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Cancer-associated thrombosis affects between 1 and 20% of all patients diagnosed with cancer and is associated with significant morbidity and a poorer prognosis. Risk assessment scores exist which include the measurement of biomarkers, and which aim to identify patients at a higher risk of developing thrombotic events, but these are poor predictors and rarely used in routine clinical practice. VEGF is a potent angiogenic factor, produced by tumour cells, and released by platelets and is essential for tumour growth and progression. It also plays a role in the promotion of thrombosis through platelet activation and adhesion, and by inducing the expression of tissue factor. Therefore, the potential of VEGF to be used as a biomarker to predict cancer-associated thrombosis requires further investigation. This study reviewed the published literature to determine whether circulating VEGF levels are associated with increased risk of venous thromboembolism in patients with cancer. PubMed and OVID databases were systematically searched according to PRISMA guidelines for relevant papers using the keywords "cancer" AND "thrombosis" AND "VEGF" up to July 2023. Inclusion and exclusion criteria were applied. Seven papers (1,528 participants) were identified and included in the meta-analysis, three of which (922 participants) measured VEGF before a thrombotic event, and the remaining four (606 participants) measured VEGF at the time of the thrombosis. Our results showed that although plasma and serum VEGF tended to be higher in those who subsequently developed thrombosis than those who did not (mean difference 70.2 pg/mL for serum, and 11.44 pg/mL for plasma VEGF, 95% CI -2.39-25.73, p  = 0.10), this was not found to be statistically significant. However, analysis of VEGF following blood sampling at the time of thrombosis showed a stronger statistically significant association between increased VEGF levels and presence of thrombosis (mean difference 117.02 pg/mL for serum, and 116.6 pg/mL for plasma VEGF, 95% CI 55.42-190.82, p  = 0.0004). Based on current studies, whilst it is increased at the time of thrombosis, VEGF is not effective as a predictive biomarker of CAT.
Chimeric antigen receptor (CAR) T cell therapy has transformed the management of hematologic malignancies, yet its clinical success is tempered by severe immune-mediated toxicities, including cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), often accompanied by CAR T cell therapy-related coagulopathy (CARAC). Converging evidence identifies therapy-related endotheliopathy as a central pathophysiological link between cytokine excess, hemostatic dysregulation, capillary leak, and organ injury. Parallel efforts aim to identify circulating biomarkers that can signal emerging toxicity before clinical deterioration. This review summarizes the biological basis of endotheliopathy during CAR T cell therapy, with particular emphasis on two interconnected regulatory systems: the von Willebrand factor (VWF)/ADAMTS13 axis, which governs platelet adhesion and microvascular thrombosis, and the angiopoietin (Ang)-tyrosine kinase receptor Tie2 signaling pathway, which regulates endothelial stability and vascular permeability. Dysregulation of these pathways drives the shift from adaptive immunothrombosis to pathological endothelial injury, characterized by loss of anticoagulant control, barrier disruption, and microvascular instability. Clinical studies show that alterations in the VWF/ADAMTS13 balance and increases in the Ang-2/Ang-1 ratio correlate with CRS and ICANS severity and may precede overt toxicity, highlighting their potential as markers of endothelial vulnerability. Defining actionable biomarker thresholds and evaluating endothelial-targeted interventions are key priorities for improving the safety and precision of CAR T cell therapy.
