Thrombosis and haemostasis最新文献

The use of low-molecular-weight heparin (LMWH) and aspirin during pregnancy is increasing, yet robust clinical trial evidence supporting their efficacy remains limited.In a multicenter prospectively enrolled cohort study, we evaluated the prescription patterns and associated maternal-fetal outcomes of antithrombotic therapy in 2,622 women admitted for delivery across three Italian obstetric centers between January 2022 and November 2023. Data were collected on conception methods, administration details (timing, dose, indication) of LMWH and low-dose aspirin (LDA), and maternal-fetal outcomes from admission to postpartum discharge. Data on prescription of antithrombotic drugs were available for 1,898 women.Among 1,898 women with available data, 157 (8.3%) received LDA (100 mg/day) and 746 (39.3%) received LMWH (49 during pregnancy and 697 in the postpartum period). Predictors of LMWH use included prior cesarean (OR 3.1, 95% CI 1.7-5.8), preterm delivery (OR 3.8, 95% CI 1.7-8.9), pregnancy loss (OR 2.7, 95% CI 1.5-4.9), and assisted conception (OR 14.6, 95% CI 2.8-76.5). LDA use was associated with pregnancy loss (OR 2.1, 95% CI 1.4-3.0), ART (OR 4.7, 95% CI 2.2-10.2), and LMWH co-administration (OR 2.5, 95% CI 1.1-5.5). Postpartum LMWH use was primarily associated with cesarean delivery. Postpartum hemorrhage occurred in 2.4% of cases, with no significant difference in those receiving LDA or LMWH.These findings reflect current real-world prescribing practices and highlight key maternal characteristics influencing antithrombotic therapy decisions. The study underscores the importance of evidence-based approaches in the use of LMWH and LDA during pregnancy, particularly in high-risk populations, to improve maternal-fetal outcomes while minimizing unnecessary exposure to therapies with uncertain benefit.

Initial thrombin (FIIa) generation is a critical trigger for the amplification and propagation phases of coagulation, driven by two distinct pathways: the tissue factor (TF)-driven and the FVIIIa/FIXa-dependent pathways. However, the clinical utility of measuring initial thrombin generation (ITG) as a marker of thrombogenicity or as a tool to monitor the efficacy of oral anti-FXa therapy remains uncertain.ITG driven by TF and the FVIIIa/FIXa complex was first measured in plasma samples from healthy adults (n = 40). This was followed by an analysis of ITG profiles in 771 consecutive patients with cardiovascular diseases to evaluate the effects of anticoagulant therapy and clinical characteristics.Of the 771 patients studied, 169 were receiving direct oral anticoagulants (DOACs). DOAC treatment significantly suppressed thrombin generation via both TF-driven and FVIIIa/FIXa-dependent pathways. Receiver operating characteristic (ROC) analysis confirmed the strong discriminatory power of both pathways for detecting DOAC use (FVIIIa/FIXa: AUC 0.863, 95% CI: 0.826-0.900; TF: AUC 0.887, 95% CI: 0.856-0.917; both p values < 0.0001). Among patients not on anticoagulants, logistic regression revealed that dialysis was associated with reduced thrombin generation through both pathways. Furthermore, chronic kidney disease and active cancer were specifically associated with diminished TF-driven thrombin generation.The ITG potentials driven by TF- and FVIIIa/FIXa-dependent pathways represent promising biomarkers for evaluating anticoagulant efficacy. Moreover, the distinct influence of pathological conditions on each pathway underscores the need to account for specific disease contexts when assessing coagulation potential.

