Background Metastasis formation is a multistep process and requires a complex interplay between tumor and host cells. Besides their key role in coagulation and maintaining hemostasis upon injury to vasculature, recent evidence indicates that platelets contribute to cancer progression. Objectives However, the molecular mechanisms of platelet activation during metastasis formation is incompletely understood. Here we aim to identify the role of platelet spleen tyrosine kinase (SYK) in the process of solid tumor metastasis. Methods To test the role of SYK in platelets, we generated platelet-specific deletion of Syk by crossing PF4-Cre and Sykflox/flox mice to obtain Pf4Cre/+Sykflox/flox animals (referred to as SykΔPlt mice), as well as used SYK-selective inhibitors, such as fostamatinib (R788), entospletinib (GS-9973) and lanraplenib (GS-9876) Results Based on our results, genetic deletion of Syk in platelets protects mice from metastasis. Administration of malignant melanoma and colorectal adenocarcinoma cells into SYK-deficient platelet-bearing SykΔPlt mice results in significantly less metastatic tumor deposits in the animals compared to wild-type mice. Further, Syk-expressing - but not SYK-deficient - platelets secrete thromboxane A2 (TXA2), that blocks the proliferation and effector function of CD8+ T cells. Pharmacological inhibition of SYK or TXA2 production in platelets results in decreased gastrointestinal tract and lung metastasis formation as well as increased number of CD8+ T cells. Conclusions Taken together, our results indicate, that SYK in platelets promotes solid tumor metastasis and tumor immunosuppression. Secretion of TXA2 by platelets is SYK-dependent and can be inhibited by SYK-selective inhibitors, which release anti-tumor CD8+ T cells from TXA2-mediated suppression.
BACKGROUND:Fibrin network architecture governs clot stability, permeability, and susceptibility to fibrinolysis, yet nanoscale characterization of hydrated fibrin structures remains challenging. Here, we apply expansion microscopy (ExM) for the first time to enable subdiffraction-resolution imaging of fixed, hydrated fibrin architecture and determine how fibrin structure varies with thrombin, regulates clot permeability, and remodels during fibrinolysis. METHODS:Fibrin structure in human platelet-free plasma clots was quantified across thrombin concentrations (0.3-1.5 U/mL) and during tissue-type plasminogen activator-induced internal fibrinolysis using ExM, with validation against confocal, stimulated emission depletion (STED), and scanning electron microscopy (SEM). Fiber diameter, pore size, porosity, and clot permeability were measured to relate nanoscale structure to macroscopic transport. RESULTS:Increasing thrombin produced progressively thinner fibers, without altering network porosity, indicating redistribution of fibrin mass at constant void fraction. In contrast, pore area decreased monotonically and exhibited a strong linear relationship with clot permeability, suggesting that pore geometry, rather than bulk porosity, is closely associated with hydraulic transport under constant fibrinogen concentration. Fiber diameters measured by ExM closely matched those by STED and were systematically larger than SEM values, consistent with preservation of hydrated ultrastructure. During fibrinolysis, ExM resolved spatiotemporal remodeling of fibrin networks, including fiber thinning during active lysis followed by increased heterogeneity, apparent fiber bundling, and marked pore enlargement in late lysis. CONCLUSIONS:These findings define a nanoscale structural sequence linking thrombin-regulated polymerization and fibrinolytic degradation to macroscopic clot function and establish ExM as a robust platform for quantitative analysis of fibrin architecture in hydrated plasma clots.
Background In the general population, ageing is associated with frailty. Screening is recommended as targeted interventions improve health outcomes. Frailty screening was incorporated within routine clinic appointments for persons with haemophilia (PWH) aged ≥50 years at our large UK Haemophilia Comprehensive Care Centre (HCCC) from 2024. Objectives To determine the prevalence of frailty in PWH aged ≥50 years. Methods Electronic health records of PWH ≥50 years were reviewed (2024-2025) to collect data on prospective Clinical Frailty Score (CFS), comorbidities, and regular medications. Results Of 116 PWH aged ≥50 years, 111 PWH had CFS documented. Frailty (CFS≥4) occurred in 24%. Amongst those aged 50-64 years (n=66), 18% were frail (11 severe and 1 moderate haemophilia); of those aged ≥65 years (n=45), 33% were frail (4 severe, 2 moderate, 9 mild haemophilia).Frailty was associated with high rates of multi-morbidity (100%) and polypharmacy (78%). Haemophilic arthropathy appeared to be a significant contributory factor in some patients. Of those with CFS≥4, falls were reported in 26%, with immobility identified in 11%, cognitive impairment in 4%, and no incontinence documented.Specific clinical actions by the haemophilia team included haemophilia physiotherapy assessment and highlighting CFS to the primary care practitioner. Conclusion PWH have higher rates of frailty than reported for the general UK population (33% vs 10% for ≥65 years), and a high incidence in younger PWH. Haemophilic arthropathy appears to be a major contributing factor in some patients. Further studies are needed to clarify the impact of identification of frailty in PWH.
