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.

Severe injuries result in acute changes in platelet number and function, but the impact of trauma and hemorrhagic shock on megakaryocytes (MKs) in the initial hours after injury have not been studied in detail. Using a murine model of trauma-hemorrhage, we identified rapid changes in MK morphology and mobilization into bone marrow sinusoids, changes that were detectable within one hour. Levels of several alpha-granule derived proteins were elevated in the bone marrow, and co-culture of naive MKs with bone marrow supernatant from injured mice resulted in similar changes to those observed in the model. These results illustrate that trauma-hemorrhage results in a hyperacute alteration in the bone marrow micro-environment that alters MK activity within an hour of injury.

IntroductionIn hemorrhagic shock, plasma resuscitation preserves vascular integrity and protects against trauma-induced coagulopathy and organ injury. Despite demonstrated clinical benefit, the endothelial mechanisms underlying plasma resuscitation remain incompletely defined. This study investigated endothelial-specific responses to plasma resuscitation to identify targetable pathways that promote vascular repair after traumatic injury. MethodsA murine model of severe polytrauma-hemorrhagic shock (PT/HS) with demonstrable vascular endotheliopathy by 24 hours was used to compare pulmonary vascular endothelial cell (EC) responses to resuscitation with lactated Ringers (LR) relative to fresh frozen plasma (FFP). Whole blood was collected for inflammatory biomarker analysis, and pulmonary vascular leak was quantified by dextran extravasation. Pulmonary EC glycocalyx (eGC) structure was assessed by transmission electron microscopy and immunofluorescence. Spatial transcriptomic profiling of pulmonary ECs was performed using a GeoMx Digital Spatial Profiler. Key transcriptomic findings related to mitochondrial biogenesis were validated by immunostaining and by treating primary human lung EC with FFP or LR. ResultsAt 24 hours after injury, FFP reduced systemic inflammatory cytokines, pulmonary innate immune cell infiltration, and PT/HS-induced vascular leak compared to LR. Plasma levels of syndecan-1, syndecan-4, and hyaluronan were decreased, consistent with enhanced pulmonary eGC expression. Although few differences in eGC-related genes were detected, pathway analysis revealed enrichment of cellular bioenergetics and metabolic recovery pathways in ECs after FFP, whereas LR was associated with oxidative stress and inflammatory signaling. FFP enhanced mitochondrial content in pulmonary EC after PT/HS and in treated human EC compared to LR-treated controls. ConclusionsFFP resuscitation after PT/HS reduces systemic inflammation and preserves pulmonary vascular barrier function, potentially through promotion of mitochondrial signaling, metabolic recovery, and endothelial stress regulation.

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.

BackgroundClassical myeloproliferative neoplasms (MPN)--essential thrombocythemia, polycythemia vera, and primary myelofibrosis--are characterized by clonal hematopoiesis, overproduction of mature blood cells, and a high burden of thromboembolic events. Although thrombosis is the leading cause of morbidity and mortality in MPN, the contribution of the vascular endothelium remains incompletely defined. We investigated patient-derived endothelial colony-forming cells (ECFCs) as a surrogate for vascular endothelium in individuals with JAK2 V617F-mutated MPN. MethodsECFCs were cultured from peripheral blood of patients with MPN and healthy controls, phenotyped for thrombo-inflammatory and adhesive markers, tested for JAK2 V617F, and profiled by bulk RNA sequencing. Functional assays assessed endothelial-dependent factor Xa generation. Transcriptomes were benchmarked against public HUVEC reference datasets processed through an identical quantification pipeline. ResultsECFCs were obtained more frequently and in greater numbers from patients with MPN than from controls, indicating enhanced endothelial regenerative or activation potential. MPN ECFCs exhibited increased von Willebrand factor and P-selectin expression and release, along with elevated endothelial cell-dependent factor Xa generation, consistent with a thrombo-inflammatory, procoagulant phenotype. JAK2 V617F was not detected in any ECFC colonies, supporting a non-clonal origin of these endothelial abnormalities. Transcriptomic analysis identified 289 differentially expressed genes in MPN versus control ECFCs, with pathway enrichment revealing coordinated dysregulation of blood coagulation, platelet activation, plasminogen regulation, vascular permeability, extracellular matrix organization, and angiogenesis. Benchmarking against HUVEC datasets confirmed strong endothelial identity of ECFC-derived cells, with MPN-associated changes reflecting endothelial activation rather than loss of endothelialness. ConclusionsECFCs from patients with JAK2-mutated MPN display functional and transcriptomic signatures of endothelial dysfunction in the absence of detectable driver mutations. These findings support a model in which a primed, thrombo-inflammatory endothelium cooperates with clonal hematopoiesis to promote the heightened thrombotic risk characteristic of MPN.