Elevated FXI levels are associated with increased risk of venous thromboembolism, yet genetic mutations contributing to elevated FXI levels have not been reported yet.We described a patient with a history of deep venous thrombosis (DVT) who tested negative on routine laboratory screening but was found to carry a novel pathogenic FXI mutation, Gly397Ser (G397S). FXI mutant protein was expressed, and functional assays were conducted in vitro to explore the underlying thrombotic mechanism.Consistent with FXI activity (FXI:C) and antigen (FXI:Ag) determined in the patient, the FXI G397S mutant exhibited a 2-fold increase of FXI:C/FXI:Ag. FXIa hydrolysis assay demonstrated that the Km value for G397S mutant was comparable to the wild-type FXIa (186.2~281.9 μM V.S. 141.6~242.6 μM), whereas the mutant FXIa displayed approximately 3-fold increases in kcat values. FXI activation by polyanions was more pronounced in the G397S mutant. Notably, this activation was more significant when triggered by thrombin compared with FXIIa in the presence of heparin. Otherwise, the G397S FXI mutant exhibited similar hydrolytic activity of FXIa against FIX, and equivalent inhibition of FXIa by Protease nexin 2 (PN2). Clot lysis assay revealed that the lysis time was longer in G397S mutant than the wild-type and the enhanced fibrinolytic resistance was thrombin-activatable fibrinolysis inhibitor-dependent. In summary, the FXI G397S mutation exhibited higher FXIa activity mainly because of the enhanced activation of FXI, which sustained thrombin generation and manifested resistance to fibrinolysis, ultimately leading to the development of DVT.

Platelet-monocyte aggregates (PMA) are modern biomarkers for the early onset of cardiovascular diseases (CVD). PMA occur in the presence of activated platelets (AP), whose incidence is higher in patients with metabolic diseases. These patients frequently benefit from anticoagulant therapy, particularly in advanced disease stages. However, the impact of these drugs on PMA occurrence is unknown.Blood samples from healthy volunteers, patients with type 1 diabetes mellitus (DMT1), type 2 diabetes mellitus (DMT2), dyslipidemia (hypercholesterolemia), and severe forms of familial hypercholesterolemia (FH) were analyzed by flow cytometry to detect both AP and PMA. Subsequently, blood samples were treated with four direct anticoagulants (rivaroxaban, apixaban, dabigatran, or argatroban, all at a final concentration of 1 µM), and PMA were again detected. Anthropological and biochemical parameters were recorded and correlated with AP and PMA.AP levels were higher solely in DMT2 patients, but PMA occurrence was significantly increased in all patient groups in comparison to that of generally healthy donors, except for FH patients. No sex-related differences were observed, but an increasing trend in AP and PMA incidence was observed with increasing age and BMI. Significant correlations of AP and PMA with anthropological and biochemical parameters were found solely in some groups of patients or in some anticoagulant-treated samples. Only argatroban and apixaban-treated samples significantly decreased PMA occurrence, and this was observed solely in the dyslipidemia patient group.These findings suggest a potential positive outcome of anticoagulant treatment for metabolic disease patients and confirm PMA as a sensitive marker in patients with metabolic diseases.

Ischemic stroke affects more than 15 million people annually, where atheroembolism from unstable atherosclerotic plaques in the carotid bifurcation contributes a significant and potentially preventable aetiology. Landmark carotid endarterectomy trials conducted decades ago proved that removing high-grade symptomatic stenoses outperforms medical therapy, yet accurately identifying unstable plaques, the true embolic source, remains elusive. Consequently, procedural efficacy is modest, and more personalized diagnostic methods are necessary for refined risk stratification and improved stroke prevention. Plaque instability and atheroembolism from rupture of the fibrous cap is driven by an enlargement of the lipid-rich necrotic core, inflammation, neovascularisation, intraplaque haemorrhage, and fibrous-cap thinning. Hypoxia-induced angiogenesis produces fragile neovessels that bleed, generating oxidative stress and further destabilisation. Biomolecules escaping into the circulation from such lesions could flag individuals at imminent risk, but imaging modalities such as magnetic resonance, computed tomography angiography, or ultrasound are necessary to identify lesions at risk. Using these methods, aligning imaging phenotypes with molecular signatures of stability or vulnerability can aid in the development of refined diagnostic methods. Integrating blood-based with quantitative imaging biomarkers and systems-biology modelling promises a personalised diagnostic pipeline to screen for circulating signals of instability to detect individuals at risk, together with noninvasive imaging to localise culprit lesions, creating the basis for tailored surgical or pharmacologic therapy. In this review, we discuss scientific advances in the development of such strategies that possess the potential to sharpen patient selection, reduce unnecessary procedures, and advance precision in stroke prevention.