Newer therapeutic options for people with severe haemophilia A (PwSHA), in addition to improved clinical and patient-reported outcomes (PROs), have offered more personalised treatment regimens. This analysis explored mental health, work productivity, and health-related quality of life (HRQoL) among PwSHA in Europe receiving a prophylactic treatment regimen. The Cost of Haemophilia: a Socio-economic Survey (CHESS) study is a retrospective cross-sectional study of adult men with haemophilia in Europe. We analysed data from CHESS participants with severe haemophilia A and no factor VIII (FVIII) inhibitors who received emicizumab or FVIII replacement prophylaxis. Data are from patient questionnaires and their treating health care providers. This analysis focused on PROs, including productivity and activity impairment via the Work Productivity and Activity Impairment, HRQoL via the EQ-5D-5L, and anxiety via the 7-item General Anxiety Disorder questionnaire (GAD-7) and depression via the 8-item Patient Health Questionnaire (PHQ-8). SHA treatment and clinical characteristics were also collected, including bleeding events, joint health, and chronic pain. All findings were analysed descriptively. A total of 350 PwSHA met the inclusion criteria, 94 (27%) of whom provided PROs. Most (68%; n  = 64) were receiving emicizumab (FVIII prophylaxis, 32%; n  = 30). Clinical characteristics were generally comparable between emicizumab and FVIII prophylaxis groups, including reported chronic pain (63% and 70%) and problem joints (61% and 63%), with on-demand FVIII use for the treatment of breakthrough bleeding events more commonly reported in the FVIII prophylaxis group (34% vs. 56%). Overall, HRQoL showed comparable EQ-5D-5L scores between the treatment groups, with a marginally higher score in the emicizumab group (0.71 vs. 0.69) compared with the FVIII prophylaxis group. Anxiety and depression scores were both numerically lower in the emicizumab group, suggesting a lower burden of disease (anxiety 7-item General Anxiety Disorder questionnaire [GAD-7] mean scores, 6.0 vs. 7.3; depression PHQ-8 mean scores, 6.8 vs. 7.8). Employed PwSHA in the emicizumab group reported a lower impact of SHA on their work impairment (31% vs. 50%), and only 19% (vs. 33%) of the emicizumab group required assistance with daily activities. More PwSHA receiving FVIII prophylaxis reported a negative impact of SHA on their ability to participate in social activities (70% vs. 56%) and on their physical activity (57% vs. 44%). Patients receiving emicizumab prophylaxis appeared to have more favourable mental health, work productivity, and HRQoL-related outcomes than those receiving FVIII prophylaxis. These findings were observed in the context of comparable clinical characteristics between emicizumab and FVIII prophylaxis despite evidence of a more complex treatment history for the emicizumab group. This analysis has limitations, including a lack of adjustment for confounding factors.
Direct oral anticoagulants (DOACs) have been widely used in patients with thromboembolism. We previously reported that among DOACs, edoxaban (EDX), a factor Xa (FXa)-specific DOAC, most effectively inhibited the growth of syngeneic non-metastatic murine colon cancer cells implanted in mice via the protease-activated receptor 2 (PAR2) pathway. This study aimed to analyze the effects and mechanism of action of DOACs targeting thrombin or FXa on the metastasis of murine melanoma B16 cells implanted in mice. B16 cells (10 6 cells in 100 μL) were implanted into the tail vein of 8-week-old female C57BL/6j mice ( n  = 5 per group), followed by daily oral administration of DOACs targeting thrombin (dabigatran etexilate [DABE], 50 mg/kg body weight [bw]) or FXa (rivaroxaban [RVX], 5 mg/kg bw; EDX, 10 mg/kg bw) for 14 days. The effects on tumor metastasis on day 15 and the inhibitory mechanism of the DOAC with the strongest inhibitory effect were analyzed. Lung metastasis of B16 cells implanted in mice was significantly suppressed in the following order: EDX > RVX ≥ DABE, compared with the saline-treated group. DOPA (3,4-dihydroxyphenylalanine)-positive cell density was significantly reduced from approximately 1,250 cells/mm 2 in the saline group to approximately 600 cells/mm 2 (RVX and DABE) and approximately 400 cells/mm 2 (EDX; p  < 0.05 or 0.01). Investigating the inhibitory mechanism of EDX revealed that inflammation-associated factors such as PAR2, interleukin 6 (IL-6), and transforming growth factor β1 (TGFβ1); angiogenic factors such as vascular endothelial growth factor A and angiopoietin-2; and epithelial-mesenchymal transition (EMT)-associated factors such as vimentin and snail, which were increased in the lungs of the saline-treated group, all significantly decreased in the EDX-treated group ( p  < 0.05 or 0.01). In contrast, intercellular tight junction factors exhibited an opposite trend. EDX also inhibited FXa-dependent production of melanin, IL-6, and TGFβ1 in in vitro cultured B16 cells. Among the tested DOACs, EDX showed the strongest inhibition of B16 cell metastasis in mice, likely via the suppression of inflammation, angiogenesis, and EMT mediated by the FXa-dependent PAR2 and TGFβ pathways in tumor and surrounding tissue cells.
