIG C_FIG

BACKGROUNDInactivation of ANGPTL3 (angiopoietin-like protein 3, A3) is a proven therapeutic strategy for lowering plasma lipid levels independently of the LDL receptor (LDLR), yet the optimal approach to inactivate A3 remains unclear. A3 is proteolytically cleaved and circulates as full-length (A3-FL), N-terminal (A3-Nter) and C-terminal (A3-Cter) fragments. The specific contribution of each form of A3, and of its paralog, ANGPTL8 (A8), in modulating circulating levels of ApoB-Containing Lipoproteins (ABCLs) remain poorly defined. Clarifying these relationships will inform next-generation A3-directed therapies. METHODSWe performed liver perfusion studies to directly compare the number and composition of VLDL particles secreted from mice with and without A3. To amplify effects on cholesterol metabolism, we generated Ldlr-/- mice expressing wildtype A3 (A3-WT), A3-FL or A3-Nter, with or without co-expression of A8, and analyzed plasma lipids, circulating A3 and A8 complexes, and intravascular lipase activities. Complementary in vitro assays and structural modeling were used to assess relative endothelial lipase (EL) inhibition by A3 alone or in complex with A8. RESULTSLiver perfusion studies revealed that A3 inactivation does not alter the rates of hepatic secretion of VLDL in wildtype or Ldlr-/- mice. Inactivation of A8 alone lowered plasma LDL-cholesterol (C) levels by [~]20%, an effect dependent upon the expression of both EL and A3. Maximal inhibition of lipoprotein lipase (LPL) required co-expression of A8 plus both A3-FL and A3-Nter, indicating that A3 cleavage, in addition to A8 expression, is essential for maximal LPL inhibition. In contrast, A8 expression, but not A3 cleavage, was required for optimal EL inhibition. CONCLUSIONSA8 acts in concert with A3 to differentially modulate LPL- and EL-mediated lipolysis, which antagonizes hepatic clearance of newly-secreted atherogenic ABCLs. This mechanistic framework refines our understanding of A3-targeted lipid lowering and highlights the therapeutic potential of dual A3- plus A8-directed strategies to treat dyslipidemia and prevent atherosclerotic cardiovascular disease. Clinical perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIInactivation of A3 lowers circulating ABCL levels without altering hepatic secretion rates of VLDL-ApoB or -TG. C_LIO_LIProteolytic cleavage of A3 is required for maximal inhibition of LPL. C_LIO_LIInactivation of A8 lowers LDL-C levels through an A3- and EL-dependent, but LDLR-independent, mechanism. C_LI What are the clinical implications?O_LICombining A8 inhibition with A3-inactivating therapies offers a strategy to achieve greater reduction in LDL-C levels and atherosclerotic cardiovascular risk. C_LI

