Journal of Thrombosis and Haemostasis最新文献

Introduction: Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterized by a severe functional deficiency of ADAMTS13. Measuring ADAMTS13 activity is crucial for diagnosing TTP (<10 IU/dL), monitoring treatments, and detecting relapses (<20 IU/dL). The Technofluor® assay allows a rapid ADAMTS13 activity measurement using the CEVERON® s100 analyzer. This study aims to evaluate the analytical and clinical performance of this new test under real-world conditions.

Materials and methods: ADAMTS13 activity was measured using two fluorometric methods: the Technofluor® ADAMTS13 activity on the CEVERON® s100 (Technoclone, Austria) and our reference FRETS-VWF73 method, in plasma samples collected under real-life conditions (01/12/2024-04/04/2024) and retrospectively selected samples from our biobank. The analytical and clinical performance of the new test was assessed, focusing on the critical low levels (<30 IU/dL).

Results: Four hundred samples were tested under real-world conditions and 100 others tested retrospectively. The Technofluor® assay showed excellent analytical performance, with a detection limit of 0.1 IU/dL, repeatability CV <11%, and reproducibility CV of 6.1% (high level [90 IU/dL]) and 7.5% (low level [40 IU/dL]). Clinical performances were strong at diagnosis (threshold: 10 IU/dL; sensitivity and positive predictive value: 1.0) and during the follow-up (threshold: 20 IU/dL; specificity: 1.0 [95% CI: 0.99;1.00], positive predictive value: 0.96 [0.90;1.01]; negative predictive value: 0.98 [0.97;1.00]). No analytical interference was observed.

Conclusion: The Technofluor® assay on the CEVERON® s100 is a fast, reliable method for measuring ADAMTS13 activity, proving effectiveness for diagnosis and monitoring of TTP patients. However, it remains crucial to interpret ADAMTS13 activity measurement with clinical context to ensure accurate diagnosis and management.

Background: Oxidative stress is one of the aging hallmarks. Small non-coding nucleolar RNAs (snoRNAs) of the ribosomal protein L13a (Rpl13a) locus are master regulators of reactive oxygen species (ROS) and oxidative stress, and their loss protects mice from diabetes. Yet, whether excess ROS in RBCs is regulated by Rpl13a snoRNAs in aging and mediate venous thrombosis (VT)/thromboembolism (VT/E) is unknown.

Objectives: We investigated if RBCs retain Rpl13a snoRNAs, and contribute to RBC ROS-mediated VT in a mid-life stage population.

Patients/methods: Blood samples were collected from healthy mid-life stage (55-68 years old) and young (21-30 years old) adults, VT/E patients, and young wild type (WT) mice at 12-24 weeks of age, and aged WT and aged Rpl3a snoRNA knockout mice at 72-96 weeks of age (equivalent to humans 21-30 and 55-68 years old, respectively).

Results: RBCs from mid-life stage adults, and VT/E patients showed higher ROS production and prothrombotic potential than the younger cohort. RBC ROS levels and prothrombotic potential were associated with abnormal Rpl13a snoRNAs levels. In aging, Rpl13a snoRNAs regulated human and murine RBC ROS levels and prothrombic activation by modulating peroxidase activity. This was due largely to Rpl13a snoRNAs-guided 2'-O-methylation on RBC peroxidasin (Pxdn) mRNA; a modification that inhibited the mRNA translation and Pxdn activity. In vivo Rpl13a snoRNA knockout in aged mice blunted RBC ROS generation, decreased thrombi size, thrombi RBC content, and RBCs-triggering prothrombin activation.

Conclusion: These findings point out to a novel role of RBC Rpl13a snoRNAs in VT by dysregulating Pxdn expression in aging.

Background: Non-small cell lung cancer (NSCLC) patients are at high risk of venous thromboembolism (VTE), especially during chemotherapy. Even though the contact system is implicated in the pathogenesis of thrombosis, limited data are available on the role of contact system activation in NSCLC-associated VTE.

Aim: In a prospective cohort of NSCLC patients starting chemotherapy, contact system activation and thrombin generation biomarkers were assessed in relation to 6-month VTE occurrence and mortality.

Methods: Prechemotherapy plasma samples of 719 newly diagnosed NSCLC patients were tested for in vivo biomarkers of contact system activation (i.e., kallikrein: antithrombin [PKa:AT], activated FXI:AT [FXIa:AT], FXIa: C1-esterase inhibitor C1Inh [FXIa:c1Inh], activated factor IX:AT [FIXa:AT]), and thrombin generation (i.e., prothrombin fragment 1+2 [F1+2], thrombin-antithrombin complex [TAT]). Clinical data, VTE, and mortality were recorded prospectively.

