Journal of Thrombosis and Haemostasis最新文献

Background: Neutrophil extracellular traps (NETs) are important in host defense but implicated in pathological thrombosis in acute ischemic stroke (AIS). NET quantification is typically done with immunostaining for NET-specific markers, but molecular techniques like mass spectrometry can provide another angle into thrombo-physiology.

Objectives: This study leverages both histological analysis of NETs, and molecular insights from proteomics, to validate using proteomics for evaluating stroke thrombus NET burden.

Patients/methods: Sections of 30 AIS thrombi were stained for myeloperoxidase (MPO), citrullinated histone H3 (H3Cit), and DNA. ImageJ measured average staining area. Mass spectrometry of sample thrombi fragments detected 5,784 proteins. Spearman correlations and Mann Whitney U-tests compared NET burden from staining to proteomic composition. Dimensional reduction of H3Cit and MPO staining created a single NET marker variable (PC1), used to compare thrombus composition between thrombi with NET marker greater (NET-rich) or lesser (NET-poor) than median.

Results: Percent coverage for the three stains was interrelated (Spearman's ρ > 0.6, p < 5E-4 for all). 531 neutrophil degranulation proteins were positively associated with all stains. PC1 explained ∼92% of the variance. Ubiquitin-proteasome degradation pathways were negatively correlated with the NET markers (q-val < 0.05 for all relevant pathways). Older patient age (median, IQR: 79, 73-85 vs 61, 57-73; Mann Whitney p = 0.03) and atrial fibrillation-associated stroke (Fisher's Exact p = 0.03) thrombi were NET-rich.

Conclusions: This study marries immunostaining with proteomics for quantifying NET burden, pointing to molecular phenotypes within NET-rich and NET-poor thrombi that may underpin NET formation and maintenance in stroke thrombi.

Background: Renal ischemia-reperfusion injury (IRI) contributes to acute kidney injury (AKI) by inducing oxidative stress, inflammation, and thromboinflammatory responses. Conventional heparins mitigate IRI, but their use is limited by bleeding and thrombocytopenia.

Objectives: To evaluate octaparin, a structurally defined synthetic sulfated octasaccharide with anticoagulant activity and minimal bleeding risk, as a novel therapeutic for IRI.

Methods: Murine renal IRI models and hypoxia/reoxygenation (H/R)-injured renal tubular epithelial cells (TCMK-1) were treated with octaparin. Renal function, histopathological changes, fibrosis, apoptosis, oxidative stress, inflammation, and coagulation parameters were systematically evaluated. DC-SIGN engagement and NF-κB signaling were analyzed through molecular docking, receptor blockade experiments, and immunoblotting.

Results: Octaparin significantly improved renal function in vivo, attenuated tubular necrosis and fibrosis, and suppressed apoptosis. It also markedly reduced oxidative stress, restored antioxidant enzyme activity, and downregulated pro-inflammatory cytokines, with reduced neutrophil infiltration. In terms of coagulation, octaparin inhibited tissue factor expression and fibrin deposition, alleviating thromboinflammatory responses. In vitro, octaparin similarly suppressed excessive ROS bursts, restored antioxidant defenses, reduced inflammatory cytokine release, and inhibited apoptosis in TCMK-1 cells. Mechanistically, octaparin bound to the DC-SIGN receptor on renal epithelial cells, inhibiting NF-κB phosphorylation and nuclear translocation, thereby disrupting ROS-driven inflammatory cascades-via a pathway distinct from classical heparin anticoagulation.

Conclusion: Octaparin exerts multifaceted renoprotective effects in IRI by modulating DC-SIGN/NF-κB signaling. Its ability to simultaneously suppress thromboinflammation, oxidative stress, and apoptosis-without the risks associated with conventional heparins-represents a transformative therapeutic strategy for AKI.

Background: The incidence of cancer-associated venous thromboembolism (CAT) has risen in recent decades, underscoring the need for novel risk biomarkers and mechanistic insights. MicroRNAs (miRNAs) have emerged as promising biomarkers for various diseases, with elevated miRNA-145-5p (miR-145-5p) levels linked to reduced venous thromboembolism (VTE) risk. We aimed to investigate the association between plasma miR-145-5p levels and risk of future VTE related to (CAT) and unrelated to cancer (non-CAT).

Methods: Plasma miR-145-5p were measured in a case-cohort derived from the Trøndelag Health Study (HUNT3; n=50,807). The study included 455 VTEs (95 CATs), occurring during 9 years of follow-up, and 1,740 randomly sampled age-weighted subcohort participants. VTEs were classified as CAT if they occurred within 1 year before or 2 years after a cancer diagnosis. Multivariable-adjusted hazard ratios (HRs) for CAT and non-CAT were estimated using weighted Cox regression, with cancer modeled as a time-varying covariate.

