As adult programs of newer anthrombotic agents, such as those directed against FXIa, are underway, there is both a pressing need and a great opportunity to optimize the design, conduct, and analysis of future pediatric trials. Recommendations for the development of new anticoagulation drugs for pediatric use were last published in 2015 on behalf of the ISTH SSC. The objective of the present manuscript is to provide updated guidance recommendations for the development of new antithrombotic drugs in children. Key recommendations include: 1) initiating pediatric pharmaceutical development (including oral formulation development) well before the intended start of pediatric clinical studies; 2) engaging multidisciplinary experts in pediatric thrombosis clinical care, clinical trials, and translational science; 3) employing extrapolation where appropriate, to assure that patient sample sizes in pediatric trials are no larger than necessary; 4) using modeling approaches to minimize patient sample sizes and optimize timing and frequency of blood sampling in pediatric PK/PD studies; 5) augmenting preclinical data from animal models (or non-animal approaches) with in vitro spiking studies using biospecimens obtained from relevant human pediatric populations; 6) addressing residual uncertainty in dose-finding, PK or PD from early-phase trials in the design of late-phase trials; and 7) committing to make all pediatric formulations found to be safe and efficacious in late-phase trials commercially available in all countries in which the trials are conducted. Additional guidance recommendations will be provided in a subsequent manuscript.

Abstract Persons with hemophilia (PwH) have lower bone mineral density (BMD) and increased fracture risk, but data for persons with von Willebrand disease (PwVWD) are limited. Biological mechanisms underlying altered bone health in bleeding disorders remain poorly defined. Primary aim: compare total BMD among PwH, PwVWD, and healthy controls. Secondary aim: characterize bone remodeling biomarkers and examine associations between BMD and simultaneous thrombin–plasmin generation (STP). In a 3-group, cross-sectional observational study of participants aged 5 to 45 years (PwH, n = 34; PwVWD, n = 45; and controls, n = 30), total BMD and bone mineral content (BMC) were assessed by dual-energy X-ray absorptiometry; trabecular and cortical parameters were assessed by peripheral quantitative computed tomography; serum procollagen type 1 N-terminal propeptide and C-terminal telopeptide of type I collagen were measured as markers of bone formation and resorption; and STP assays quantified coagulation and fibrinolytic activity. Associations with BMD z scores were evaluated using linear and multivariable regression. PwVWD had significantly lower BMD z scores compared with controls, whereas PwH showed a similar but nonsignificant trend. Both PwH and PwVWD had significantly lower BMC. In multivariable models, higher thrombin generation was associated with higher BMD z scores, whereas higher plasmin generation was associated with lower BMD z scores. In multivariable models, STP parameters were associated with BMD and accounted for a greater proportion of variance than models including clinical factors or bone turnover markers. PwH and PwVWD exhibit measurable deficits in bone health. Observed associations between thrombin-plasmin dynamics and BMD are hypothesis-generating and support further longitudinal and mechanistic studies of hemostasis-bone relationships.

STUDY QUESTION:To describe the blood product utilization during neonatal and pediatric extracorporeal membrane oxygenation (ECMO) and to determine whether its usage correlates with clinical outcomes of bleeding, thrombosis or mortality. DESIGN:Prospective observational study of neonatal and pediatric ECMO patients from September 2016, until January 2022. SETTING:Tertiary center for pediatric and neonatal ECMO. PATIENTS:One hundred and ten runs of ECMO, nine patients had two runs of ECMO during the same hospital admission. RESULTS:All patients received at least one red blood cell (RBC) transfusion. RBCs, as well as fresh frozen plasma (FFP), platelets and cryoprecipitate transfusion, as well as total donor exposure did not predict increased mortality in ECMO patients. Increased volume of platelets was associated with patients who bled, but not those who had a thrombosis or died. DISCUSSION:This prospective cohort study failed to demonstrate that the volume of red blood cells, FFP and platelets or total donor exposure was predictive of increased mortality. Of particular importance is the lack of increased volume of red blood cells transfused to patients who experienced major bleeding events compared to those who did not, suggesting that the trigger for transfusions may not be based on the clinical status of bleeding and resultant Hb in isolation.