Thrombin-antithrombin complex (TAT), a2-plasmininhibitor-plasmin complex (PIC), thrombomodulin (TM), tissue plasminogen activator-plasminogen activator inhibitor complex (t-PAIC) has been increasingly applied in clinical practice in recent years, especially in the diagnosis and treatment of diseases associated with thrombosis and hemorrhage. However, there is no universally accepted evaluation standard for the performance verification of these four indicators currently. Therefore, we designed experiments to verify the precision, trueness, carryover, linearity, and reference intervals of these four indicators. This study is expected to provide references for subsequent research in terms of data and experimental methods. According to the Clinical and Laboratory Standards Institute (CLSI) guidelines EP15-A2, EP06-A, and C28-A, the precision, trueness, carryover, linearity, and reference intervals were evaluated. The within-laboratory CVs of TAT, PIC, TM, and t-PAIC were 3.67, 6.51, 3.64, and 2.46% on Control L and 4.68, 4.67, 5.08, and 3.87% on Control H. The assigned value of calibrations of TAT, PIC, TM, and t-PAIC were all included in the verification intervals. The biases of the four items of Calibration 1 were -6.67, -0.90, -3.58, and -6.78% and biases on Calibration 2 were -2.70, 1.63, 2.66, and -1.16%, respectively, compared with the assigned value provided by the manufacturer. The carryover rate of each indicator was less than 1%. Within the range that meets clinical use, the best fit curves of the four indicators were linear, and the correlation coefficients of all indicators were greater than 0.99. The reference intervals provided by the manufacturer were appropriate in our laboratory. The performance of HISCL-5000 analyzer for TAT, PIC, TM, and t-PAIC analysis were acceptable and the systems were suitable for clinical analysis.
Recurrent thrombosis poses a clinical challenge in patients with antiphospholipid syndrome (APS). There are limited data on risk factors due to its rarity. This study aimed to study the association between cardiovascular (CV) and APS-related risk factors and recurrent thrombosis and evaluate the adjusted Global Anti-Phospholipid Syndrome Score (aGAPSS). This retrospective cohort study comprised APS patients at Karolinska University Hospital, Sweden, from 2014 to 2020 with follow-up until the last medical visit or death. Multiple thrombotic events per patient were included. Cox proportional hazard model estimated hazard ratios (HRs) and 95% confidence intervals (CIs). Logistic regression and Poisson regression were conducted to further examine the relation between risk factors and recurrent thrombosis. The cohort included 250 patients (67% women and 52% primary APS) with a median age of 44.5 (35-59) years. Forty-nine recurrent thrombotic events occurred in 36 patients, yielding an incidence of 4.46 (95% CI 3.30-5.90) per 100 person-years. Thrombocytopenia was associated with recurrent thrombosis (HR 2.57 [95% CI 1.01-6.02]). Although CV risk factors were not consistently significant for recurrent thrombosis, chronic kidney disease (CKD) indicated an increased probability (OR 2.55 [95% CI 1.01-6.26]). For each point of aGAPSS, the HR for recurrent thrombosis increased by 10% (1.10 [95% CI 1.01-1.19]). Notably, inadequate anticoagulation triggered recurrence in almost a quarter of cases. Thrombocytopenia was confirmed as a major risk factor for recurrent thrombosis. CKD warrants closer attention in future assessment. Although an increase in aGAPSS was associated with recurrent thrombosis, further evaluation is required. Improving anticoagulation treatment is essential to reduce recurrence.
New therapies for haemophilia A have created momentum for enhancing protection against bleeding. The ultimate objective is to address remaining unmet needs and promote health equity. In this initiative, 14 haemophilia A experts from Europe ( n  = 13) and North America ( n  = 1) were involved to identify unmet needs in people with haemophilia A (PwHA) and measures to address these needs (e.g., normalization of factor VIII [FVIII] levels) to facilitate health equity. It combined online workshops, a modified Delphi process to develop consensus statements on remaining unmet medical needs (consensus was defined as ≥70% of panellists who gave a rating of 4 [agree] or 5 [strongly agree] using a 5-point Likert scale), and development of policy recommendations. Among the panellists who voted, consensus was reached on 25/26 statements, including 13 with 100% agreement. The experts outlined a need to optimize prophylaxis in all eligible PwHA, aiming for high-sustained FVIII levels or normalized haemostasis. The panel also highlighted a need to prevent all bleeds (including microbleeds) that can occur despite prophylaxis, and to address chronic pain which is highly prevalent. Overall, 23 policy recommendations were finalized, which included the wider use of prophylaxis to reduce the burden of disease on PwHA and/or provide normalized haemostasis, the expansion of multidisciplinary teams and the establishment of networks of expertise, and sharing of specialist knowledge. This initiative lays an ambitious foundation to rigorously evaluate unmet needs in PwHA and the determinants of health equity, offering a comprehensive set of recommendations to overcome barriers and achieve the possibility of normalization of haemostasis.