Results: The 6-month VTE and mortality cumulative incidences were 11% and 27%, respectively. Basal levels of FXIa:AT complexes, F1+2, and TAT levels were higher in patients who developed VTE than VTE-free patients. Differently, PKa:AT, FIXa:AT, and TAT were lower in survivors compared to non-survivors. The multivariable analysis identified FXIa:AT (HR 1.17, 95%CI 1.00-1.37) and TAT (HR 1.28, 1.10-1.50) as VTE-independent risk factors during chemotherapy. A score based on these biomarkers was generated, able to discriminate patients at significantly higher rates of VTE and mortality.

Conclusions: Elevated in vivo contact pathway activation and thrombin generation were observed in NSCLC patients who developed VTE. Furthermore, a score based on both FXIa:AT and TAT levels was developed to identify those patients at higher risk of VTE and mortality.

Anti-FXI/FXIa anticoagulants under development include antisense oligonucleotides, monoclonal antibodies and small molecules. They do not require routine monitoring, but knowledge of their impact on coagulation tests is essential in view of their expected widespread use. A concentration-dependent prolongation of activated partial thromboplastin time has been shown but varies according to reagents, and the lack of comprehensive data makes interpretation of this test difficult. Measurement of FXI clotting activity is relevant only in case of treatment with antisense oligonucleotides. Measurement of contact pathway factors, if required, should be performed after multiple dilutions of the plasma sample to overcome any inhibitory effect of the anticoagulant. All other tests used in clinical trials (FXIa, FXI antigenic method, specific thrombin generation assay) are not implemented in clinical laboratories. More comprehensive information on the effect of anti-FXI/FXIa anticoagulants on coagulation tests is urgently needed to anticipate the use of these drugs once they are approved.

Background: 2-Methoxybenzoic acid (2MOA) is a natural compound with potential salicylate-like effects; however, its impact on arterial thrombosis remains unclear. This study aims to investigate the effects of 2MOA on thrombogenesis and its underlying mechanisms.

Methods: FeCl₃-induced carotid artery injury and laser-induced cremaster artery injury thrombosis assays were utilized to explore the effect of 2MOA on thrombogenesis in vivo. Various ex vivo platelet function assays were conducted to evaluate the impacts of 2MOA on platelet activity. In addition, untargeted metabolomics analysis was performed to identify the alterations in intraplatelet metabolites following 2MOA treatment.

Results: We found that 2MOA significantly ameliorated thrombosis in a dose-dependent manner, without affecting the normal hemostasis in C57BL/6J mice. 2MOA suppressed platelet reactivity as indicated by decreased spreading, retraction, and aggregation in both mouse and human platelets. Metabolomics analysis revealed significantly alterations in purine metabolism following 2MOA treatment, which increased cyclic guanosine monophosphate (cGMP) production in platelets. Mechanistically, 2MOA inhibited the activity of carbonic anhydrase, leading to elevated intra-platelet cGMP level, and subsequent suppression of cytosolic phospholipase A2 phosphorylation.

Conclusion: Our study illustrates that 2MOA efficaciously inhibits platelet reactivity and alleviates thrombogenesis via suppressing carbonic anhydrase activity, which should be a promising reagent in the prevention and treatment of arterial thrombotic events.

Background: Newly produced platelets are thought to be more functional than their older counterparts. However, recent work suggests that murine platelets formed following immune thrombocytopenia possess a transient glycoprotein VI (GPVI) signalling defect.

Objectives: In this study we explored if other models of stress thrombopoiesis would generate platelets that display a functional defect.

Methods: Platelet function was assessed by light transmission aggregometry and/or flow cytometry in genetic and disease models of thrombocytopenia and after chemotherapy-induced thrombocytopenia.

Results: We evaluated platelet function in mice bearing a point mutation in Bcl-x and in two cancer models, all presenting with thrombocytopenia and a high proportion of reticulated platelets. Flow cytometric analysis of platelet degranulation and integrin activation revealed a significantly diminished response to the GPVI agonist convulxin in all models, but not thrombin. Likewise, platelet aggregation and Syk phosphorylation downstream of GPVI, in response to convulxin, was significantly reduced. Furthermore, a rebound from carboplatin- or immune-thrombocytopenia, caused a transient GPVI defect. The Mpl^-/- model of thrombocytopenia (with a normal proportion of reticulated platelets) was included as a negative control. In response to convulxin, Mpl^-/- platelets exhibited normal degranulation and integrin activation.

Conclusion: Here, we report a functional defect in platelet GPVI signalling present in multiple models of thrombocytopenia that are accompanied by an increased proportion of rapidly generated young platelets. These results indicate that during stress thrombopoiesis, the GPVI receptor becomes entirely functional only after spending some time in circulation.