BACKGROUND:Discovered in 1938, haptoglobin is a liver-produced α2-glycoprotein that binds free hemoglobin, mitigating its toxic effects, and serves as a biomarker for hemolysis. The current study, under the HAPPI Kids initiative, aimed to define paediatric reference intervals for haptoglobin through the collection of samples from healthy children and neonates, with the overall aim to improve diagnostic accuracy in young patients. METHODS:Blood was collected from 335 children aged 30 days to 18 years and 20 neonates from February 2015 through December 2016. Samples were analysed on 3 automated analysers: a) Roche Cobas c501/c502, b) Roche Cobas c701, and c) Beckman Coulter Unicel DXL 600/800. RESULTS:Age and sex specific haptoglobin reference ranges were reported with a 95% confidence interval. CONCLUSION:We report continuous age-specific reference intervals for serum haptoglobin in neonates and children. The data suggests that haptoglobin levels have limited clinical value in children under 13-15 years, as they are often naturally low and may fall below the assay detection threshold.

Objectives: Acute hypoxemic respiratory failure is a leading cause of death and disability in critically ill children. Pulmonary capillary barrier dysfunction, in large part, drives severity. We aimed to define how blood and tracheobronchial lavage (TBAL) fluid differentially affect pulmonary capillary barrier function and, as a second exploratory aim, identify protein mediators. Design: We performed a secondary analysis in April and May of 2024 of blood and TBAL samples from a previously completed observational cohort study of mechanically ventilated children collected from October 2018 to February 2020. Setting: Single PICU. Subjects: Stored blood and TBAL samples from 65 children requiring mechanical ventilation collected at 24 and 4872 hours after intubation. Interventions: None. Measurements and Main Results: We quantified inflammatory proteins in plasma and TBAL using Olink Target 48 Cytokine multiplex. We assessed changes in barrier function of cultured human pulmonary microvascular endothelial cells (HPMECs) using electrical cell-substrate impedance to measure transendothelial electrical resistance. Plasma samples from critically ill children significantly enhanced HPMEC barrier function and contained lower concentrations of proinflammatory cytokines compared with TBAL. Over time, the effects of plasma on barrier integrity diminished, while TBAL samples significantly improved barrier function and had reduced abundance of several proinflammatory cytokines. Notably, TBAL samples from children with severe lung injury augmented HPMEC barriers more than those from children without lung injury, highlighting distinct compartmental and disease-specific influences on HPMEC barriers. Conclusions: These unexpected findings reveal that blood and lung compartments contain differentially abundant proteins that exert distinct and evolving influences on pulmonary endothelial integrity in critically ill children. These results may inform on the timing and compartment-specific assessment of biomarkers, and eventually, delivery of therapies.

Abstract Background: Sickle cell disease (SCD) is a multi-system, life-threatening disorder that leads to a wide spectrum of clinical complications. It is characterized by a complex thromboinflammatory state that affects all components of Virchow's triad. The interplay between inflammation and coagulation leads to microvascular thrombosis, which is at the core of several complications associated with the disease including episodes acute chest (ACS), and vaso-occlusive (pain) crisis (VOC). However, patients with SCD exhibit substantial variability in disease phenotype and severity, and genotype-based stratification cannot fully capture the complexity of disease phenotype in this population. Biomarker data to inform predictions of disease phenotype and burden in children with SCD are lacking. Aim: To identify plasma markers of inflammation and endothelial damage predictive of the development ACS and VOC, and of recurrent (>1) hospital admissions for these complications. Methods: Clinical data and plasma samples from a multicenter, prospective, inception cohort study with biobanking of children with SCD were analyzed. Children (<21 years old) with SCD of any genotype admitted (March 2023 – April 2025) with an acute episode of ACS or VOC were included. Plasma samples were collected at the time of acute presentation (enrollment), during the convalescent period (6 weeks – 3 months after admission) and at the time of readmission for ACS and/or VOC in those with >1 admission during follow up. Clinical data were collected prospectively including demographic data, prior history of SCD severity and therapies, signs and symptoms at the time of presentation, and treatment received for the acute event. Inflammatory cytokines and cell adhesion molecules were detected in plasma using custom ProcartaPlex panels, on a Luminex™ 200 instrument (Invitrogen, Waltham, MA). Descriptive statistics were used to summarize the data. Biomarkers predictive of ACS, VOC or recurrent admissions were evaluated via univariate logistic regression, using odds ratios (OR) and 95% confidence intervals (CI). Results: A total of 43 children with SCD were included (ACS n= 22, VOC n=21). Mean age was 11.5 years, 35% were female, and most frequent SCD genotype was Hb-SS (72%). Children with ACS were younger compared to those with VOC (7.4 years vs. 12 years; p<0.001). They also had a higher frequency of Hb-SS/Sβ0thalassemia (81% vs. 71%), prior recurrent episodes of ACS (32% vs. 14%) and VOC (23% vs. 14%), and a higher frequency of asthma (41% vs. 29%), but these differences were not statistically significant. Children presenting with ACS had higher median plasma levels of C-X-C motif chemokine ligand 10 (CXCL10) at enrollment and readmission (8 ng/mL vs. 3.5 ng/mL, p=0.002; 6.7 ng/mL vs. 4.2 ng/mL, p=0.009, respectively), vascular endothelial growth factor A at enrollment (VEGFA; 45 ng/mL vs. 26 ng/mL, p=0.004) and vascular cell adhesion molecule-1 at readmission (VCAM-1; 324 ng/mL vs. 162 ng/mL, p=0.013) compared to those presenting with VOC. In logistic regression analysis, CXCL10 at enrollment remained independently associated with the development of ACS (OR= 1.13, 95%CI 1.01, 1.27; p=0.038). Sixty-three percent (n=27) of children had a single admission, 33% (n=14) had 2-3 admissions and 5% (n=2) had >3 admissions during follow up. Children with >1 admission more frequently had SS/Sβ0thalassemia genotype (94% vs. 70%, p<0.001) and received chronic blood transfusions (44% vs. 17%, p=0.032) compared to those with a single admission. Compared to children with a single admission, children with >1 admission had higher median plasma levels of several biomarkers including E-selectin, P-selectin and interleukin-6 but these differences were not statistically significant. Conclusions: In this analysis, we identified higher median plasma levels of CXCL10, VEGFA and VCAM-1 at the time of presentation with an acute episode of ACS compared to VOC in children with SCD. Elevated plasma CXCL10 was independently associated with the development of ACS in pediatric SCD. Mass spectrometry proteomics and functional coagulation assays are currently being conducted to further identify molecular markers predictive of SCD complications in children.

