Journal of Thrombosis and Haemostasis最新文献

Beta-2-Glycoprotein I (β2GPI) is the main autoantigenic target of antiphospholipid syndrome (APS) with antibodies leading to clinical manifestations. There are two known structural isomers of β2GPI, a J shape and a circular shaped one. The transition between these structures is incompletely understood, with the functional implications unknown. β2GPI is a substrate of the protease plasmin, which cleaves within the fifth domain of β2GPI leading to altered cellular binding. Very little is currently known regarding the structure and function of this protein variant. We present the first comprehensive structural characterisation plasmin-clipped β2GPI and the associated implications for pathogenic antibody binding to this protein.

Methods: β2GPI was purified using an adapted acid-free process from healthy control plasma and cleaved with plasmin. Cleavage was confirmed by SDS-PAGE. Structural characterisation was undertaken using dynamic light scattering (DLS), small angle X-ray scattering (SAXS), ion mobility mass spectrometry (IMMS) and molecular dynamics simulation (MD). Activity was tested using inhibition of β2GPI ELISAs with patient samples and cleaved β2GPI in the fluid phase and cellular binding by flow cytometry using HUVEC cells.

Results: DLS revealed a significantly smaller hydrodynamic radius for plasmin-clipped β2GPI (p=0.0043). SAXS and MD analysis indicated a novel S-like structure of β2GPI only present in the plasmin-clipped sample whilst IMMS showed a different structure distributions in plasmin clipped compared to non-clipped B2GPI. The increased binding of autoantibodies was shown for plasmin-clipped β2GPI (p=0.056), implying a greater exposure of pathogenic epitopes following cleavage.

Conclusions: Cleavage of β2GPI by plasmin results in the production of a unique S-shaped structural conformation and higher patient antibody binding. This novel structure may increase the production of antibodies and explain the loss of binding to phospholipids described previously for plasmin-clipped β2GPI.

Background: Although acute minor stroke often presents with mild symptoms, such as unilateral limb weakness, mild aphasia, dizziness or mild cognitive impairment, untreated outcomes could be poor, and optimal treatment methods are still debated. We aimed to identify the optimum treatment for minor strokes with a network meta-analysis.

Method: Studies from Embase, Ovid, and Cochrane Library were considered. Randomized controlled trials and prospective cohort studies with ischemic stroke with a National Institutes of Health Stroke Scale (NIHSS) no more than five, explicit intravenous thrombolysis or antiplatelet therapy were included. Efficacy outcome was measured by three-month modified Rankin scale (mRS), with primary outcome defined as mRS 0-1 and secondary outcome as mRS 0-2. Safety outcomes included symptomatic intracranial hemorrhage (sICH) and mortality at three months.

Findings: Nine studies encompassing 10,665 patients were meta-analyzed. Aspirin plus clopidogrel (n=4,283) was more strongly associated with primary outcome than aspirin (n=2,128, OR 1.26, 95%CI 1.04-1.54) and rt-PA (n=1,840, OR 1.23, 95%CI 1.00-1.50). Aspirin plus clopidogrel (n=3,933) also had a lower sICH risk than rt-PA (n=2,538, OR 0.11, 95%CI 0.04-0.30) and tenecteplase (n=194, OR 0.15, 95%CI 0.03-0.68), as well as a lower mortality than aspirin alone (n=830, OR 0.27, 95%CI 0.10-0.71). Patients treated with aspirin (n=815) also had a lower sICH risk than rt-PA (n=2538, OR 0.20, 95%CI 0.04-0.95).

Discussion and conclusion: Dual antiplatelet therapy based on aspirin and clopidogrel offers balanced efficacy and safety, positioning it as a potentially optimal treatment for minor stroke. rt-PA showed comparable efficacy, while its associated risks were more pronounced.

Background: Venous stasis, which can occur with prolonged sedentary behavior (SB), is associated with venous thromboembolism (VTE) risk, but VTE risk associated with accelerometer-measured SB has not been quantified.

Objectives: To evaluate accelerometer-based measures of SB in relation to incident VTE.

Methods: We included 5,591 participants, aged 63-99 years, of the Women's Health Initiative Objective Physical Activity and Cardiovascular Health cohort study without prior VTE. Between May 2012-2014, participants wore the ActiGraph GT3X+ accelerometer at the hip for 7 days. Three SB measures were classified using the Convolutional Neural Network Hip Accelerometer Posture algorithm: total sitting time, mean sitting bout duration, and total time spent in prolonged (≥30 minute) sitting bouts. VTE events were centrally adjudicated. Multivariable-adjusted Cox models estimated hazard ratios (HRs) for each SB and VTE risk. Women were censored at first VTE, death, loss to follow-up, or February 2023. Mediation by body mass index (BMI) was evaluated.

Results: Over a mean follow-up of 8.2 years, 229 women experienced a VTE. In adjusted models, longer mean sitting bout duration was associated with greater incident VTE risk (HR per 5-minute increase=1.15; 95% CI: 1.04, 1.28). BMI mediated approximately 30% of this association (p<0.01). We found no significant evidence that total sitting time or total time spent in prolonged sitting bouts were associated with VTE.