Hemophilia A (HA) is an X-linked disorder characterized by a deficiency of factor VIII (FVIII). Approximately 30% of patients with severe HA (baseline FVIII levels < 1 IU/dL) develop antibodies that neutralize the activity of FVIIIs. Emicizumab, a bispecific monoclonal antibody that has FVIII mimetic activity, is effective at preventing bleeding in patients with HA and has become a standard prophylactic therapy for hemophilia patients with inhibitors. However, breakthrough bleeding and perioperative management still require additional bypassing agents, which increases the risk of thrombosis in these patients. This in vitro study compares the hemostatic effect of adding factor IX (FIX) to the plasma of HA patients on emicizumab prophylaxis with the addition of bypassing agents. Blood from 24 HA patients on emicizumab was collected, and plasma samples were spiked with increasing concentrations of recombinant factor VIIa (rFVIIa), activated prothrombin complex concentrate (aPCC), recombinant FIX (rFIX), and plasma-derived FIX (pdFIX). Thrombin generation (TG) was assessed using calibrated automated thrombography. Plasma emicizumab and FIX activity were measured. TG capacity improved significantly with increasing concentrations of rFVIIa ( p  < 0.0001), aPCC, rFIX, and pdFIX ( p  < 0.005). In contrast to high doses of aPCC and rFVIIa, TG parameters obtained with the addition of FIX did not cross the upper limit of normal physiologic ranges. Our data suggest a possible increased risk of thrombosis with higher concentrations of rFVIIa and aPCC, while adjunctive FIX therapy appears to be safe and effective in improving coagulation in HA patients with inhibitors on emicizumab prophylaxis. Future clinical trials are needed to confirm these results.
Incomplete thromboxane suppression with 81 mg aspirin daily is common and predicts higher vascular risk. We compared the effects of aspirin in controls and patients with cardiovascular disease and tested strategies to improve thromboxane suppression. Two-phase randomized, open-label pharmacodynamic study. In Phase 1, controls and patients received enteric-coated (EC) aspirin 81 mg daily for 3 weeks. In Phase 2, the best responders within each cohort were randomized to EC aspirin 81 mg daily, EC aspirin 81 mg alternate daily, or soluble aspirin 81 mg daily, and the poorest responders to EC aspirin 81 mg daily, 325 mg daily, or 162 mg twice daily. Where results with EC and soluble aspirin 81 mg daily were similar, these arms were pooled for comparisons of daily versus alternate daily dosing. Outcome measures were light transmission aggregation in response to arachidonic acid, adenosine diphosphate, and collagen; serum thromboxane B2 (sTXB2); and urinary 11-dehydro-thromboxane B2 (uTXB2; creatinine-normalized). We enrolled 136 controls and 115 patients. Among controls, EC aspirin 81 mg daily suppressed platelet aggregation and reduced sTXB2 from 238.4 to 2.7 ng/mL and uTXB2 from 112.0 to 39.0 ng/mmol creatinine (all p  < 0.0001). After 3 weeks, aggregation and serum thromboxane were similar in controls and patients but uTXB2 remained higher in patients (46.5 vs. 39.0, p  = 0.004). In Phase 2, different aspirin formulations, doses, and dose frequencies did not materially affect platelet aggregation. In the best responders, EC and soluble aspirin had similar effects on thromboxane, but sTXB2 levels were higher with alternate daily versus daily dosing (pooled EC and soluble) in both controls (8.1 vs. 2.2 ng/mL, p  = 0.005) and patients (5.2 vs. 1.7 ng/mL, p  = 0.048). In the poorest responders, EC aspirin 162 mg twice daily produced the greatest suppression of both sTXB2 and uTXB2, with intermediate suppression with EC aspirin 325 mg daily and the least with EC aspirin 81 mg daily (all trend P values <0.05). Daily low-dose aspirin suppresses aggregation and serum thromboxane, but urinary thromboxane metabolites are less completely suppressed in patients with established cardiovascular disease. Alternate daily dosing attenuates thromboxane suppression, whereas divided dosing improves suppression in the poorest responders.