BACKGROUNDThe vascular endothelium is a dynamic tissue central to vascular homeostasis and disease, with endothelial cells (ECs) exhibiting plasticity that drives adaptive remodeling. Reelin, a secreted extracellular matrix glycoprotein critical for neuronal migration via ApoER2/VLDLR-DAB1 signaling, may also modulate vascular function and inflammation. However, its direct role in EC biology remains unclear. We investigated Reelin as a context-dependent signaling modulator in ECs, assessing its engagement of non-canonical pathways and regulation of endothelial plasticity relevant to cardiovascular pathology. METHODSHuman endothelial cells were stimulated with recombinant Reelin and analyzed by immunoblotting, immunofluorescence, and functional assays. Time-course studies assessed signaling, including phosphorylation of FAK, AKT, and DAB1 by Western blotting, while wound-healing assays quantified endothelial migratory capacity in vitro systems. RESULTSReelin rapidly robustly activated noncanonical signaling in endothelial cells, increasing FAK and AKT phosphorylation in a time-dependent manner consistent with cytoskeletal remodeling. Canonical DAB1 activation was limited. Functionally, Reelin enhanced migration, upregulated Endoglin/CD105, and induced a remodeling-associated phenotype. Reelin silencing altered endothelial phenotype, clearly indicating a role in homeostasis. Signaling was independent of VEGFR2 interaction. Overall, Reelin preferentially engages FAK/AKT pathways to drive partial phenotypic modulation without full endothelial-to-mesenchymal transition. CONCLUSIONWe show that Reelin is a previously unrecognized regulator of endothelial signaling and plasticity, acting via non-canonical FAK- and AKT-dependent pathways. By partially and dynamically modulating endothelial phenotype, Reelin promotes a remodeling-permissive state without triggering full mesenchymal transition. These findings identify Reelin as a novel modulator of endothelial function with potential implications for vascular remodeling and cardiovascular disease. What Are the Clinical Implications?Our findings identify Reelin as a modulator of endothelial signaling with a clear bias toward non-canonical FAK- and AKT-dependent pathways that regulate endothelial plasticity and remodeling. This signaling profile is highly relevant to vascular diseases in which endothelial dysfunction is driven by maladaptive cytoskeletal reorganization, altered migration, and persistent activation rather than complete loss of endothelial identity. The ability of Reelin to promote partial and dynamically regulated phenotypic modulation suggests that it may operate at early and potentially reversible stages of vascular pathology. In this context, dysregulated Reelin signaling could contribute to pathological vascular remodeling, including processes underlying atherosclerosis, fibrosis, and microvascular dysfunction. These results also raise the possibility that circulating or locally produced Reelin may serve as an indicator of endothelial activation state, providing a novel biomarker for vascular disease progression. Importantly, the identification of a signaling bias toward FAK- and AKT-dependent pathways highlights potential therapeutic targets downstream of Reelin that could be selectively modulated to limit maladaptive endothelial remodeling while preserving essential endothelial functions. Collectively, this study positions Reelin signaling as a previously unrecognized and potentially actionable pathway in the regulation of endothelial behavior, with direct implications for the development of targeted strategies aimed at preventing or attenuating cardiovascular disease progression

G protein-coupled receptors rely on dynamic conformational changes to coordinate G protein activation and recruitment of regulatory transducers such as G protein-coupled receptor kinases and {beta}-arrestins. The chemotactic receptor GPR183 has been implicated in a context-dependent role in hematological malignancies. Here, we investigated the impact of A338V mutation located within the C-terminal tail of GPR183. This mutation is associated with acute myeloid leukaemia. Using bioluminescence resonance energy transfer-based assays in HEK293A cells, we assessed receptor-proximal signaling events. The A338V variant displayed preserved agonist potency and comparable agonist-induced Gi activation relative to wild type, although constitutive activity towards Gi was modestly reduced. In contrast, recruitment of GRK2 and {beta}-arrestin2 was consistently impaired across multiple assay configurations. These differences were not attributable to altered receptor abundance, as the C-tail untagged mutant exhibited increased plasma membrane expression despite reduced regulatory transducer engagement. While intramolecular conformational biosensor measurements revealed subtle differences in global receptor conformation between WT and A338V, extensive molecular dynamics simulations supported the altered conformational sampling of the C-terminal tail in the A338V variant. Together, these data support a model in which the A338V substitution selectively alters C-terminal structural dynamics, impairing GRK2 and {beta}-arrestin2 recruitment while preserving G protein activation.