Conclusion: Longer mean sitting bout duration was associated with greater VTE risk, with substantial mediation by BMI. Behavioral efforts to reduce sedentary bout length in older women may reduce their VTE risk.

Background: Clinically significant extracranial bleeding, defined as bleeding at any site other than the brain or spinal cord requiring either a hospital admission >24 hours, red blood cell transfusion, surgery for hemostasis, or resulting in death, is a common side effect of antithrombotic agents. Compared to intracranial bleeding, the impact of clinically significant extracranial bleeding on long-term outcomes, including functional independence, has been poorly studied.

Objective: To determine if clinically significant extracranial bleeding impacts the development of functional disability in healthy older adults.

Methods: We performed a secondary analysis of the Aspirin in Reducing Events in the Elderly (ASPREE) trial. The primary outcome of this study was incident dependence in the Katz Activities of Daily Living (ADL), defined as being unable to perform or requiring assistance with any ADLs or being admitted to a long-term care facility.

Results: 18,982 participants were included in the analysis, of which 547 (2.9%) developed clinically significant extracranial bleeding during study follow-up. In adjusted analyses, clinically significant extracranial bleeding was significantly associated with the development of incident ADL dependence (HR 2.46, 95% CI 1.97-3.07). This finding was similar for gastrointestinal (HR 2.29, 95% CI 1.72-2.08) and non-gastrointestinal extracranial bleeds (HR 2.68, 95% CI 1.96-3.69). The association with increased risk of incident ADL dependence remained significant in secondary analysis of groups randomized to either aspirin (HR 2.15, 95% CI 1.57-2.94) or placebo (HR 2.84, 95% CI 2.09-3.86).

Conclusion: Clinically significant extracranial bleeding was associated with the development of incident ADL dependence in otherwise healthy older adults.

Unfractionated heparin (UFH) remains the anticoagulant of choice in critically ill patients. However, its laboratory monitoring and clinical management are particularly challenging. Between November 2023 and February 2024, we surveyed 142 clinicians and laboratory medicine specialists from 15 countries involved in the care of patients receiving therapeutic-intensity UFH. Our objective was to describe current practices and variations among centers. UFH monitoring was based on an anti-Xa assay or on activated partial thromboplastin time in 54% and 46% of respondents, respectively. Different therapeutic ranges were used depending on local protocols and indications; the 0.3-0.7 IU/mL anti-Xa range was commonly used, except for patients on mechanical circulatory support with a lower range, mostly 0.3-0.5 IU/mL. Most respondents managed therapeutic UFH administration with weight-based dosing (88%), while fewer used a nomogram (57%) for dose adjustment. When a nomogram was used, it was primarily based on anti-Xa monitoring (86%). The situations when respondents administered antithrombin varied widely; 22% reported using it when antithrombin levels were below 60IU/dL(%) and 20% reported never using it. Our survey results revealed considerable heterogeneity in UFH management approaches, reflecting a knowledge gap and a paucity of evidence to guide decision-making. Key issues requiring well-designed up-to-date studies were identified that include optimal approaches to heparin monitoring, assays and reagents to be used, therapeutic range based on indications, the use of weight-adjusted nomograms for initial dosing and titrating of UFH infusion, and indications for antithrombin supplementation. Survey results provide a strong rationale for the development of international guidance addressing these issues.

Background: Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin, associated with increased risk of thromboembolic complications. Intravenous immunoglobulins (IVIG) have been used as a therapeutic for HIT and are believed to alleviate thrombocytopenia and reduce thrombosis risk. Yet the anti-thrombotic effects of IVIG in HIT remain underexplored.

Objective: To investigate the effect of IVIG on thrombus formation in an ex vivo model of HIT-IgG-induced thrombosis.

Methods: Microfluidic channels were coated with a confluent monolayer of human umbilical vein endothelial cells (HUVECs) that were primed with TNF-α to induce an activated, inflammatory state. Whole blood was exposed to unfractionated heparin, with or without IVIG before subjecting to treatment with a monoclonal HIT-like antibody (K070), or HIT-patient-IgG. Recalcified blood was perfused over HUVECs at a venous shear stress. Thrombus structure and dynamics were investigated by immunofluorescence microscopy.

Results: HIT-patient-IgGs and K070 induced thrombus formation in the presence of prophylactic heparin exposure, over TNF-α-treated, inflamed endothelial cells. HIT thrombi were enriched in fibrin, phosphatidylserine-bearing platelets, and leukocyte aggregates. We observed thrombi being formed on adherent platelets, which gradually recruited leukocytes into a three-dimensional thrombus structure. Pre-treatment of blood with IVIG significantly reduced cellular adhesions and prevented thrombus formation.

Conclusions: Our endothelialized ex vivo flow chamber system effectively recapitulates the immunothrombotic phenotype of HIT and offers a reliable tool to urgently validate the efficacy of IVIG intervention against HIT-IgG-induced thrombosis in patients.

Background: Damaging STAB2 gene variants are associated with increased venous thromboembolic risk. STAB2 encodes stabilin-2, a clearance receptor, expressed by the liver and spleen. Given its function, it's likely that the prothrombotic state associated with stabilin-2 deficiency is due to reduced procoagulant protein clearance, but the identity of these ligands is unknown.