Results: Increasing miR-145-5p levels were associated with lower risk of CAT (HR per 1 standard deviation: 0.70, 95% CI: 0.52-0.96) and non-CAT (HR: 0.81, 95% CI: 0.72-0.91). Individuals in the highest miR-145-5p quartile had lower risk of both CAT (HR: 0.55, 95% CI: 0.25-1.20) and non-CAT (HR: 0.48, 95% CI: 0.33-0.69) compared to those in the lowest quartile.

Conclusions: High plasma miR-145-5p levels were associated with lower risk of CAT and non-CAT, suggesting that miR-145-5p has the potential to serve as a risk biomarker for CAT.

Background: Human platelets express 2 thrombin-activated G protein-coupled receptors (GPCRs), protease activated receptor (PAR)1 and PAR4. Previous studies have demonstrated PAR1 activation to be fast on-fast off while PAR4 activation was slower but more sustained. However, how PARs regulate second messengers such as cyclic adenosine monophosphate (cAMP), a negative regulator of platelet activation, remain unknown.

Objectives: This study investigated how PAR1 and PAR4 activation by thrombin regulates cAMP levels and platelet activation.

Methods: PAR1 and PAR4 on washed human platelets were inhibited with vorapaxar or BMS-986120, respectively, in the presence or absence of prostaglandin (PG)I₂. Platelets were stimulated with various thrombin concentrations, and calcium flux and platelet aggregation were measured. PAR regulation of cAMP in megakaryocytes (MKs) was also studied by genetic disruption of PAR1 or PAR4 using CRISPR/Cas9 editing.

Results: In the presence of PGI₂, cAMP was significantly reduced after thrombin stimulation when platelets or MKs were activated through PAR1, but not PAR4. Calcium flux was highly associated with cAMP level changes. High concentrations of cAMP were associated with low calcium flux with PAR4 stimulation. In contrast, stimulating PAR1 decreased cAMP levels, and this was associated with a more pronounced calcium flux. Phosphodiesterase (PDE)3A is involved in regulating cAMP levels. Blocking PDE3A with a pharmacological inhibitor in platelets or genetic knockout in MKs reversed PAR1-induced decrease of cAMP level, while PAR4 was not affected by PDE3A inhibition.

Conclusion: The data suggest differences between PAR1 and PAR4 induced thrombin activation are due, in part, to cAMP regulation by PDE3A.

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare blood disorder that leads to microvessel clots and brain injury. While modern treatments have improved survival during initial disease onset, long-term complications such as cognitive decline are now a prominent concern for reasons that are not yet fully understood.

Objectives: This prospective study aimed to investigate the effects of iTTP on the brain in the post-remission phase to address a critical gap in our understanding of iTTP-brain pathophysiology.

Patients/methods: We followed 22 iTTP survivors over one year. Participants underwent advanced brain MRI to assess white matter integrity, CT-perfusion to evaluate cerebral blood perfusion and blood-brain barrier (BBB) integrity, and cognitive testing.

Results: At baseline, most patients (82%) showed brain abnormalities on MRI, and all presented with compromised BBB integrity (mean permeability surface product: 0.43±0.13 mL/min/100 g). After 12 months, BBB function improved (0.37±0.10 mL/min/100 g, p = 0.04) but remained compromised. Over this period, MRI revealed progressive brain volume loss and diffusion imaging showed reduced white matter integrity in tracts related to memory and verbal processing. Cognitive scores remained lower than those of controls, especially in the memory and verbal domains.

Conclusions: These findings suggest that brain injury and cognitive dysfunction in iTTP patients are not limited to the acute phase but continue to progress after remission. This progression may be underpinned by persistent vascular injury, including BBB dysfunction and reduced perfusion. Long-term brain monitoring and early neuroprotective strategies may be needed to improve the quality of life of iTTP survivors.

Background: Rapid and accurate coagulation analysis is essential for managing critical care and surgical patients, patients with coagulation disorders, and cardiovascular patients on anticoagulant therapy. Traditional assays often require large sample volumes, posing iatrogenic risks in vulnerable populations, including pediatric and elderly patients. Integrated quasi-static acoustic tweezing thromboelastometry (i-QATT^TM) offers a comprehensive, non-contact assessment of coagulation using a 4-6 microliter drop of blood that minimizes diagnostic errors associated with sample-container surface interactions.