Background The coagulation factor V (FV) variant derived from the venom of the Australian snake Pseudonaja textilis (ptFV) has several unique procoagulant adaptations that circumvent the normal regulatory mechanisms. Notably, ptFV comprises a significantly extended A2 domain C-terminus (A2T), a region of which the role in human FV biology is poorly understood. Objectives In this study, we generated chimeric FV variants to elucidate the functional relevance of this extended structural region. Methods The ptFV A2T was exchanged for the homologous human FV region and vice versa, thereby generating ptFV-hA2T and hFV-ptA2T. Results Surprisingly, our findings demonstrate that enhanced procoagulant activity of ptFV is mediated by the A2T. Using this structural element, ptFV bypasses the need for membrane binding by facilitating productive interactions with P. textilis venom FXa (ptFXa) and prothrombin in solution. Substitution of the human A2T for the corresponding region in ptFV enabled human FVa to function in the absence of membranes, in a similar fashion to ptFVa, although this required complex formation with ptFXa, suggesting the presence of an essential element in the venom protease. In addition, we observed that the ptFV A2T region significantly contributes to the functional activated protein C-resistance. Unlike in mammalian FVa, a ptFVa variant comprising the human FV A2T retained structural integrity despite loss of cofactor function. Conclusions Taken together, the ptFV A2T represents an exceptional structural element driving the enhanced procoagulant functions that are at the basis of the snake venom’s extreme toxicity.
Background Antiphospholipid syndrome (APS) is a thrombotic autoimmune disease associated with persistent presence of antiphospholipid antibodies. A range of mechanisms have been implicated in the clinical manifestations of APS, but it is unclear which processes are dominant in vivo, although all mechanisms take place in the circulation. Exploring the plasma protein profile may provide leads for further research. Objective To assess plasma protein abundances in clinically stable APS patients compared with controls without APS across two cohorts. Methods The discovery cohort included 61 APS patients and 19 controls, and the verification cohort included 77 APS patients and 49 controls. Quantitative protein mass spectrometry was used to measure 159 plasma proteins, including proteins involved in blood coagulation, complement pathway, transport, apolipoproteins, and immunity. Proteins were quantified in plasma with use of stable isotope-labeled peptide standards. Data were analyzed using a Welch’s t-test. Ten proteins with the lowest p-value from the discovery cohort were selected for validation in the verification cohort. Results Of the ten selected proteins, six were differentially abundant between APS patients and controls without APS in the verification cohort and showed consistent fold changes across both cohorts: alpha-1-acid glycoprotein 1, beta-2-microglobulin, complement C2, insulin-like growth factor-binding protein complex acid labile subunit, leucine-rich alpha-2-glycoprotein 1, and cystatin-C. Conclusions We identified six plasma proteins that were differentially abundant in APS patients compared with controls without APS and confirmed these associations in an independent verification cohort. Collectively, these proteins may reflect subclinical inflammation and renal involvement in APS.
We report the first case of emicizumab prophylaxis in refractory acquired von Willebrand syndrome (AVWS) secondary to multiple myeloma (MM). A 72-year-old woman with IgG kappa MM and gastrointestinal angiodysplasia suffered recurrent life-threatening hemorrhage unresponsive to multimodal therapies, including factor concentrates, intravenous immunoglobulin, plasmapheresis, and endoscopic interventions. Subcutaneous emicizumab combined with octreotide, ongoing anti-myeloma therapy, and previous endoscopic control achieved complete cessation of bleeding despite persistently undetectable von Willebrand factor activity. Over 12 months of follow-up, the patient maintained a very good partial response and remained transfusion-free with an ECOG performance status of 0. This case suggests that emicizumab-mediated restoration of secondary hemostasis can provide sustained bleeding control in refractory AVWS when combined with a multidisciplinary approach, offering a valuable therapeutic option when conventional hemostatic measures fail.