Background and Aims Despite treatment, patients with established atherosclerotic cardiovascular disease (ASCVD) are at high risk of recurrent events. Existing clinical risk scores for recurrence provide only moderate predictive performance and rely largely on the same conventional risk factors used to predict disease onset. Proteomics is a promising source of new biomarkers but the technologies need focused use cases in order to achieve utility and implementation. We aimed to determine whether plasma proteomics improves prediction of recurrent cardiovascular events beyond established clinical risk models in secondary prevention in a population-scale cohort. Methods Plasma proteomic profiles from ~9,300 participants in the UK Biobank with established ASCVD at baseline were analysed using machine learning methods to derive and evaluate proteomic predictors of recurrent cardiovascular events. The top performing model comprised proteins with non-zero weights (full protein score). Predictive performance of the proteomic predictors, an established clinical risk score (SMART2), and their combination was evaluated across six pre-defined testing datasets representing multiple ethnic and geographic groups. A parsimonious set of proteins with existing clinical-grade enzyme-linked immunosorbent assays (ELISAs) available was then derived. Results The full protein score achieved higher performance for recurrent ASCVD than the SMART2 risk score across all ethnic and geographic subgroups (mean C-index 0.743 vs 0.653). Adding the full protein score to SMART2 improved discrimination, with the largest increase in White Irish participants ({Delta}C-index, 0.140; 95% CI, 0.074-0.205; P<0.001). However, adding SMART2 to the protein score provided minimal additional value. The parsimonious score preserved most of the discrimination of the full protein model with C-indices of the recurrent ASCVD risk model comprising age, sex and the parsimonious protein score being nearly identical to the full protein model in the largest testing set (0.723 vs 0.728 for White British in England and Wales). The parsimonious protein score showed a marked gradient of risk with the top, middle and bottom quintiles showing 10-year recurrent ASCVD rates of ~27.4%, ~9.6% and ~2.4%, respectively. Conclusions In patients with established ASCVD, plasma protein measurements substantially improved prediction of recurrent events beyond conventional clinical risk factors, supporting their potential as a complementary tool to guide secondary prevention of cardiovascular disease.

Polycomb repressive complexes PRC1 and PRC2 regulate diverse developmental processes, including the fetal-to-adult switch in hemoglobin production, a process whose reversal is a goal for the treatment of sickle cell disease and {beta}-thalassemia. PRC inhibitors show promise for various disorders, but use is limited because of pleiotropic PRC activities. We explored whether fetal hemoglobin (HbF) can be reactivated in adult erythroid cells by selective perturbations of PRC1 or PRC2 components without complete loss of PRC function. A high-density CRISPR-Cas9 mutagenesis screen identified a region in the EZH2 subunit where Cas9 induced exon 14 skipping (EZH2{Delta}14). EZH2{Delta}14, which lacks a portion of the CXC domain, relieves HbF repression while largely maintaining cellular fitness. EZH2{Delta}14 retains H3K27 methylation and repression of a PRC target gene subset. Experiments in cells derived from mice bearing human {beta}-globin genes confirm that pathways mediating EZH2 control of HbF expression can function in a mouse model of HBG switching. These findings demonstrate that partial disruption of PRC can yield selective phenotypes, highlighting the therapeutic potential of targeting non-enzymatic domains within chromatin-modifying complexes. Key PointsO_LICRISPR-Cas9 screen across PRC1 and a saturating mutagenesis screen of PRC2 found the EZH2 CXC domain a desirable target for HbF induction C_LIO_LIthe EZH2-CXC domain leads to exon 14 exclusion, resulting in de-repression of HbF but maintenance of cell fitness. C_LI

BackgroundIndividuals with JAK2V617F-mutant myeloproliferative neoplasms or clonal hematopoiesis of indeterminate potential have a markedly increased risk of cardiovascular disease, yet the mechanisms by which mutant blood cells drive vascular and cardiac dysfunction remain incompletely understood. Although the thrombopoietin (TPO) receptor MPL is central to hematopoiesis and is expressed in vascular endothelial cells (ECs), its role in JAK2V617F-associated cardiovascular complications is unknown. Methods and ResultsWe generated chimeric mice with JAK2V617F-mutant blood cells and wild-type endothelium by bone marrow transplantation and challenged them with a high-fat/high-cholesterol diet to model cardiometabolic stress. These mice developed a distinct cardiovascular phenotype characterized by microvascular disease, increased left ventricular mass, and relatively preserved left ventricular ejection fraction. Histological analysis revealed coronary arteriole stenosis, perivascular fibrosis, reduced microvascular density, and endocardial injury, without evidence of epicardial coronary stenosis or myocardium infarction. Single-cell RNA sequencing revealed activation of inflammatory, stress-response, and endothelial-to-mesenchymal transition gene signatures in ECs, most prominently within the endocardial ECs. Immunohistochemistry identified MPL expression predominantly in endocardial ECs. TPO/MPL signaling was upregulated in endocardial ECs in mice with JAK2V617F-mutant hematopoiesis, and treatment with an anti-MPL neutralizing antibody markedly improved cardiac pathology, restored endocardial integrity, and increased coronary microvascular density despite persistent systemic inflammation. ConclusionsJAK2V617F-mutant hematopoiesis induces coronary microvascular dysfunction. Endocardial ECs represent a key cellular target under cardiometabolic stress, and endocardial MPL signaling constitutes a potential targetable pathway in JAK2V617F-associated cardiovascular disease. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/715884v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1b0c2d7org.highwire.dtl.DTLVardef@1c7da20org.highwire.dtl.DTLVardef@1c19af9org.highwire.dtl.DTLVardef@1a588b3_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIJAK2V617F-mutant hematopoiesis induces cardiac microvascular disease C_LIO_LIMPL is expressed in endocardial ECs and MPL inhibition restores endocardial integrity and improves cardiac microvascular function C_LI