Venous thromboembolism (VTE) can considerably limit patients' functioning and quality of life. Using patient-reported outcome measures (PROMs), the full impact of VTE on individual patients can be captured. To evaluate the experiences of patients and healthcare professionals with the routine use of PROMs for VTE patients visiting the outpatient clinic, a mixed-methods study was performed at Leiden University Medical Center, the Netherlands. VTE PROMs were incorporated into routine care since March 2023, through a digital application sending patients invitations to complete PROMs. Quantitative and qualitative data were obtained from semi-structured interviews with patients and involved healthcare professionals. The NoMAD (normalization measure development) questionnaire was used to assess the implementation process from the professionals' perspective. Patients aged ≥18 years who experienced VTE and completed PROMs at two follow-up time points during ≥3 months follow-up and VTE patients who did not complete PROMs at both time points were asked to participate. Eight patients (five completed PROMs; three did not) and four professionals were interviewed. Both patients and professionals experienced the use of PROMs as neutral to predominantly positive (lower limit 3 on a scale of 1-5). All professionals valued the effects of PROMs on their work. Most patients felt the questionnaires contained too many questions. Suggestions to improve the completion rate, accessibility, PROMs content, and the digital tool were shared. PROMs were believed to provide additional value during preparation for the appointment and during the consultation. The first experiences of patients and professionals, tending toward positive, can be used to improve PROMs application and support implementation in routine thrombosis care.
The development of inhibitors to von Willebrand factor (VWF) is a rare but potentially serious complication of VWF replacement therapy in patients with von Willebrand disease (VWD). Patients who develop VWF inhibitors may become unresponsive and/or may develop severe anaphylactic reactions to VWF concentrates. Data on inhibitor development and management in VWD remain limited, and better understanding of inhibitor development is an important goal in VWD management. The WIL-31 study demonstrated the efficacy and safety of prophylaxis with wilate, a plasma-derived VWF/factor VIII (pdVWF/FVIII) concentrate, in children and adults with VWD of all types. The annualized bleeding rate (ABR) was reduced by 84% with wilate prophylaxis compared with on-demand treatment, and prophylaxis was well tolerated. No inhibitors developed during the WIL-31 study. Here, we report two brothers with type 3 VWD who at the 6-month visit were found to have VWF inhibitors, which on further investigation were found to have already been present before the study. Despite the presence of inhibitors, neither patient showed any clinical symptoms, and prophylaxis with wilate led to a ≥85% reduction in ABR in both boys compared with on-demand treatment.
To describe an innovative anticoagulation strategy in a 20-year-old woman with innate jejunal atresia and ultrashort bowel syndrome who was dependent on long-term parenteral nutrition and suffered from multiple venous thrombotic events and bleeding complications since infancy. Single-patient case report. Dresden University Hospital, Dresden, Germany. Being fully CVC-dependent since birth, our patient repeatedly developed catheter-related thrombosis (CRT) since infancy and was treated with daily low-molecular-weight heparin injections for more than 15 years. Despite this, clotting, severe gastrointestinal bleeding, and osteoporosis remained a persistent problem, causing numerous hospitalizations over the years, significant developmental delays, and a decline in the patient's body mass index (BMI). A short period of rivaroxaban treatment had to be stopped owing to acute gastrointestinal bleeding. After the failure of all approved anticoagulant concepts, compassionate use access was granted to the investigational drug osocimab, a human monoclonal antibody inhibitor of factor XIa. Hereditary FXI deficiency as well as FXI inhibition in animal models have been shown to reduce arterial and venous thrombosis without increasing bleeding. Consistent with this, short-term osocimab treatment has shown clinical efficacy in preventing postoperative venous thromboembolism after knee replacement surgery and in reducing dialysis conduit clotting compared with placebo in patients undergoing hemodialysis, without increasing the rate of clinically relevant bleeding versus comparators. After initiating osocimab, the patient experienced no further clotting complications, and bleeding decreased in frequency and severity. The patient's BMI decline immediately stopped; her weight increased by over 10% in the subsequent 20 months, and menstruation started 3 months later without signs of menorrhagia. Now, with 2.5 years of uninterrupted exposure outside of a clinical trial, this patient has experienced the longest duration of factor XIa inhibition to date. She continues to receive osocimab under the compassionate use program and maintains a positive change in her well-being and quality of life.