Leukocyte-platelet aggregates (LPAs) play a crucial role in the pathogenesis of inflammatory diseases, linking pathological immune responses with thrombosis.The levels of LPAs, their composition, and cellular reactivity were determined in patients with distinct inflammatory conditions, namely coronavirus disease 2019 (COVID-19), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE), compared with healthy controls. Flow cytometry was used to identify cell types and measure LPA levels in the blood. The ability of platelets, neutrophils, and monocytes to form additional LPAs in response to hyperstimulation with phorbol-12-myristate-13-acetate (PMA) was assessed. Coaggregation of isolated neutrophils and platelets in vitro was visualized using scanning electron microscopy. Blood tests included coagulation, hematology, biochemistry, and immunology.LPA levels were significantly higher in all patient groups compared with controls, with variations in the composition: neutrophil-platelet aggregates predominated in the COVID-19 patients, whereas monocyte-platelet aggregates prevailed in the blood of RA and SLE patients. Platelet-to-leukocyte ratios within aggregates varied in a broad range with a substantial prevalence of platelets over leukocytes. Morphological analysis revealed coaggregation of platelets with neutrophils, including relatively large homotypic platelet aggregates associated with one or two neutrophils. In PMA-treated pathological blood samples from COVID-19, RA, and SLE patients, the ability to form additional LPAs over the patients' baseline level was reduced compared with normal blood samples, indicating impaired reactivity (exhaustion) of neutrophils and monocytes in all patient groups.This study highlights distinct changes in the number and composition of LPAs in inflammatory diseases of various etiologies associated with altered functionality of the innate immune cells.

Human platelets express protease-activated receptor (PAR) 1 and 4 on the platelet membrane. PAR1 activation is crucial for hemostasis, while PAR4 activation produces a prolonged pro-inflammatory platelet response. Once activated, the PAR receptors cause platelet activation and aggregation. The PARs are activated by proteolytic cleavage of their N-terminus, revealing a new N-terminus called the tethered ligand. This tethered ligand folds back and activates the receptor. PAR1 and PAR4 differ in their tethered ligand sequence as well as their signaling cascades; however, they are both activated by various serine proteases, specifically thrombin.Much is known about PAR4 activation via thrombin activation; however, little is known about activation by other proteases. Thrombin cleavage of PAR4 and subsequent activation leads to G proteins, Gα_q and Gα_12/13, and β-arrestin signaling. The objective of this study is to characterize trypsin activation of PAR4 on human platelets.Platelets were isolated from the whole blood of healthy human volunteers by centrifugation. Platelets were utilized in flow cytometry assays, calcium mobilization assays, and western blot experiments to characterize trypsin activation of human platelets.Here, we demonstrate that trypsin activates human platelets solely through PAR4. Trypsin activation of PAR4 signaling through both Gα_q and Gα_12/13 is as effective as α-thrombin activation of PAR4.Thrombin may be the main protease to activate PAR4 under typical conditions; however, under pathological conditions such as acute pancreatitis (AP), trypsin activation of PAR4 may be a main driver of platelet activation and disease progression.