BACKGROUND:CYCS variants cause a rare autosomal dominant nonsyndromic thrombocytopenia: thrombocytopenia 4 (THC4). OBJECTIVES:This study aimed to determine the genetic basis of thrombocytopenia in a Chinese family and to summarize the clinical spectrum of CYCS-related thrombocytopenia. METHODS:Clinical data and laboratory findings, including complete blood counts, thromboelastography, platelet aggregation, and surface glycoprotein expression, were collected. Whole-exome sequencing followed by Sanger sequencing was performed to identify disease-associated variants. In silico pathogenicity prediction analyses were conducted. A systematic literature review of previously reported CYCS-related thrombocytopenia was also performed. RESULTS:Four affected individuals presented with isolated mild-to-moderate thrombocytopenia (platelet counts ranging from 54 to 83 × 109/L) with normal platelet size and morphology. Thromboelastography revealed decreased maximum amplitude. Mildly reduced responses to ADP, arachidonic acid, collagen, and ristocetin were observed in patients I-1 and II-2. Surface glycoprotein expression was normal. A heterozygous CYCS missense variant, c.292T>C (p.Y98H), cosegregated with thrombocytopenia across 3 generations. Structural prediction suggested protein destabilization. Individuals harboring variants within the central Ω-loop of cytochrome c had significantly higher platelet counts than those carrying variants in other domains. CONCLUSION:This study reports a Chinese pedigree with CYCS-associated thrombocytopenia, adding detailed clinical and laboratory data to the rare CYCS p.Y98H variant and expanding the known phenotypic and geographic spectrum of THC4. Domain-specific genotype-phenotype correlations in CYCS-related thrombocytopenia suggest a potential structural influence of mutation location on disease severity. Intrafamilial heterogeneity underscores the potential role of secondary genetic modifiers in modulating individual phenotypic traits, which warrants further scrutiny.
Background FIX exhibits significant extravascular distribution and binds type IV collagen in the subendothelial extracellular matrix in the extravascular space, which influences its function and pharmacokinetics (PK). Many hemophilia B patients carry missense F9 mutations that produce circulating, dysfunctional FIX protein (cross-reacting material; CRM). In murine hemophilia models, the presence (CRM+) or absence (CRM-) of dysfunctional FIX modifies the PK and hemostatic efficacy of exogenous FIX. We hypothesized that the PK parameters of EHL FIX products that have a significant (Fc-FIX) or negligible (PEG-FIX) extravascular distribution might be differently influenced by the presence of endogenous FIX antigen in the vascular and extravascular space. Aim To investigate the association of F9 variants and individual PK profile of patients treated with EHL FIX concentrates. Methods Individual PK profiles were extracted from the WAPPS-Hemo database, and causal F9 mutations were sourced from linked databases. Differences in PK parameters between CRM+ and CRM- groups were analyzed. Results Seventy individuals treated with EHL FIX, with known PK profile and gene mutation were included. Among rFIX-Fc recipients, CRM+ patients had significantly lower Clearance, longer Terminal half-life, higher In vivo recovery, and smaller Volume of distribution. No significant PK differences were found between CRM+ and CRM- patients receiving N9-GP. Conclusions The pharmacokinetics of EHL FIX products are influenced by their ability to access the extravascular space. For products with substantial extravascular distribution, patient CRM status further modulates pharmacokinetic behaviour.
BACKGROUND:GNE-related thrombocytopenia (GNE-RT) is a very rare disorder caused by biallelic variants in GNE, encoding a key enzyme for the sialic acids biosynthesis. Patients usually present severe thrombocytopenia and excessive bleeding. Knowledge about this condition is still poor. There are no recognized tools for diagnosis. Previous study of one patient showed reduced platelet half-life as a mechanism of thrombocytopenia; however, it is still unclear if the sialylation defect also impairs platelet biogenesis. OBJECTIVES:To gain insights into clinical aspects and platelet biogenesis in GNE-RT. METHODS:We investigated 4 novel GNE-RT patients (3 families). A recently standardized flow cytometry assay was applied to characterize platelet sialylation. Patients' megakaryocytes were cultured to study megakaryopoiesis and proplatelet formation. A validated three-dimensional bone marrow model was exploited to investigate platelet production. RESULTS:We characterized 3 novel GNE variants, demonstrating the pathogenicity of 2 VUS. In all individuals, platelet flow cytometry detected an obvious increase in RCA-1 and ECL lectins binding and decreased MAL-II binding. Sialylation of serum transferrin did not show straightforward alterations. Although presenting the sialylation defect, patients' megakaryocytes showed preserved differentiation, maturation, and proplatelet formation. Megakaryocytes generated ex-vivo a normal number of normal-sized platelets. Two patients received Eltrombopag, achieving a durable clinical response (38- and 72-month follow-up). CONCLUSIONS:The profound megakaryocyte sialylation defect induced by GNE variants does not affect platelet biogenesis. Platelet flow cytometry for RCA-1 and MAL-II binding is a reliable and simple assay for diagnosis of GNE-RT. Based on literature review, 55% of GNE-RT patients responded to thrombopoietin-mimetics.