Objective To assess the effectiveness of recombinant factor VIII Fc fusion protein (efmoroctocog alfa; referred to herein as rFVIIIFc) prophylaxis in people with hemophilia A (PwHA) stratified by age, body mass index (BMI), disease severity, and inhibitor history included in the A-SURE (NCT02976753) and PREVENT (NCT03055611) real-world studies. Methods PwHA receiving at least one prescription of rFVIIIFc and ≥3 months of follow-up during the prospective periods were included. Post hoc analyses were performed for the overall analysis population and subgroups based on age, BMI, disease severity, and those with and without previous inhibitors. Effectiveness endpoints were annualized bleeding rate (ABR), annualized joint bleeding rate (AjBR), injection frequency, and factor consumption in the prospective period. Results Overall, 333 PwHA were analyzed. ABRs and AjBRs, generally, were low across the different subgroups (range of medians, 0.0-0.6 for both ABRs and AjBRs, except for ABRs in PwHA aged ≥65 years). Injection frequencies ( n injections/week) were comparable across subgroups (range of medians, 2.0-2.3), and factor consumption was generally similar (range of medians, 68.3-100.0 international units/kg/week), although with slightly higher factor consumption in pediatric PwHA and lower consumption in the overweight or obese BMI groups. Conclusion These post hoc analyses support the effectiveness and, therefore, use of rFVIIIFc prophylaxis in PwHA across all ages, BMI categories, severities, and for those with a history of inhibitors.
Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone encoded by the SERPINH1 gene, has emerged as a groundbreaking focus in thrombosis research. Recent findings published in "Science" have revolutionized our understanding of thrombosis, identifying HSP47 as a critical mediator in a new thrombosis target for treatment. This discovery not only unveils a novel pathway in thrombosis but also opens new avenues for therapeutic intervention. HSP47's significance extends beyond thrombosis, influencing pathological processes such as fibrosis and cancer. In fibrosis, its upregulation promotes collagen deposition, while its dysregulation in osteogenesis imperfecta (OI) Type X underscores the protein's indispensable role in collagen biosynthesis. The therapeutic challenge lies in balancing HSP47 inhibition to reduce fibrotic burden without impairing its essential physiological functions. In cancer, HSP47 plays dual roles. It supports tumor progression through collagen stabilization and metastasis facilitation while contributing to tissue repair under hyperthermia treatment combined with radiotherapy or chemotherapy. However, its overexpression can exacerbate tumor aggressiveness via mechanisms such as angiogenesis and epithelial-mesenchymal transition. This review emphasizes the pivotal discovery of HSP47's thrombogenic role and its broader implications in disease biology. These findings mark a paradigm shift in thrombosis research and underscore the potential of HSP47 as a target in diverse pathological contexts, from platelet-driven diseases to fibrotic and oncological disorders.
Platelets and certain immune cells contain releasable granules that play essential roles in hemostasis and immune regulation. However, the regulatory mechanisms governing degranulation of platelets versus immune cells remain incompletely understood. This study aimed to elucidate the distinct regulatory mechanisms underlying granule release in platelets and immune cells. Mouse platelets were freshly isolated from peripheral blood, neutrophils were purified from bone marrow, and mast cells were generated from bone marrow progenitors by in vitro differentiation. The effects of phorbol ester (PMA), ionomycin, and their combination on degranulation of platelets, neutrophils, and mast cells were examined. In addition, pharmacological inhibitors targeting key components of multiple signaling pathways were used to investigate the molecular mechanisms regulating degranulation of these three cell types. PMA and ionomycin strongly induced α-granule release of platelets, whereas only moderately stimulating dense-granule secretion. For neutrophils, PMA and ionomycin each triggered moderate azurophilic-granule release, whereas their combination markedly enhanced degranulation. In contrast, PMA alone failed to induce mast cell degranulation, ionomycin robustly stimulated granule release, and their combination further amplified ionomycin-induced response. Mechanistically, thrombin-induced platelet degranulation was inhibited exclusively by phospholipase C (PLC) and protein kinase C (PKC) inhibitors. Zymosan-induced neutrophil degranulation was suppressed by inhibitors of phosphoinositide 3 kinase, spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), mitogen-activated protein kinase 1/2 (MEK1/2) and P38 but was enhanced by PLC and PKC inhibition. Mast cell degranulation induced by IgE and antigen was significantly inhibited by the inhibitors targeting SYK, BTK, PLC, and PKC. These findings reveal substantial differences in the regulatory pathways controlling degranulation in platelets and immune cells. Such distinctions highlight the opportunities for the development of cell type-selective inhibitors to modulate degranulation, providing potential therapeutic strategies for thrombotic diseases and immune-related diseases.