Objectives: This study aims to assess the preliminary clinical utility of the i-QATT^TM technique.

Methods: Using blood samples from healthy volunteers and liver transplant patients as well as control plasmas, we established the reference ranges for i-QATT^TM coagulation parameters and validated them across several abnormal conditions, including single-factor deficiencies, abnormal fibrinogen levels, unfractionated heparin (UFH) anticoagulation, and coagulopathy.

Results: i-QATT^TM identified coagulation abnormalities within 3-8 minutes. Its parameters showed strong correlations with gold-standard assay data, e.g., aPTT (r ≥ 0.81), INR (r ≥ 0.74), TEG CK channel parameters (R, K, α, MA; r ≥ 0.65), TEG CRT (MA, r = 0.64), and TEG CFF (MA, r ≥ 0.64). i-QATT^TM data demonstrated a near-linear response to UFH concentrations spanning the prophylactic and therapeutic ranges (0-1 IU/mL, R² = 0.98), and the anticoagulation reversal by heparinase was reliably detected. i-QATT^TM accurately assessed the functional level of fibrinogen and platelet activity in normal and abnormal blood samples.

Conclusions: This study positions i-QATT^TM as a highly sensitive, rapid, and ultra-low-volume alternative to conventional coagulation assays, with strong potential to transform point-of-care diagnostics across a wide range of clinical settings.

Introduction: Sphingosine-1-phosphate (S1P) is a bioactive signalling sphingolipid secreted by platelets during activation. Platelets express distinct S1P receptors on their surface, and a comprehensive understanding of their effects is yet to be achieved. Here, we describe the regulation of fibrinogen binding in activated platelets via S1P receptor type 4 (S1PR₄).

Methods: Thrombus formation and platelet function in an S1pr₄^-/- background were assessed in an ex vivo flow chamber setting and by rotational thromboelastometry. In vivo coagulation was studied in a murine model of polymicrobial abdominal sepsis. Fibrinogen binding was assessed by FACS in the murine and human system.

Results: In the Colon Ascendens Stent Peritonitis (CASP) model, an increased incidence of disseminated intravascular coagulation (DIC) was observed in S1PR4-deficient mice. Murine platelets from S1PR4-deficient mice showed excessive fibrinogen binding and disorganised thrombus formation compared to wild-type platelets. Rotational thromboelastometry confirmed this result, showing a shortened clotting time and an increased clot size in S1PR₄-deficient mice. Flow cytometric analysis of glycoprotein IIb/IIIa (GPIIb/IIIa) expression implicated an increased low- to-high-affinity switch for increased fibrinogen binding in S1PR₄-deficient platelets. Analysis of human platelets using a specific antagonist showed that S1PR₄ signalling impacted fibrinogen binding.

Conclusion: These findings strongly suggest that S1P signalling via S1PR₄ acts as a negative regulator of fibrinogen binding in activated platelets.

Background: Emerging evidence suggests that platelet activation and aggregation are common factors in both metabolic syndrome (MetS) and severe COVID-19, emphasizing the need to investigate their biological connection.

Objectives: We hypothesized that enhanced platelet aggregation mediates the association between MetS and severe COVID-19. This study aimed to determine whether platelet aggregation serves as a mechanistic link between MetS and COVID-19 severity and to develop an image-based biomarker capable of predicting severe disease.

Methods: We conducted massive image-based profiling of circulating platelets in a retrospective cohort of 327 COVID-19 patients (63.9% male; median age, 59.0 years) with metabolic records. Morphologic features of platelets, including shape, density and radial distribution, and texture, were extracted. A machine learning model was developed to construct the COVID-19 Platelet Aggregate Formation Index (CoPAFI), which could quantitatively reflect platelet aggregation and predict severe COVID-19. Mediation analysis was then performed to quantify the extent to which platelet aggregation mediated the association between components of MetS and severe COVID-19.

Results: The CoPAFI predicted severe COVID-19 with an area under the curve of 0.82 and an odds ratio of 3.76 (95% CI, 2.63-5.38). The CoPAFI was strongly associated with a hypercoagulable state and served as a reliable indicator for assessing the risk of severe COVID-19. In patients with components of MetS, platelet aggregation, as measured by the CoPAFI, accounted for approximately 25% of the increased severity of COVID-19.

Conclusion: Enhanced platelet aggregation partially mediates the impact of components of MetS on COVID-19 severity, accounting for approximately 25% of the association, emphasizing the need for integrated metabolic and coagulation management in COVID-19 treatment.