ObjectivePost-thrombotic syndrome (PTS), a common complication of deep vein thrombosis, lacks objective diagnostic biomarkers and its molecular mechanisms remain poorly understood. This study aimed to identify plasma biomarkers and clarify pathways using integrated multi-omics and machine learning. MethodsProteomic and metabolomic profiling of 75 PTS patients and 75 controls was performed. Differential expression analysis, pathway enrichment, and protein-metabolite network analysis were conducted. A multi-algorithm machine learning with 8 feature selection methods prioritized biomarkers. Validations and 14 models were assessed. Results1,104 proteins and 1,891 metabolites were differentially expressed. Citrate cycle and unsaturated fatty acid biosynthesis were enriched. Three proteins, namely DIP2B, KNG1, and SUCLG2, were consistently selected as core biomarkers. All of these proteins were significantly downregulated in PTS and externally validated. A random forest model utilizing these proteins achieved an accuracy of 97.7% in independent testing, with SUCLG2 being the most influential predictor. ConclusionThis study identifies a novel three - protein biomarker panel for the diagnosis of PTS and reveals an immunometabolic axis in the pathogenesis of PTS, which links inflammatory regulation with mitochondrial energy metabolism. These findings provide valuable insights into the development of diagnostic tools and targeted therapeutic approaches.

Thrombotic diseases are the major worldwide cause of cardiovascular death. Platelets prevent blood loss following injury (haemostasis), but inappropriate and excessive platelet activation can lead to thrombosis. Platelet activation must be tightly controlled. Pro-coagulant platelets expose phosphatidylserine (PS), enabling coagulation complex assembly, enhancing thrombin generation and thrombosis. PS is normally restricted to the inner leaflet of the plasma membrane by flippase (aminophospholipid translocase) activity. However, the flippase protein(s) responsible for this crucial activity in platelets remains unidentified. The P4 ATPases ATP11A and ATP11C, regulated by their obligatory partner CDC50A, flip PS at the plasma membrane in a range of different cell types. To investigate platelet flippases, human induced pluripotent stem cells (hiPSCs) were forward-programmed into CD41+/CD42+ megakaryocytes, the platelet precursor. Wildtype (WT) forward-programmed megakaryocytes showed similar flippase activity to human platelets with internalisation of NBD-PS that could be inhibited by high cytosolic Ca2+ or N-ethylmaleimide (NEM). We then generated CDC50A, ATP11A or ATP11C single knockout and ATP11A/11C double knockout (DKO) hiPSCs using CRISPR-Cas9. CDC50A-KO, ATP11A-KO, ATP11C-KO and DKO hiPSC clones successfully formed CD41a+/CD42a+ mature megakaryocytes. CDC50A-KO megakaryocytes bound Annexin V when unstimulated and had no remaining NEM-sensitive flippase activity indicating the involvement of a P4-ATPase. Although ATP11A-KO and ATP11C-KO megakaryocytes had similar flippase activity to WT clones, DKO clones had inhibited NBD-PS internalisation compared to WT and had no remaining NEM-sensitive flippase activity. This indicates that the CDC50A-regulated P4-ATPases ATP11A and ATP11C act together at the megakaryocyte plasma membrane and are responsible for PS flippase activity and therefore likely responsible in human platelets.