Terminal erythroid differentiation involves dramatic cellular remodeling that culminates in the expulsion of the nucleus, a process known as enucleation. While enucleation is conserved across mammals and is crucial for the generation of fully functional erythrocytes, the mechanisms governing this process have remained largely unknown, in part because the absence of genetic material in mature, enucleated red blood cells hinders genetic experimentation. Here, we performed a pooled, forward-genetic CRISPR-Cas9 screen in enucleated red blood cells derived from primary human hematopoietic stem cells to identify genes required for enucleation. We found that Chloride Intracellular Channel 3 (CLIC3) and Vesicle-associated membrane protein 8 (VAMP8) are both necessary for terminal erythroid differentiation, yet likely act through different mechanisms. Knockdown of CLIC3 led to a delay in erythroblast differentiation, culminating in impaired enucleation. We found that the knockdown cells had increased p53 and p21 and exhibited cell cycle alterations, suggesting CLIC3 plays a crucial role in coordinating cell cycle progression during erythropoiesis. In comparison, VAMP8-depleted cells initially appear to undergo accelerated differentiation but then display a specific defect in enucleation. Transcriptional analysis of the VAMP8-knockdown cells suggested dysregulation of pathways for vesicle trafficking and actin binding, and imaging of late-stage erythroblasts revealed impaired nuclear polarization and disorganized actin. This work provides a new approach for functional genomics in enucleated cells and reveals novel factors important for terminal erythroid differentiation and enucleation. Key pointsO_LIA CROPseq-based CRISPR-Cas9 screen enables functional genomics in enucleated primary human red blood cells. C_LIO_LIChloride Intracellular Channel 3 (CLIC3) and Vesicle Associated Membrane Protein 8 (VAMP8) were identified as critical for terminal erythroid differentiation and enucleation, likely acting through two distinct mechanisms. C_LI

There are few therapeutic options to treat patients with sickle cell disease (SCD), a blood disorder caused by mutations in the {beta}-globin gene that affects >7M individuals worldwide. Combining human genetics and high-throughput proteomics can help identify new drug targets. Here, we present results from a proteogenomic analysis of the plasma proteome in SCD patients. We measured the levels of 5,411 plasma proteins and tested their associations with common genetic variation in 343 SCD patients. After conditional analyses, we identified 560 protein quantitative trait loci (pQTL), including 58 (10%) that are novel. Many of these pQTL are not specific to SCD patients and associate with clinically relevant traits in non-SCD African Americans from the Million Veteran Program (e.g. hemoglobin concentration, triglycerides). The effect sizes of the pQTL is largely concordant between SCD and non-SCD individuals, although we found examples (e.g. APOL1, haptoglobin) with evidence of heterogeneity that suggests an interaction between the plasma proteome and the SCD genotype. Finally, we combine pQTL and genome-wide association study results for fetal hemoglobin (HbF) in a Mendelian randomization analysis to prioritize five proteins that may increase HbF production (ENPP5, LBP, NAAA, PT3X, ZP3).

