Thrombosis and haemostasis最新文献

Hereditary antithrombin (AT) deficiency is a rare autosomal dominant disorder that predisposes to the development of recurrent venous thromboembolism (VTE). Diagnosis is based on the measurement of reduced AT activity in plasma. However, not all commercial AT activity assays are equally suited for diagnosis since they differ in sensitivity toward different AT mutations.The aim of this study was to compare the ability of commonly used AT activity assays to diagnose inherited antithrombin deficiency, with a focus on type II AT deficiencies. We used samples from genetically confirmed AT deficient subjects (n = 76) and from patients with request for AT activity measurement for diagnostic purposes (n = 152). AT activity was measured with five commercial assays on three analyzers.All reagent/analyzer combinations showed specificities varying from 87 to 100%, except for 1 assay (56.5% specificity). Diagnostic sensitivity varied widely between assays, from 36.8% to 100%. All reagent-analyzer combinations correctly identified variants causing type I AT deficiencies but only one variant responsible for type II heparin binding site (HBS) deficiency: p.Arg79Cys. For one of the assays, we observed a large difference in sensitivity (82.9 versus 55.3%) toward antithrombin type II HBS variants depending on the coagulation analyzer on which it is performed, suggesting that incubation time of sample and substrate could be a crucial driver of the sensitivity toward variants causing type II antithrombin deficiency. Our results suggest that inherited antithrombin deficiency is probably underestimated and improved diagnostic strategies are mandatory.

Protein C (PC) is an important physiological anticoagulant factor in humans. Activated protein C (APC) is generated from the PC zymogen through proteolytic activation by thrombin. APC inhibits thrombin generation by inactivating activated factors V and VIII via limited proteolysis. In addition to its anticoagulant function, APC also exhibits potent cytoprotective and anti-inflammatory properties. We have identified a young male with multiple-site thrombosis, who carries a heterozygous mutation c.1151A > G,p.Asn384Ser(N384S) in PC. Although this mutation has been previously documented, limited functional research has been conducted to elucidate its pathogenesis.To elucidate the functional alternations of the N384S mutant protein C and delineate the molecular mechanism underlying thrombosis in the patient carrying this mutation.We expressed the recombinant PC-N384S in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems.The expression level of the PC-N384S was reduced to approximately 7% of that observed for PC-WT. The activation of PC-N384S by thrombin or thrombin-thrombomodulin (TM) complex was significantly impaired, although the addition of TM exhibited a slight enhancement in the activation process. In terms of cleaving a chromogenic substrate, the catalytic efficiency reduced to approximately 50% of that observed in the wild type. In addition, in comparison with APC-WT, APC-N384S demonstrated a pronounced decline in amidolytic activity following an extended incubation period at 37°C. APC-N384S exhibited slightly impaired anticoagulant activity in either FVa inhibition assay or plasma-based assay systems. Furthermore, anti-inflammatory activity of APC-N384S was dramatically impaired as determined by evaluating the barrier-protective effect.The Asn384Ser mutation impairs both the anticoagulant and barrier-protective activities of protein C, thereby increasing the thrombosis risk in the heterozygous young male.

ST elevation myocardial infarction (STEMI) patients display endothelial dysfunction. We investigated whether endothelial glycocalyx thickness is affected in STEMI patients and may predict left ventricular performance post event.We examined 278 STEMI patients and 140 matched controls. We measured: (a) perfused boundary region (PBR) of the sublingual microvessels (range 4 to 25 μm; increased value indicates reduced endothelial glycocalyx integrity) at baseline; (b) left ventricular ejection fraction (LVEF) and global longitudinal strain (LVGLS), at baseline and at 12 months, (c) the percentage change of left ventricular end-systolic volume (ΔLVESV) at 12 months.Compared with matched controls, STEMI patients had higher PBR4-25 (2.11 ± 0.17 μm vs. 1.98 ± 0.20 μm, p < 0.001). In a model including age, sex, hypertension, diabetes, hyperlipidemia, smoking, family history of coronary artery disease, number of diseased vessels, location of STEMI medication, and high-sensitivity troponin T (hs-troponin), PBR4-25 was independently associated with LVEF and LVGLS at 48 hours post-MI (for LVEF: unstandardized β coefficient: -4.71, 95% CI: -8.53 to -0.71, p = 0.019 and for LVGLS: 2.89, 95%CI: 1.63-4.16, p < 0.001). Using multivariable analysis, PBR4-25 remained a significant predictor of the percentage change in LVEF, LVGLS, and ΔLVESV at 12-month follow-up (LVEF change: unstandardized β coefficient: -1.38, 95% CI: -1.80 to -0.96, p < 0.001; for LV GLS change: -0.66, 95% CI: -1.14 to -0.18, p = 0.007 and for ΔLVESV: 1.42, 95% CI: 0.06-2.93, p = 0.039). A PBR4-25 cut-off value of 2.29 μm could detect LV EF less than 45% at 48 h as well as at 12 months (AUC: 0.82, p < 0.001 and AUC: 0.80; p < 0.001).Endothelial glycocalyx assessment is associated with myocardial performance after STEMI.