OBJECTIVES:To determine if a priori standardization of outcome hemostatic definitions alone was adequate to enable useful comparison between two cohorts of pediatric extracorporeal membrane oxygenation (ECMO) patients, managed according to local practice and protocol. DESIGN:Comparison of two separate prospective cohort studies performed at different centers with standardized outcome definitions agreed upon a priori. SETTING:General and cardiac PICUs at the Royal Children's Hospital (RCH) in Melbourne, Australia, and the Sophia Children's Hospital (SCH) in Rotterdam, The Netherlands. PATIENTS:Children (0-18 yr old) undergoing ECMO. INTERVENTIONS:None. MEASUREMENTS AND MAIN RESULTS:Although outcome definitions were standardized a priori, the interpretation of surgical interventions varied. The SCH study included 47 ECMO runs (September 2019 to April 2023), and the RCH study included 97 ECMO runs (September 2016 to Jan 2022). Significant differences in patient populations were noted. RCH patients biased toward frequent cardiac ECMO indications, central cannulation, and cardiopulmonary bypass before ECMO. The frequency of outcome ascertainment was not standardized. CONCLUSIONS:This international comparison shows that standardizing hemostatic outcome definitions alone is insufficient for sensible comparison. Uniform interpretation of definitions, consistent frequency of outcome ascertainment, and stratification based on patient populations and ECMO practices are required. Our results highlight the granularity of detail needed for cross-center comparison of hemostatic outcomes in pediatric ECMO. Further work is needed as we move toward potential multicenter trials of pediatric ECMO.

Rationale: Extreme premature neonates (born ≤28 weeks of gestation) have the highest risk of prolonged invasive mechanical ventilation and lung injury due to extubation failure. Current prediction models do not incorporate clinical data and biomarkers to predict the risk of extubation failure. This study explored differences in the proteome profiles of extremely premature neonates who succeeded or failed their first extubation attempt. We hypothesized that protein abundance before the extubation would differentiate neonates according to extubation success or failure. Methods: We conducted a pilot prospective cohort study in a level IV NICU. Neonates born ≤28 weeks of gestation or with birth weight ≤1000 grams intubated within the first week of life were included. Neonates with congenital anomalies were excluded. Informed parental consent was obtained for the collection of blood samples on days 0, 1, 2, 3, 7, and 14 of the intubation period. The primary outcome was extubation failure, defined as reintubation within 7 days of extubation. Plasma samples were analyzed using Tandem Mass Tag (TMT)-labeled liquid chromatography-mass spectrometry (LC-MS) proteomics. The Mass Dynamics platform was utilized to identify differentially abundant proteins with a fold change ratio > 1.5 and a False Discovery Rate threshold (adjusted P-value) < 0.05. Results: 17 participants were enrolled, with 13 completing sample collection. 7 (54%) had extubation success, 4 (31%) had extubation failure, and 2 (15%) were not extubated within the study time frame. We analyzed plasma from 4 neonates with extubation success and 2 with extubation failure, matched by gestational age and timing of extubation. The median gestational age was 27 weeks for the success group and 26 weeks in the failure group. Average birth weight was lower in the failure group at 645 grams compared to 844 grams of the success group. A total of 1041 unique proteins were observed in 24 samples from 6 subjects. We identified 43 proteins with decreased abundance and 30 with increased abundance in extubation failure compared to extubation success. Conclusions: We identified a proteomic signature differentiating between extubation failure and success that has the potential to predict extubation failure and targets for new therapies. Although this protein signature needs to be validated in a larger cohort to confirm our findings, proteins of interest were previously implicated in lung injury and bronchopulmonary dysplasia, with some (e.g. DLK1, TUBB4B, XPO1) being targets for drugs approved by the FDA.