PurposeSepsis-associated immunothrombosis significantly contributes to high mortality, yet the role of N-glycosylation in this process remains poorly understood. This study aimed to comprehensively profile the plasma N-glycosylation landscape in sepsis and elucidate how its specific reprogramming in the complement and coagulation cascades influences immunothrombotic balance and patient outcomes. MethodsWe performed in-depth 4D-DIA proteomic and N-glycomic analyses on plasma from 43 sepsis patients and 9 healthy controls. Differential expression, weighted gene co-expression network analysis (WGCNA), and protein-glycosylation correlation analyses were used to characterize molecular features. Clinical relevance was assessed via correlation and survival analyses. ResultsExtensive N-glycosylation reprogramming was observed in sepsis plasma,with marked enrichment in complement and coagulation pathways(KEGG p=7.76x10- {superscript 2}{superscript 1}).Pro-coagulant proteins(eg,vWF,fibrinogen)showed increased abundance together with enhanced site-specific glycosylation,potentially amplifying their activity.In contrast,key anticoagulant proteins(eg,SERPINC1)displayed unchanged glycosylation at critical sites despite abundance changes,which may impair function.Survival analysis revealed distinct prognostic values of glycoproteins and specific glycosylation sites.For instance,high vWF protein levels predicted mortality(HR=2.83),whereas elevated glycosylation at vWF N211 was associated with improved survival(HR=0.135),suggesting a negative regulatory role.These glycosylation markers correlated closely with disease severity and prognosis,representing potential early-warning biomarkers independent of current clinical coagulation indicators. ConclusionOur study demonstrates widespread reprogramming of the plasma proteome and N-glycome in sepsis.We propose that decoupling of protein function from abundance through N-glycosylation in the complement-coagulation network contributes to immunothrombotic imbalance.Specific N-glycosylation sites may serve as novel prognostic biomarkers,offering new perspectives for early risk stratification and glycosylation-targeted therapies in sepsis. Key PointsO_LISepsis plasma exhibits specific N-glycosylation reprogramming overwhelmingly focused on the complement and coagulation cascade. C_LIO_LIA dominant "glycosylation-dominated co-upregulation" mode in procoagulant factors, coupled with a "silent" glycosylation state in key anticoagulants, drives prothrombotic imbalance. C_LIO_LISite-specific N-glycosylation levels provide prognostic information distinct from, and often superior to, their carrier protein abundance, offering novel early-risk biomarkers. C_LI

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.

Von Willebrand factor (VWF) is an essential contributor to hemostasis through its interaction with the platelet glycoprotein (GP) Ib receptor. VWF is cleaved by ADAMTS13 to limit its prothrombotic properties. Failure to do so can result in platelet-VWF complexes that occlude the microcirculation, as seen in thrombotic thrombocytopenic purpura (TTP). In this setting, plasmin becomes active to cleave VWF, forming a distinct plasmin-generated cleavage product of VWF (cVWF) that is detectable during acute attacks in patients with TTP and following therapeutic plasminogen activation in a mouse model of TTP. However, it remains unclear whether plasmin-mediated proteolysis of VWF alone accounts for the breakdown of platelet-VWF complexes. Using ristocetin-induced platelet agglutinations, we show that plasmin cleavage of VWF does not impair its platelet-binding capacity, whereas plasmin-mediated cleavage of GPIb reduces the ability of platelets released from agglutinates to bind VWF. Furthermore, platelets in suspension are relatively resistant to plasmin cleavage. We therefore propose that VWF binding may enhance GPIb cleavage by recruiting plasmin(ogen) to the platelet surface. In a TTP mouse model, plasminogen activation led to a VWF-dependent reduction in GPIb detectability, although to a lesser extent than observed in vitro. In patients with acute TTP, soluble GPIb levels were elevated, indicating increased GPIb shedding during attacks of thrombotic microangiopathy, although the extent to which this is plasmin-mediated remains unclear. Together, our findings demonstrate that plasmin cleavage of GPIb drives the disruption of ristocetin-induced agglutinates, while its contribution to the breakdown of platelet-VWF complexes in vivo appears limited.