Background Intracranial aneurysm rupture is the leading cause of spontaneous subarachnoid hemorrhage and is associated with substantial mortality and long term neurological disability. Emerging evidence suggests that the gut microbiome may influence vascular inflammation and endothelial integrity through immune and metabolic pathways, yet human evidence linking gut microbial alterations to intracranial aneurysm remains fragmented and inconsistent. Objective This systematic review and meta analysis aimed to synthesize available human evidence on the association between gut microbiome alterations and intracranial aneurysm formation or rupture, with a primary focus on microbial dysbiosis and differences in gut microbial alpha diversity. Methods This study was conducted according to PRISMA 2020 guidelines and the protocol was prospectively registered in PROSPERO (CRD420261360785). A comprehensive search of PubMed, Scopus, Web of Science, Embase, and Cochrane CENTRAL was performed from database inception until April 1, 2026, with additional screening of grey literature sources. Observational human studies evaluating gut microbiome characteristics in patients with intracranial aneurysm were included. Mendelian randomization (MR) studies investigating genetically predicted microbial taxa and aneurysm outcomes were also reviewed. Random effects meta analysis using standardized mean differences (SMD) was performed for alpha diversity outcomes. MR taxa reported in at least two independent studies were quantitatively synthesized using inverse variance weighting of log odds ratios. Results The systematic search identified 396 records. After removal of duplicates and eligibility screening, 20 studies met inclusion criteria, including 12 observational clinical studies and 8 Mendelian randomization analyses. Meta analysis of three microbiome sequencing studies demonstrated significantly reduced gut microbial alpha diversity in patients with ruptured intracranial aneurysms compared with controls. Sensitivity analyses confirmed the robustness of pooled estimates. In addition, MR evidence identified several microbial taxa, including Ruminococcus1, Bilophila, Fusicatenibacter, and Porphyromonadaceae, as potentially protective factors against aneurysm related outcomes. Across observational studies, gut dysbiosis was frequently associated with inflammatory pathways and alterations in microbial metabolites implicated in vascular dysfunction. Conclusion Current human evidence suggests a potential association between gut microbiome dysbiosis and intracranial aneurysm pathophysiology, particularly in relation to aneurysm rupture. Reduced microbial diversity and specific microbial taxa may influence vascular inflammation and aneurysm wall stability. However, existing evidence remains limited and heterogeneous. Large prospective cohorts and mechanistic studies are required to clarify causal relationships and evaluate whether microbiome targeted interventions could contribute to aneurysm risk stratification or prevention strategies.

Objective: To assess ethnic disparities in time to hospital presentation, use of acute reperfusion therapies, and in-hospital treatment times among patients presenting with stroke in a Dutch emergency department. Methods: In this single-centre observational cohort study, we included patients with a first-ever ischemic stroke between September 2020 and September 2021. Patients were categorized by ethnicity (with or without migration background). Demographic and stroke characteristics were compared between groups. Outcomes included: rates of presentation outside therapeutic time window, acute reperfusion therapy (intravenous thrombolysis (IVT) and endovascular thrombectomy (EVT)), and, when applicable, door-to-treatment time (DTTT), with a door-to-needle time (DTNT) and door-to-groin time (DTGT) for IVT and EVT respectively. Univariable and multivariable linear and logistic regression analyses were performed, adjusted for age, sex, and NIHSS at presentation, where appropriate. Results: A total of 232 patients were included, of whom 62 (26.7%) had a migration background. These patients were younger (66.6 vs 71.2 years) and more frequently had diabetes (27.4% vs 15.9%). Sex distribution was similar (59.7% vs 60.6% male). Stroke etiology differed between groups with less cardio-embolism (4.8% vs 15.3%) and more small vessel disease (69.4% vs 48.2%) among patients with a migration background. These latter patients presented more often outside the therapeutic time window (53.2% vs 37.1%; OR 1.90; 95% CI 1.05-3.45). EVT was less frequently performed in patients with a migration background compared to those without (8.1% vs 22.4%; OR 0.28; 95% CI 0.10-0.75). There were no significant differences in treatment times (DTTT 38min vs 30min, DTNT 35min vs 26min, DTGT 64min vs 54min). Conclusion: Patients with a migration background were more likely to present outside the therapeutic time window and had a lower rate of EVT. In order to improve access for these patients, more insight into prehospital and within hospital barriers and facilitators for appropriate management are needed.