Platelets serve not only as crucial hemostatic components but also as pivotal regulators of inflammatory responses, capable of interacting with diverse cell types and secreting abundant extracellular factors. Accumulating evidence demonstrates that high mobility group box 1 (HMGB1), a DNA-binding protein and critical inflammatory mediator, plays multifaceted roles in disease progression, with platelets being one cellular source of circulating HMGB1. Under pathological conditions, platelets release HMGB1 into the extracellular matrix, establishing bidirectional communication between platelets and other immune cells. Moreover, HMGB1 reciprocally activates platelets through Toll-like receptors (TLRs) and receptor for advanced glycation end-products (RAGE), facilitating platelet activation and subsequent release of regulatory factors that drive inflammation-associated pathological thrombosis. In this review, we systematically characterize the HMGB1-platelet axis and elucidate its context-dependent roles in specific disease states. The mechanistic interplay between HMGB1 signaling and platelet pathophysiology is discussed, particularly its implications for disease progression. Furthermore, we critically evaluate therapeutic strategies targeting HMGB1 developed over the past decade, while proposing future directions for dual-target interventions that simultaneously modulate HMGB1 and platelet activity to combat inflammation-driven thrombotic disorders.

Comprehensive characterization of platelets requires various functional assays and analytical techniques, including omics disciplines, each demanding a separate aliquot of the given sample. Consequently, sample material for each assay is often highly limited, necessitating the downscaling of methods to work with just a few micrograms of platelet protein.Here, we present a novel sample preparation platform for proteomics analysis using only 3 μg of purified platelet protein, corresponding to 2 × 10⁶ platelets, which can be obtained from approximately 2 to 8 μL of blood from a healthy individual (1.5 × 10⁵-4.5 × 10⁵ platelets/μL) or approximately 100 μL of blood from a patient with severe thrombocytopenia (<2 × 10⁴ platelets/µL).Using this platform, we detected a significant fraction of key players in the platelet activation cascade and, most importantly, identified 36 clinically relevant platelet disease markers even with a non-state-of-the art instrument. This makes LC-MS-based proteomics a highly attractive alternative to conventional assays, which often require milliliters of blood. Our platform transitions from our previously established 96-well proteomics workflow (PF96), which has been successfully employed in numerous platelet proteomics studies, into the 384-well format. This transition is accompanied by (1) a more than two-fold increase in sensitivity, (2) improved reproducibility, (3) a four-fold increase in throughput, allowing 1,536 samples to be processed per lab worker per week, and (4) reduced sample preparation costs.Thus, LC-MS-based platelet proteomics offers a compelling alternative to immunoaffinity assays (which depend on antibody availability and quality), as well as to genomic assays (which can only reveal genotypes). In summary, in conjunction with recent advances in LC-MS instrumentation, our platform represents a highly valuable tool for rapid phenotyping of platelets in research with extraordinary potential for future employment in companion or routine diagnostics.

Atrial fibrillation (AF) is linked to an elevated risk of thromboembolic events. Despite the use of guideline-recommended direct anticoagulants (DOACs), a significant proportion of AF patients show a residual risk of thromboembolic events, driven by mechanisms that are not fully understood.We conducted a pilot study to characterize the platelet function in DOACs-treated AF patients, to explore whether an association between platelets and the residual thromboembolic risk exists.Within the Age-It project of the National Recovery and Resilience Plan, we examined by flow cytometry the platelet phenotype, reactivity, and mitochondrial function and quantified 12 inflammatory cytokines of individuals with DOACs-treated permanent AF without a history of stroke (n = 18, 66 ± 13 years, 39% females), compared with an age-, sex-, and comorbidity-matched control group without AF (n = 18, 65 ± 11 years, 39% females).Unstimulated circulating platelets of DOACs-treated AF displayed a low-adhesive phenotype compared with matched controls. Upon stimulation, platelets of DOACs-treated AF were hyporeactive to ADP and PAR1 stimulation, but hyper-reactive to GPVI stimulation (adjusted p < 0.01). The lower responsiveness to ADP correlated with increased plasmatic concentrations of IFN-γ (r = - 0.539; p < 0.05) and TNF-α (r = - 0.472; p < 0.05). The higher reactivity to GPVI associated with an increased mitochondrial function, which positively correlated with TNF-α levels.Individuals with AF treated with DOACs exhibit low-grade inflammation and an altered platelet reactivity, suggesting a potential mechanism behind their residual thromboembolic risk. Further well-powered studies are warranted to test whether the observed platelet phenotype is implicated with the residual thromboembolic events in DOACs-treated AF patients.