AbstractHereditary Hemorrhagic Telangiectasia (HHT) is a genetic vascular disorder characterized by distinct vascular malformations, including deep organ arteriovenous malformations (AVMs) and mucocutaneous telangiectasias. People with HHT inherit monoallelic pathogenic variants in members of the TGF{beta} signaling cascade (ACVRL1, ENG and SMAD4), resulting in a loss of gene function and dysangiogenesis. While these heterozygous inactivating mutations are present in all cells, malformations develop locally, indicating a focal trigger of onset. Indeed, recent human sequencing studies revealed that second-hit somatic mutations, resulting in complete bi-allelic loss of gene function, are linked to lesion formation in the three major types of HHT (HHT1, HHT2, JP/HHT). To model the loss of heterozygosity (LOH) associated with HHT patients, we generated new Eng and Smad4 HHT mouse models whereby endothelial cell-specific, somatic LOH mutations are induced within a heterozygous loss of function background (HHT-iEC-LOH). The HHT-iEC-LOH models recapitulate the mosaic makeup of patient malformations and indicate that multiple, distinct secondary somatic mutations can contribute to AVM onset. Utilizing immunofluorescent staining, blue latex vasculature casting, weighted tracer perfusions, and lineage tracing studies, HHT-iEC-LOH models were phenotypically assessed and compared to traditional inducible endothelial cell knockout (HHT-iECKO) HHT models. Overall, HHT-iEC-LOH mice exhibit increased malformation frequency and vascular phenotypes that are comparable or exceed the severity of iECKO models. Significantly, HHT-iEC-LOH mice can be induced early in development and live into adulthood, displaying persistent cerebrovascular phenotypes. The heightened patient representation offered by these newly developed models enables the study of long-term disease progression and testing of therapeutic interventions.

BackgroundActivated platelets release polyphosphate (polyP), a linear polymer of inorganic phosphate residues, from dense granules. Experiments performed under no-flow conditions show that polyP alters the kinetics of tissue factor (TF) pathway reactions, accelerating FXI activation by thrombin and FV activation by FXa and thrombin, and may impact inhibition by tissue factor pathway inhibitor (TFPI). How polyP influences this pathway in conjunction with platelet deposition under flow remains understudied. ObjectivesTo investigate how polyP-mediated acceleration of FV and FXI activation modulates thrombin generation under flow in TF-initiated coagulation. MethodsWe extended a previously validated mathematical model of platelet deposition and coagulation under flow to examine polyP-mediated effects following a small vascular injury during intravascular clotting. Simulations varied the surface density of TF exposed, wall shear rate, and plasma TFPI concentration. ResultsPolyP shifts the threshold TF density for a thrombin burst to lower TF densities. For TF densities above this threshold, polyP shortens the lag time to thrombin generation in a TF- and shear-rate-dependent manner. Although no explicit effect of polyP on TFPI function was included in the model, thrombin generation was much less sensitive to TFPI concentration with polyP, in a TF-dependent manner. Relative contributions of accelerations of FV and FXI activations depend on incompletely known enhancements by polyP. ConclusionsThe experimentally observed influence of polyP on TFPI function depends on TF density and may arise indirectly from accelerated FV and FXI activation, with the dominant effect arising through accelerated thrombin-mediated conversion of FV to FVa.

BackgroundSickle Cell Anaemia (SCA), a genetic blood disorder caused by a single mutation in the beta globin gene, displays a highly variable clinical course. Hydroxyurea (HU), an effective treatment, has an unclear mechanism of action. Plasma proteins can act as biomarkers for understanding disease states and response to HU treatment in SCA patients. MethodsPlasma proteome profiling of 31 healthy individuals and 76 SCA patients, including those with and without HU treatment, was performed using a high-performance liquid chromatography system and Orbitrap mass spectrometer. Statistical analysis was performed to identify differentially abundant proteins (DAPs) between SCA patients and healthy controls. Subgroup analyses were performed to look at the impact of HU treatment on plasma proteome. ResultsOur analysis yielded 43 DAPs in the plasma of SCA patients. Global correlation and protein-protein network analysis revealed that these proteins are part of a robust interaction network. Proteins showing higher abundance (LBP, ORM1 and TFRC) were primarily associated with immune response whereas those with reduced abundance (FBLN1 and F13B) were linked to blood coagulation and proteolysis. Differential abundance of several proteins such as CD14, FCN3, LFALS3BP, LAP and TGFBI was observed in either male or female patients indicating influence of gender. Importantly, HU treatment was associated with elevated levels of haptoglobin (HP) and hemopexin (HPX), key proteins involved in free hemoglobin scavenging. Notably, DAPs such as F10, LPA, and FCN3 overlapped with proteins previously reported to be differentially abundant in beta-thalassemia patients. Moreover, multiple proteins, including APOL1, AZGP1, FBLN1, GPLD1, HPX, LGALS3BP, and TFRC correlated with clinical parameters, such as blood transfusion frequency and, vaso-occlusive crisis, and WBC and platelet counts. ConclusionsThis study identifies novel differentially abundant plasma proteins in SCA, expanding the current repertoire of disease-associated biomarkers and proteins modulated by hydroxyurea therapy. The observed overlap with beta-thalassemia associated signatures reinforces shared pathophysiological mechanisms between these hemoglobinopathies. Several of these proteins show significant correlations with key clinical parameters and disease complications, offering insights into disease mechanisms and potential utility in disease management. Collectively, these findings provide a strong foundation for translational validation in larger, independent cohorts.