Central venous catheter (CVC) insertion is a cornerstone procedure in hospitalized and critically ill adults. However, many patients requiring CVCs have coagulopathy, thrombocytopenia, liver disease, or hematologic malignancies, raising concerns about bleeding risk. The true incidence of hemorrhagic complications and the value of preventive measures in these populations remain uncertain. The objective of this study is to systematically evaluate the incidence of bleeding related to CVC placement in adults at increased hemorrhagic risk and to assess the effectiveness of periprocedural preventive strategies. PubMed, Embase, Cochrane Library, and Web of Science were searched from January 2000 to March 2025. Randomized trials and observational studies involving adults with elevated bleeding risk undergoing CVC placement were included. Data extraction and risk of bias assessment (RoB 2 and Newcastle-Ottawa Scale) were performed independently by two reviewers. Certainty of evidence was rated using GRADE (Grading of Recommendations Assessment, Development, and Evaluation), and random-effects meta-analyses were conducted when appropriate. Forty-one studies encompassing 7,603 patients and 8,796 CVC insertions were analyzed. Major bleeding occurred in 0.57% of procedures and minor bleeding in 8.1%. The pooled incidence of any bleeding across 22 studies was 6.8% (95% confidence interval, 3.7-10.7%). Bleeding was more frequent among patients with hematologic malignancies, severe thrombocytopenia, or critical illness. Ultrasound guidance markedly reduced complications compared with landmark technique. Platelet transfusion was effective only below 30 × 10 9 /L, whereas fresh-frozen plasma showed no clear benefit. CVC placement in adults with coagulopathy or thrombocytopenia is generally safe. Ultrasound guidance, restrictive transfusion thresholds, and thromboelastography-guided assessment enhance procedural safety and reduce unnecessary transfusions.
DNA methylation modifies nucleotides, regulating gene expression without sequence change. The effects of DNA methylation within blood cell lineages on the development and function of endomitotic high DNA copy megakaryocytes (MKs) to anucleate platelets remain poorly understood. We sought to characterize this potential relationship by investigating associations between platelet function and methylation in circulating nucleated blood cells. Data were measured in the Framingham Heart Study Third Generation cohort ( n = 1,314). Five bioassays assessed platelet function in response to up to seven agonists in whole blood and platelet-rich plasma, and the Illumina 450K array was used to conduct an epigenome study of blood DNA methylation. In adjusted statistical association models, we found 46 significant associations (false discovery rate-adjusted p < 0.05) across 36 genomic DNA methylation sites, including cg24267699 in a putative regulatory site -742/-743 bases upstream from the ABO transcription start site associated with ristocetin platelet agglutination ( β = 0.21, standard error = 0.03, p < 1.04E-11). The 36 sites collectively reside within genes acting as transcription factors, genes implicated in granule release and exocytosis, cytoskeletal functions, mitochondrial function, and platelet function. The set of associated cytosine-phosphate-guanines was enriched in MKs for markers of regulatory activity, including DNase-I hypersensitivity sites and histone activity. Overall, we report in the first such epigenome scan that blood cell DNA methylation appears to be significantly associated with several platelet reactivity traits, and may drive the regulation of key genes involved in those processes presumably at the upstream level of hematopoietic stem cells or megaerythroid lineage cells.
Although the association between platelet characteristics and the risk of developing atherosclerosis (AS) has been acknowledged, the specific role of platelets in AS development and progression remains unclear. Therefore, the aim of this study was to identify platelet characteristics in patients with and without AS to enhance the understanding of their pathophysiological functions and discover more sensitive biomarkers for AS diagnosis. We conducted a cross-sectional study involving AS patients and healthy controls (N). Based on the Chinese guidelines for diagnosing carotid and vertebral artery AS and the 2010 American College of Cardiology Foundation/American Heart Association (ACCF/AHA) guidelines, we defined AS using carotid ultrasound to measure intima-media thickness (IMT). General information, including sex, age, height, and weight, was collected upon enrollment. A series of examinations, including physical exams, serum lipid profiles, blood glucose tests, liver and kidney function tests, platelet aggregation assays, and carotid artery ultrasounds, was performed. Platelets were extracted from plasma for RNA-seq analysis. No statistically significant differences in age, sex, body mass index, or blood pressure were observed between the groups. Total triglyceride, total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B, red blood cell count, hemoglobin concentration, cholesterol levels, and carotid IMT were significantly greater, and vascular endothelial function was significantly lower in the AS group than in the N group. Using RNA-seq, we identified 784 differentially expressed genes-141 downregulated and 643 upregulated-with Gene Ontology enrichment showing significant associations with blood coagulation pathways, among others. Weighted correlation network analysis revealed four hub genes related to IMT: Integrin Subunit Alpha 2b (ITGA2B), Transforming Growth Factor Beta 1 (TGFB1), Platelet Factor 4 (PF4) , and Glycoprotein IX Platelet (GP9) . Our findings indicate moderate correlations of elevated ITGA2B ( r  = 0.327, p  = 0.004), TGFB1 ( r  = 0.362, p  = 0.001), PF4 ( r  = 0.240, p  = 0.038), and GP9 ( r  = 0.302, p  = 0.008) levels with increased IMT, suggesting that these genes may serve as predictive biomarkers for AS.