The International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT) is widely used to screen inherited bleeding disorders. Adult reference ranges are age- and sex-specific. However, a single reference range (0-2) is currently applied to all individuals under 18 years. Adolescence' developmental changes and global differences in healthcare access may influence bleeding scores, necessitating validation in a large international pediatric cohort. We aimed to evaluate age- and sex-specific ranges for ISTH-BAT scores in individuals<18 years using international dataset. Under the auspices of two ISTH Scientific Subcommittees, healthy participants <18 were recruited from 7 countries across 5 continents. ISTH-BAT assessments were completed by trained investigators and standard protocol. Participants were stratified by age quartile, sex, country, and World-Bank-income. Reference ranges were defined using the 2.5th-97.5th percentile and assessed by 95% Confidence Interval. A total of 1,168 participants were included (mean age 7.7±5.2 years; 50.5% female). The overall reference range was 0-2. Quartile differences weren't significant (p = 0.065), but a significantly positive age-related trend of increase in BAT score >0 was observed (p < 0.001). Females more often had a bleeding score >0 (p = 0.02), with significant sex differences observed only among Q4 (p=0.003). The 2.5th-97.5th percentile for Q4 females was 0-3. Scores varied by country and income classification (p ≤ 0.001), but reference ranges remained stable. We conclude ISTH-BAT reference range of 0-2 is appropriate for individuals <18 years. For females (12-17 years), 0-3 may be considered and interpreted in conjunction with clinical context when guiding referral decisions.
BACKGROUND:Medical devices, such as central venous catheters and mechanical heart valves (MHVs), are associated with an increased risk of thrombosis. Clotting on these devices is initiated by contact with the system surface. Adsorbed factor (F)XII undergoes autoactivation and activates FXI, which then propagates coagulation. Abelacimab, asundexian, and milvexian are FXI or FXIa inhibitors currently undergoing or having completed phase 3 evaluation. OBJECTIVES:To compare the effects of abelacimab, asundexian, and milvexian on activated partial thromboplastin time (APTT), catheter-induced clotting, and device-induced thrombin generation. METHODS:The effects of abelacimab, asundexian, and milvexian on APTT (measured with HemosIL APTT-SP reagent), catheter-induced clotting, and thrombin generation induced by catheter segments or MHV leaflets were assessed in human platelet-poor plasma. Thrombin generation was monitored over time using a fluorogenic substrate, and peak thrombin and endogenous thrombin potential were quantified. RESULTS:Abelacimab, asundexian, and milvexian prolonged APTT and catheter-induced clotting time and attenuated device-induced thrombin generation in a concentration-dependent manner. At clinically relevant concentrations, abelacimab and milvexian had more pronounced effects on APTT, catheter-induced clotting, and device-induced thrombin generation than asundexian. Even at the highest concentration tested, asundexian showed less inhibition of device-induced thrombin generation than the other inhibitors. CONCLUSION:At the concentrations achieved by the dosing regimens evaluated in the phase 3 trials, abelacimab and milvexian had greater effects on APTT, catheter-induced clotting, and thrombin generation induced by catheters or MHVs than asundexian.
BACKGROUND:The risk of venous thromboembolism is significantly increased during the postnatal period, and pharmacological thromboprophylaxis remains the cornerstone of venous thromboembolism preventive measures. It is well recognized that many women struggle with the parenteral nature of low-molecular-weight heparin. OBJECTIVES:The aim of this phase 4 pharmacokinetic clinical trial was to characterize the maternal and breastmilk pharmacokinetics of rivaroxaban and edoxaban at steady state in volunteer women within 12 weeks of delivery. METHODS:Volunteer breastfeeding women were recruited and, following informed written consent, took rivaroxaban 20 mg daily or edoxaban 60 mg daily for 3 consecutive days, with matched maternal plasma and breastmilk samples obtained over 6 days. Noncompartmental pharmacokinetic analysis was conducted, including computation of the milk:plasma ratio at steady state. Additionally, physiologically based pharmacokinetic analysis was conducted, allowing simulation of expected feeding-infant plasma concentrations following maternal ingestion. ISRCTN registry: ISRCTN5783139. RESULTS:Four women took part, with 2 taking edoxaban and 2 taking rivaroxaban. Rivaroxaban distributed into human breastmilk at low concentrations; the mean breastmilk area under the concentration-time curve at steady state on day 3 was 583 ng/mL.h, and the milk:plasma ratio 0.2. Edoxaban distributed into human breastmilk mirrored the maternal plasma concentration; the mean breastmilk area under the concentration-time curve at steady state on day 3 was 1654 ng/mL.h, and the milk:plasma ratio was 0.9. Physiologically based pharmacokinetic simulations for both drugs show low infant plasma concentrations in fully breastfed infants. CONCLUSION:Our findings provide compelling evidence for using rivaroxaban in breastfeeding women from a feeding-infant's safety perspective. Testing rivaroxaban against low-molecular-weight heparin in a postnatal thromboprophylaxis trial should be considered to establish maternal efficacy and safety. Edoxaban use in breastfeeding women merits further investigation.