Objectives: Identify plasma stabilin-2 ligands using proximity biotinylation proteomics.

Methods: Cells stably expressing stabilin-2-TurboID were incubated with human plasma and biotin to initiate TurboID labeling of plasma ligands in endocytic vesicles. Biotinylated proteins were purified and identified using mass spectrometry. Candidate plasma ligands with roles in hemostasis were fluorescently labeled and incubated with stabilin-2 expressing and control cells. Flow cytometry assessed ligand surface binding and confocal microcopy assessed colocalization with stabilin-2 and lysosomes. Furthermore, plasma levels of ligands were measured in Stab2 deficient mice and littermate controls.

Results: Twenty-eight stabilin-2 specific ligands were identified. Interactions with von Willebrand factor (VWF), fibrinogen, pro(thrombin), heparin cofactor II (HCII), high molecular weight kininogen (HMWK), plasminogen and C4b binding protein (C4bp) were probed. HCII, HMWK, plasminogen and fibrinogen showed binding to stabilin-2 using flow cytometry (>2-fold higher than controls). Confocal microscopy demonstrated stabilin-2 dependent colocalization of all ligands with lysosomes. In Stab2 deficient mice, ligand levels were not significantly increased, suggesting in mice stabilin-2 is not their main clearance receptor.

Conclusion: These results confirm the value of proximity labeling proteomics in identifying receptor ligands and suggest damaging STAB2 variants may increase venous thromboembolic risk potentially through altered hemostatic protein clearance.

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) presents challenges due to its complex pathobiology. Although numerous studies have reported heterogeneous cell types by single-cell RNA sequencing, the atlas and characteristics of plasma cells remain poorly understood.

Objectives: To identify the altered phenotype and differentiation patterns of plasma cells in CTEPH.

Methods: We performed single-cell RNA sequencing on pulmonary endarterectomy tissue from 5 patients and 6 normal pulmonary arteries. Serum immunoglobulins (Igs) were measured using protein electrophoresis among 273 CTEPH patients, 259 idiopathic pulmonary arterial hypertension (IPAH) patients, and 251 healthy controls.

Results: The percentage of plasma cells was significantly increased from less than 1% in healthy controls to 15% in CTEPH patients. We identified 1 B cell cluster and 5 distinct mature plasma cell clusters, including IGHG1, HSPA1A, AHNAK, IGLC3, and IGKV4. Notably, the AHNAK and IGLC3 subclusters are newly identified. GeneSwitches analysis indicated early activation of IGHG1 and early deactivation of HLA-DPA1. The trajectory of AHNAK cluster was earlier than that of IGLC3 cluster, with an enrichment for pathways responsive to lipopolysaccharide. The IGLC3 cluster revealed lower differentiation potential and was predominantly associated with Ig production. Furthermore, Igα2 levels in CTEPH patients were lower than in controls but higher than in IPAH patients. Significantly, Igγ levels were markedly elevated in CTEPH patients compared with IPAH patients and controls, better distinguishing CTEPH patients from controls and IPAH patients.

Conclusion: Plasma cells of CTEPH had a distinctive landscape and heterogeneity. The newly identified clusters represented excessive Ig production but lacked immune response function. These findings highlight that targeted plasma cells can be used to develop novel CTEPH treatments.

Background: The impact of central localization of pulmonary embolism (PE) on clinical outcomes is uncertain.

Objectives: To compare clinical presentation, risk factors, and outcomes between patients with central pulmonary embolism (cPE) and non-cPE.

Methods: We retrospectively analyzed 597 patients with acute PE from the prospective SWITCO65+ cohort between 09/2009-12/2013. cPE was defined as an embolus in the pulmonary trunk or the left or right pulmonary artery. We compared baseline clinical characteristics and outcomes at 3 months (recurrent venous thromboembolism [VTE], overall/PE-related mortality, PE-related quality of life) and over the entire follow-up (recurrent VTE, overall/PE-related mortality) between patients with cPE vs. non-cPE. We examined the association between PE localization and recurrent VTE and overall mortality, adjusting for multiple confounders including thrombolysis and periods of anticoagulation, and competing risk of non-VTE-related death if appropriate.

Results: Overall, 217 (36.3%) patients had cPE. Symptoms/signs of respiratory distress, right-ventricular dysfunction, and myocardial injury were more prevalent in those with cPE. VTE recurrence, overall/PE-related mortality, and PE-related quality of life at 3 months did not vary by PE localization. After a median follow-up of 29.6 months, patients with cPE had a higher risk of fatal PE (5.5% vs. 2.1%; P=0.033). After adjustment, cPE was associated with recurrent VTE (SHR 2.22, 95%CI 1.25-3.91) but not with overall mortality (HR 0.74, 95%CI 0.45-1.21) during follow-up.

Conclusion: cPE was associated with a 2.2-fold increased risk of recurrent VTE compared to non-cPE. Whether an extended anticoagulation duration could reduce the recurrence risk following cPE should be further examined.