Limited data are available on the relationship between bleeding outcomes and physical activity, and the quality of daily life (QoL), in children with haemophilia A (HA) receiving emicizumab prophylaxis. TSUBASA evaluated physical activity, bleeding events, safety, and QoL in Japanese people with HA initiating emicizumab prophylaxis. This paper reports the results from the final analysis, focusing on children and adolescents with HA without factor VIII inhibitors, and their caregivers. TSUBASA was a prospective, multicentre, observational study conducted across 50 medical institutions in Japan. Participants received emicizumab for 97 weeks. Bleeding events and physical activity data were obtained using an electronic patient-reported outcomes application; activity intensity was collected by wearable activity trackers worn over five 8-day monitoring periods. Adverse events (AEs) were documented on the electronic case report form and QoL was assessed using questionnaires. A total of 46 participants aged <18 years were enrolled; most (84.8%) had severe HA. Over a median observation period of 674 days (quartile 1-quartile 3: 665-690), the mean annualized bleed rate was 0.86 (standard deviation: 1.26). In all Twenty-six participants experienced 66 AEs, of which 2 were injection-site reactions deemed related to emicizumab. J-KIDSCREEN-52 questionnaire scores were maintained from baseline onwards. Of the completed caregiver questionnaires ( n  = 32), 43.8% reported increased activity and 56.3% reported unchanged activity. Additionally, 56.3% reported decreased anxiety about bleeding and 37.5% reported unchanged anxiety about bleeding. A total of 172 events of physical activity were recorded by 19 participants; 44 were high risk, 70 were moderate risk, and 42 were low risk. One activity-related traumatic bleed resulting from the impact of a basketball occurred, and more than 25 different types of physical activity were performed without bleeding. Children and adolescents with HA who receive prophylaxis with emicizumab may be able to engage in consistent physical activity, with a low risk of experiencing bleeds. Additionally, the questionnaire responses from caregivers on physical activity and caregiver experience provide rare insights into the real-world impact of emicizumab prophylaxis. These findings present a more comprehensive view of the benefits of emicizumab. No new safety signals were observed and QoL was maintained for 2 years after emicizumab initiation.
Approximately 20% of patients with cancer will have cancer-associated venous thromboembolism (CAT), which is associated with significant morbidity and mortality. Despite its clinical importance, CAT awareness in cancer patients and caregivers remains low. We sought to assess the patients' knowledge of CAT through a national survey. A survey assessing knowledge of different aspects of CAT was developed by a steering committee including four clinicians with expertise in CAT and a patient partner with lived experience. Survey dissemination among patients with cancer occurred through the Environics network, the Thrombosis Canada member network, the Thrombosis Canada social media platforms, and was advertised through Instagram and Facebook, and the Canadian Cancer Survivor Network newsletter. Out of the 312 patients with cancer or survivors who responded to the survey, 179 (57.4%) were female, and 118 (37.8%) were over 65 years old. Overall, 119 patients (38.1%, 95% confidence interval [CI]: 37.7-49.8%) reported having no knowledge of CAT. Only 84 (26.9%, 95% CI: 22.1-32.2%) and 94 (30.1%, 95% CI: 25.1-35.6%) patients reported receiving education about their underlying risk of CAT or education about signs and symptoms of venous thromboembolism, respectively. A total of 66 (21%, 95% CI: 16.8-26.1%) patients reported being informed by a health care professional about considering thromboprophylaxis. Patients were interested in learning more about the risk of CAT, its associated risk factors, and the benefits and potential side effects of thromboprophylaxis. Many patients with cancer lack awareness or knowledge of CAT. Our results highlight ongoing education and awareness of the CAT burden.