Insufficient lipid-lowering therapy is associated with residual cardiovascular risk. Low-density lipoprotein (LDL) is well known to promote atherosclerosis and cardiovascular disease (CVD) and during lipoprotein metabolism, free fatty acids (FFA) are generated. Besides this, residual thrombotic risk occurs in patients with impaired pharmacodynamic response to aspirin-so-called high on-treatment platelet reactivity (HTPR). Until now, it is not known if there is a mutual link.For in vitro analyses, blood from healthy donors was used and incubated with different concentrations of LDL, FFA, and acetylsalicylic acid. Arachidonic acid (AA)-induced light transmittance aggregometry (LTA), thromboxane (TX) formation, and cyclooxygenase (COX)-1 activity were measured. In a cross-sectional analysis, aspirin antiplatelet effects, and LDL and FFA concentrations were measured in 612 aspirin-treated CVD patients. In vitro administration of LDL and FFA impaired aspirin antiplatelet effects. In patients, FFA levels were associated with HTPR to aspirin. FFA levels and plasma LDL correlated with AA-induced platelet aggregation. Statin medication improved aspirin antiplatelet effects. AA-induced platelet aggregation was decreased in patients with statin medication.In this study, we were able to demonstrate a link between residual lipid and thrombotic risk in patients with cardiovascular disease. We could show that LDL and FFA impair pharmacodynamic response to aspirin at the level of COX. Statin therapy improved aspirin antiplatelet effects.

Chromogenic anti-Xa assay is currently used in the management of patients on unfractionated heparin (UFH). It has been shown that inter-assay variability in anti-Xa levels can be explained in part by the presence or absence of dextran sulfate (DXS) in the reagents. DXS has the ability to dissociate UFH from neutralizing proteins, including platelet factor 4 (PF4).Investigate whether PF4 plasma levels along with the presence/absence of DXS in anti-Xa reagents are associated with variations in UFH anti-Xa levels in different clinical situations.In the prospective multicenter study DEXHEP-NCT04700670, critically ill patients on UFH therapy (four groups) were recruited. Blood was collected into citrate and CTAD tubes. Chromogenic anti-Xa levels were assessed using seven reagent/analyzer combinations including two without DXS. Plasma PF4 was measured by ELISA (Zymutest-PF4-Hyphen-Biomed).A total of 144 patients were analyzed: average PF4 levels in citrate plasma samples were consistently higher than in CTAD ones (206 vs. 46 ng/mL, p < 10^-4), regardless of the patient group. Using a linear mixed-effect model, we found a significant effect of both DXS and PF4 on anti-Xa level, with a significant interaction term (p < 10^-4). Considering the 0.3 to 0.7 IU/mL therapeutic range, agreement between anti-Xa values (Liquid-anti-Xa/DXS-free vs. Biophen-LRT/DXS) was observed in roughly two-thirds of the patients.PF4 levels slightly affects anti-Xa levels, the use of CTAD tubes minimizing the effect. However, PF4 levels do not fully explain the differences of anti-Xa levels observed in the presence or absence of DXS, which has a greater effect. Anti-Xa assays require better standardization.

Inflammation and thrombosis are traditionally considered two separate entities of acute host responses to barrier breaks. While inciting inflammatory responses is a prerequisite to fighting invading pathogens and subsequent restoration of tissue homeostasis, thrombus formation is a crucial step of the hemostatic response to prevent blood loss following vascular injury. Though originally designed to protect the host, excessive induction of either inflammatory signaling or thrombus formation and their reciprocal activation contribute to a plethora of disorders, including cardiovascular, autoimmune, and malignant diseases. In this state-of-the-art review, we summarize recent insights into the intricate interplay of inflammation and thrombosis. We focus on the protective aspects of immunothrombosis as well as evidence of detrimental sequelae of thromboinflammation, specifically regarding recent studies that elucidate its pathophysiology beyond coronavirus disease 2019 (COVID-19). We introduce recently identified molecular aspects of key cellular players like neutrophils, monocytes, and platelets that contribute to both immunothrombosis and thromboinflammation. Further, we describe the underlying mechanisms of activation involving circulating plasma proteins and immune complexes. We then illustrate how these factors skew the inflammatory state toward detrimental thromboinflammation across cardiovascular as well as septic and autoimmune inflammatory diseases. Finally, we discuss how the advent of new technologies and the integration with clinical data have been used to investigate the mechanisms and signaling cascades underlying immunothrombosis and thromboinflammation. This review highlights open questions that will need to be addressed by the field to translate our mechanistic understanding into clinically meaningful therapeutic targeting.