BackgroundTranscatheter aortic valve replacement has transformed the management of aortic stenosis; however, adverse outcomes such as leaflet thrombosis and hypoattenuating leaflet thickening remain clinically significant concerns. Flow disturbances resulting from valve canting may alter local hemodynamics and promote thrombogenic conditions. We investigated how modest transcatheter heart valve canting alters cusp-specific sinus flow and washout and promotes localized thrombogenic microenvironments associated with leaflet surface thrombus formation using particle image velocimetry, a physiologic blood loop, and tissue analysis. MethodsA patient-derived aortic root model was used to evaluate the hemodynamic and thrombogenic effects of THV canting at -10{degrees} (anti-curvature), 0{degrees} (neutral), and +10{degrees} (along-curvature). High-resolution particle image velocimetry quantified sinus flow fields and washout characteristics, and complementary whole-blood loop experiments enabled histologic assessment of leaflet-associated thrombus formation. ResultsCanting redistributed systolic jet orientation and sinus recirculation in a direction-dependent manner while preserving global hemodynamic measurements. The most spatially constrained cusp showed the largest increase in stasis and the slowest washout. In the right coronary cusp, anti-curvature canting increased the fraction of sinus area with velocity magnitude <0.05 m/s to 92% versus 43% in neutral and 10% in along-curvature deployments, and prolonged neo-sinus (T90) washout to 4.7 cycles versus 2.9 and 1.8 cycles, respectively. Histology localized surface-adherent platelet/fibrin thrombus to these poorly washed regions, most prominently on the right coronary cusp leaflet in anti-curvature deployments. Left and noncoronary cusp responses shifted with tilt direction, indicating redistribution rather than uniform worsening of thrombogenic conditions. ConclusionsEven modest noncoaxial deployment is sufficient to create sinus-resolved throm-bogenic microenvironments that are not captured by global gradient or effective orifice area. Deployment configuration is therefore a modifiable determinant of post-TAVR leaflet throm-bosis risk and may contribute to HALT.

A prominent radiological manifestation of cerebral amyloid angiopathy (CAA) is enlargement of perivascular spaces (EPVS), which is suggested to result from fluid stagnation due to impaired perivascular clearance. Here, we report a novel observation of hypointense rims in cerebral white matter surrounding EPVS near haemorrhages on in vivo 7T Gradient Echo MRI. We hypothesised that the observed black rim pattern denotes iron accumulation that may be caused by incomplete clearance following bleeding. We investigated the occurrence and localisation of this marker on in vivo and ex vivo MRI and examined its histopathological correlates. From MRI data of the prospective longitudinal natural history study of hereditary Dutch-type CAA (D-CAA) at Leiden University Medical Centre, we selected the first 20 consecutive patients who underwent 7T imaging and assessed the presence of black rims on MRI. Post-mortem material was available from one donor with black rims on in vivo scans. Formalin-fixed coronal brain slabs were scanned at 7T MRI, including a high-resolution T2*-weighted sequence. Guided by ex vivo MRI, tissue blocks from representative areas with black rims were sampled for histopathological analysis. Serial sections were stained for iron, calcium, myelin, and general tissue morphology. On in vivo 7T MRI, 9 out of 20 participants exhibited one or several black rims, all located close to a haemorrhage. In the D-CAA donor, ex vivo MRI signal loss matched the in vivo contrast changes. Thirty-six vessels with ex vivo-observed black rims were retrieved and histopathologically examined, showing iron accumulation surrounding perivascular spaces, but the pattern and severity of iron deposition varied. Across groups, vessels displayed microvascular degeneration, including hyaline vessel wall thickening, adventitial fibrosis, and perivascular inflammation. We identified black rims on in vivo 7T MRI and confirmed their correspondence on ex vivo imaging. Iron deposition was determined as the underlying correlate of black rims, but the histopathology appears heterogeneous. The preferential deposition of iron around EPVS may indicate incomplete clearance of iron-positive blood-breakdown products after bleeding. The varied pattern of iron accumulation and microvascular alterations may reflect different pathophysiological mechanisms related to the formation and maintenance of black rims in D-CAA.