Intracerebral haemorrhage (ICH) is a severe form of stroke with high morbidity and mortality rates. For survivors, acute haematoma expansion strongly determines neurological outcome. Although blood pressure reduction is widely investigated as a strategy to limit haematoma growth, the haemodynamic mechanisms regulating haemorrhage development remain poorly understood. Zebrafish provide a tractable in vivo model to study cerebrovascular biology and spontaneous ICH, yet the contribution of vascular regulation to haemorrhage onset and expansion has not been explored in this species. Here, we investigated whether pharmacological modulation of vascular dilation influences ICH development in zebrafish larvae. We first characterised vascular changes during the developmental window in which spontaneous ICH occurs and observed increased heart rate and progressive reductions in arterial diameter between 2 and 3 days post-fertilisation, suggesting increased vascular resistance. We then tested whether vasoconstriction promotes haemorrhage using angiotensin II, which induced systemic and cerebrovascular vasoconstriction but did not increase ICH incidence or haematoma size in two independent ICH models. In contrast, pharmacological vasodilation using sodium nitroprusside or isoproterenol significantly reduced haematoma size in a high-incidence model of atorvastatin-induced ICH. Live imaging of cerebral blood flow revealed that vasodilation was associated with the confinement of red blood cells around affected vessels rather than dispersing into the brain ventricles. Together, these findings indicate that vascular dilation modulates haemorrhage progression in zebrafish ICH and establish this model as a platform to investigate haemodynamic mechanisms regulating haematoma expansion.

BackgroundEndovascular thrombectomy (EVT) has transformed the treatment of acute ischemic stroke (AIS). However, a substantial proportion of AIS patients experience poor outcomes despite successful recanalization, often due to severe neurological deterioration or life-threatening complications. Early identification of these high-risk patients remains a major unmet need. In this study, we developed and validated machine-learning (ML) models that integrate automated quantitative cerebrovascular morphology and collateral grading with demographic, clinical, laboratory, and imaging variables to predict major post-EVT complications and early neurological outcomes. MethodsUsing a prospectively collected database of 727 AIS patients that underwent EVT, we developed ML models to incorporate patient-specific vascular morphometry with conventional clinical, laboratory, and imaging data to predict emergence of early neurological deterioration (END), symptomatic intracranial hemorrhage (sICH), malignant brain edema (MBE) requiring surgical decompression, and neurogenic respiratory failure and dysphagia requiring tracheostomy/gastrostomy (TC/PEG). ResultsOur analysis of morphological features, including increased tortuosity and reduced vessel diameter, showed strong associations with complications. Morphology-informed (MI) models consistently outperformed baseline-clinical (BC) models for patients with END (AUROC 0.81 for MI model vs. 0.73 for BC), sICH (AUROC 0.68 MI vs. 0.56 BC model), MBE (AUROC 0.67 MI model vs. 0.56 BC), or patients who underwent TC/PEG (AUROC 0.66MI vs. 0.58 BC model). Statistical testing confirmed significant AUROC improvements for END, sICH and mRS (p < 0.05), Finally, patient-specific calibrated probability profiles enabled individualized, multidimensional risk stratification, revealing distinct complication-specific risk patterns across patients. ConclusionsThese findings demonstrate that cerebrovascular structure--an often overlooked yet physiologically fundamental determinant of ischemic injury and reperfusion dynamics--provides significant predictive information that is not captured by standard clinical or visual imaging assessments. Automated vascular segmentation and collateral grading techniques enable rapid and objective integration of cerebrovascular metrics into prognostic models, offering a scalable tool for precision risk stratification, supporting earlier intervention, targeted monitoring, and improved post-EVT management.

Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) is a lifesaving intervention used to manage non-compressible torso hemorrhage by temporarily occluding the aorta to minimize blood loss and preserve perfusion to vital organs. Partial REBOA (p-REBOA) has been proposed to mitigate ischemic injury associated with full-REBOA (f-REBOA). However, implementation of p-REBOA clinically has been challenging due to our limited understanding of the acute hemodynamics with p-REBOA particularly in relation to cardiac, carotid, and renal perfusion. In this study we developed and utilized a novel porcine model to continuously measure cardiac, carotid, renal and systemic hemodynamic responses to varying degrees of hemorrhagic shock and aortic occlusion. Yorkshire pigs (N=54) underwent instrumentation for continuous hemodynamic monitoring and hemorrhage was induced for 30 minutes to achieve 10%, 20%, or 30% blood volume loss (n=18/group), followed by randomized treatments of either no occlusion, p-REBOA, or f-REBOA occlusion strategies (n=6/group) for 30 minutes. After occlusion, shed blood was re-transfused over 15 minutes, and REBOA balloons were deflated and removed. This was followed by a 3-hour automated resuscitation and critical care period. Renal and carotid perfusion decreased progressively with hemorrhage severity. Interestingly, 30 minutes of f-REBOA resulted in significant ischemia-reperfusion injury where renal perfusion was profoundly suppressed to 40% of baseline renal flow. On the other hand, p-REBOA yielded superior renal perfusion, while maintaining cardiac function and carotid perfusion. p-REBOA also required less fluid and vasopressors. This translational pig model offers new opportunities to assess acute cardiovascular hemodynamics during interventions for the management of hemorrhagic shock.

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.

Rationale: Fibrinolysis resistance in sepsis associates with thrombotic burden, multi-organ failure and death. The degrees and dynamics of resistance that associate with mortality in acute sepsis are unknown, and a simple tool to aid clinician interpretation of fibrinolysis measurements is lacking. Objectives: To establish a point of care grading tool of fibrinolysis resistance that aligns with scoring systems for disease acuity, is substantiated by plasma fibrinolysis markers and enables rapid investigation of the fibrinolysis state at the point of care. Methods: Prospective observational study of 116 adult sepsis/septic shock patients with sequential measurements of fibrinolysis resistance during Intensive Care Unit (ICU) admission using tissue plasminogen activator (tPA) enhanced viscoelastic testing (VET). The clot lysis time (TPA-LT) adjusted for fibrin clot amplitude (TPA-LT/FIBA10, sec/mm) underwent cluster analysis and was evaluated against disease severity scores, standard pathology, clinical outcomes and fibrinolysis markers. Measurements and Main Results: Three clusters of progressively increasing fibrinolysis resistance were identified (Grades 1-3). At admission, Grade 3 associated with the highest disease severity, organ failure, haematological and biochemical perturbations, fibrinolysis marker inhibitory profile and mortality (42% versus 24% and 15% in Grade 2 and Grade 1, respectively) with a 3.9-fold [95% CI 1.4-11] increased hazard ratio for death at 28 days compared to Grade 1. Transitions between grades were frequent over 7 days with a reduced Grade associated with decreased risk of death. Conclusions: Grading of fibrinolysis resistance in sepsis enables rapid identification of patients at greatest mortality risk with any dynamic improvement corresponding to favourable clinical outcomes.

BackgroundCompetitive transplantation is essential for defining intrinsic repopulating capacity of murine hematopoietic stem and progenitor cells (HSPCs), yet comparable assays for human cells have been limited by the lack of a robust in vivo platform. MethodsHere, we describe a novel competitive transplantation method in humanized NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice that enables simultaneous engraftment and longitudinal tracking of distinct human grafts within a shared microenvironment. ResultsUsing human leukocyte antigen-mismatched donor CD34+ cells, this method facilitates standard flow cytometry panels to track multiple donor cell chimerism, lineage output, and HSPC composition. The experimental framework may be adapted to different mouse models, conditioning strategies, donor sources, and treatments. ConclusionsOverall, this humanized competitive repopulation assay fills a critical translational gap and offers a flexible foundation for advancing mechanistic discovery in human hematopoietic biology and improving clinical strategies for stem cell transplantation.