Background: The development of inhibitory antibodies (inhibitors) is a serious complication in the treatment of hemophilia A with clotting factor VIII (FVIII) replacement therapy. Inhibitor formation critically depends on T cell help and modulation by regulatory T cells (Tregs).

Objective: In this study, we evaluated the F5111 immunocytokine (IC), a single chain fusion between the human interleukin-2 (IL-2) cytokine and an IL-2 antibody that biases cytokine activity towards cells with high IL-2 receptor alpha (IL-2Rα) expression, leading to extended IL-2 half-life and selective expansion of Tregs.

Methods: A transient F5111 IC administration regimen was applied to a hemophilia A murine model of FVIII replacement therapy. Inhibitory antibody development to FVIII was monitored longitudinally by Bethesda assay and ELISA.

Results and conclusion: F5111 IC failed to stimulate cell types that predominantly express the dimeric IL2Rβγ receptor complex such as effector T and NK cells. Potent and highly transient Treg expansion was associated with suppression of effector T cells and in vivo conversion into Tregs. When tested in the hemophilia A model, F5111 IC completely prevented the formation of inhibitors against FVIII for up to 4 months, long after Treg numbers returned to baseline levels. These results demonstrate that F5111 IC induces a superior and prolonged tolerogenic response compared to an unbiased control immunocytokine. Overall, this study presents a novel and effective strategy for preventing inhibitory antibodies that hinder the effectiveness of FVIII replacement therapy in hemophilia A.

Background: For every man diagnosed with hemophilia, approximately 1.6 women are expected to be carriers. Carriers are classified based on their Factor VIII (FVIII) levels and symptoms, ranging from asymptomatic to mild, moderate or severe symptoms. Close monitoring is critical for carriers with low FVIII levels or bleeding symptoms, as bleeding risk is difficult to assess due to inconsistent correlations with routine one-step assay (OSA) measurements.

Objective: This study hypothesized that the chromogenic FVIII assay (CSA) may provide valuable information for estimating bleeding risk in some hemophilia carriers.

Patients/methods: This retrospective study included 109 hemophilia A carriers from two centers.

Results: Among them, 23% had FVIII levels below 40 IU/dL using OSA, while 41% showed discrepancies when assessed using CSA. VWF activity and antigen levels were normal, with mean values of 84 IU/dL and 107 respectively. There was a significant correlation between OSA and CSA FVIII results, although 20 women had discordant results between the two methods. Bleeding events were reported in 49 women, including 18 surgical complications, 1 joint bleeding episode, and 30 cases of heavy menstrual bleeding, all occurring with normal VWF levels. There were 157 pregnancies in which 14 cases of postpartum hemorrhage were observed, 3 of which required transfusion or surgery.

Conclusion: This study highlights the significant discrepancies between OSA and CSA in FVIII results, with implications for diagnosis and bleeding risk assessment. It emphasizes the need to use both methods to identify women at higher risk of bleeding, especially before surgery.

Background: Patients with cirrhosis develop multiple hemostatic alterations. Although fibrinolysis is also affected by liver disease, studies have produced conflicting results, highlighting the need for a reliable fibrinolysis assay. Assessing the kinetics of plasmin generation (PG) is a new method to study the fibrinolytic state of cirrhosis patients.

Objectives: This study aimed to compare fibrinolysis between patients with cirrhosis and healthy subjects.

Methods: This single-center cohort study included cirrhosis patients from the Padova University Hospital. Fibrinolysis and hemostasis were assessed with PG, thrombin generation (TG), and clot lysis time (CLT). To quantify malalignment between TG and PG, ratios were calculated.

Results: In total, 101 patients with cirrhosis (Child-Pugh A/B/C: 36/24/41) were included and 20 healthy subjects. Compared with healthy subjects, patients showed a significantly lower endogenous plasmin potential (EPP) and plasmin peak. PG capacity decreased with liver disease severity. The lag-time to PG was prolonged in patients. No differences in endogenous thrombin potential (ETP) and lag-time were found when comparing TG profiles. Patients had a shorter CLT. Increased TG/PG ratios for the EPP and plasmin peak were found in patients, compared to controls. TG/PG ratios increased with liver disease severity.

Conclusions: Patients with cirrhosis have a complex fibrinolytic profile, with a delayed and decreased capacity to generate plasmin and a more rapid clot lysis. A disbalance was found between coagulation and fibrinolysis, with a normal to increased TG capacity and a decreased PG capacity. These results support the theory that cirrhosis patients are in a prothrombotic state.