The risk of venous thromboembolism (VTE) is high after cancer surgery and is reduced by antithrombotic prophylaxis.We conducted a systematic review and network meta-analysis to evaluate the effectiveness and safety of direct oral anticoagulants (DOACs) for VTE prophylaxis after cancer surgery. The primary study outcome was 30-day VTE after surgery. Pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were estimated.Five randomized controlled trials (RCTs; two gynecological, one abdominal, one neurosurgical, and one thoracic; 1,694 patients) and seven observational studies (three urological, two abdominal, and two gynecological; 2,042 patients) were included. DOACs reduced the incidence of 30-day (OR 0.52, 95% CI 0.27-0.98, I2 22.7%) and 90-day VTE (OR 0.51, 95% CI 0.28-0.92, I2 0%) compared to LMWH. No difference was observed between DOACs and LMWH in 30 and 90-day bleeding outcomes. In random effect analyses, apixaban (OR 0.31, 95% CI 0.11-0.84) and not rivaroxaban reduced the risk of 30 day-VTE compared to LMWH (OR 0.69, 95% CI 0.35-1.34) without increasing the risk of bleeding at 30 or 90 days. No difference in the risk of VTE or bleeding was observed between DOACs and placebo/no treatment, but these analyses were probably underpowered. Subgroup analyses were conducted on LMWH pre-treatment, extended prophylaxis, duration of surgery, and type of surgery.Our study supports apixaban and rivaroxaban as promising alternatives to LMWH in post-operative prophylaxis of VTE after cancer surgery. Further high-quality data are needed in specific surgical settings.

In comparative effectiveness research (CER), ensuring internal, construct, and external validity is crucial. Internal validity determines whether observed outcomes are causally linked to an intervention; construct validity assesses whether a study measures what it intends to; and external validity relates to generalizability in routine practice. Double-blind randomized trials optimize internal validity by minimizing bias and confounding, while construct validity is strengthened through pre-specified protocols and standardized data collection. However, controlled conditions limit external validity. Pragmatic RCTs improve generalizability but may compromise internal validity due to open-label designs. Observational CER studies-including observational studies following the target trial emulation framework-offer broader external validity and feasibility in less time and at lower cost. However, due to lack of random assignment, these studies are susceptible to measured and unmeasured confounding. Several techniques help mitigate these concerns, including a detailed pre-specified protocol, tools such as propensity score matching to balance measured confounders, falsification endpoint testing for assessing the presence of unmeasured confounders, and quasi-experimental designs (including instrumental variable analysis), which may be able to address both. Pre-specified sensitivity analyses and triangulation with complementary data sources further enhance robustness. Construct validity in observational CER depends on accurate patient profiling and validated computational phenotypes for identifying patients, exposures, and outcomes. Thoughtful study design and analytic rigor are essential for balancing these validity considerations. This brief review highlights these issues with examples from thrombosis research.

Emicizumab is an antibody that mimics the function of factor (F)VIII and has been approved for prophylaxis in hemophilia A patients. However, the development of anti-drug antibodies (ADA) against emicizumab, although rare, can impair its efficacy. In cases with low drug levels or bleeding events, differentiating between ADA- and adherence-related issues can be challenging.We aimed at evaluating the effectiveness of a modified bridging ELISA (Valsecchi et al, JTH 2021) in detecting ADA in patients suspected of developing this response. Clinical and laboratory data were retrospectively collected from six patients with suspected ADA and one with a confirmed case. The modified ELISA was performed blindly to identify potential ADA presence. After a new ADA case was confirmed, it was characterized by assessing its expression over time and neutralizing effect.Five patients had emicizumab levels ≤1 µg/mL, while two had higher levels (13 and 15 µg/mL). Among the patients, two experienced spontaneous bleeding, and four had traumatic bleeding. ADA was detected in two patients, including the one with a known ADA. In ADA-negative patients, emicizumab levels increased following adjustments for compliance or administration issues. The newly identified ADA was neutralizing, blocking emicizumab's binding to factors IX and X. Its pattern of expression was similar to that of the known ADA case, peaking 3 months after the loss of emicizumab efficacy and remaining positive for over a year after emicizumab discontinuation.In bleeding patients with low emicizumab levels, the modified bridging ELISA may effectively differentiate ADA patients from those with other issues leading to decreased emicizumab concentration.