Congenital heart disease (CHD) is a risk factor for thromboembolism (TE). Data describing the rate of, and risk factors associated with, recurrent TE in children with CHD are limited. We prospectively evaluated TE recurrence risk in children with CHD and acute TE and investigated clinical risk factors associated with recurrent TE. Patients < 21 years of age with CHD and acute TE were enrolled in a single-institutional prospective inception cohort study (July 2013-April 2024). Descriptive statistics summarized variables including CHD and thrombus characteristics, antithrombotic regimens, bleeding, and recurrent TE. Multivariable logistic regression determined risk factors for recurrent TE. Among 40 children with CHD and acute TE, 13 (33%) developed >= 1 recurrent TE (arterial n = 1 [6%], venous n = 15 [83%], venous + arterial n = 2 [11%]) at a median time of 86 (interquartile range, 45-112) days postdiagnosis of the index TE. One-year cumulative incidence of recurrent TE was 38%. Twelve (67%) recurrent TE events were central venous catheter (CVC)-related. In univariable analyses, immobility (46% vs. 7%, p = 0.01), the presence of a CVC (69% vs. 30%, p = 0.02), and lower extremity index venous TE (89% vs. 41%, p = 0.04) were associated with TE recurrence. After adjustment for other potential risk factors via multivariable logistic regression, immobility (adjusted odds ratio [OR] 13.2, 95% confidence interval [CI] 1.16-151.3, p = 0.04) and the presence of a CVC (adjusted OR 5.28, 95% CI 1.03-27.1, p = 0.05) remained as independent risk factors for recurrent TE. The 1-year risk of TE recurrence was high among pediatric patients with CHD and acute TE. Immobility and the presence of CVC were independent risk factors for recurrent TE. Multicenter prospective cohort studies are warranted to substantiate and expand upon these important findings.

Background: Antithrombin deficiency represents one of the most severe inherited thrombophilias. Albeit a rare disorder, available knowledge suggests that antithrombin deficiency is underestimated due to the limitations of current diagnostic algorithms. The high clinical variability of this patient population may be another cause of under-diagnosis. Heterozygous type I (quantitative) variants are normally associated with a severe thrombophilic phenotype, while heterozygous type II (qualitative) variants are heterogeneous, including heparin-binding site defects, which are mild/moderate and the most prevalent. Antithrombin Budapest 3 (p.Leu131Phe) is the most frequent type II/heparin-binding site deficiency in Europe, particularly in the Roma population, with a remarkable existence of homozygous subjects. Objectives: To determine the clinical features, diagnostic procedures, and management of patients with severe antithrombin deficiency, leveraging the study of cases homozygous for the antithrombin Budapest 3 variant. Methods: Patients were selected from 699 subjects with antithrombin deficiency and recruited over 25 years from reference centers in Spain, Belgium, and Hungary. Results: Guided by 2 illustrative cases with homozygous antithrombin Budapest 3, we report the spectrum and clinical management of patients with this disorder. These cases, with very low antithrombin activity (<20%) and juvenile and recurrent venous thromboembolism, recapitulate numerous issues that one might encounter when treating patients with antithrombin deficiency. In addition, special clinical scenarios for which no formal evidence-based guidelines exist might be found more frequently in these patients, including heparin resistance, vena cava anomalies, and obstetric complications. Conclusion: Expert proposals on the optimal management of these controversial areas, as well as future perspectives, are also formulated.

BACKGROUND:Data on the characteristics and antithrombotic treatments of venous thromboembolism (VTE) among children with congenital heart disease undergoing cardiac surgery are limited. OBJECTIVES:We aimed to evaluate the features and rates of recurrent thromboembolism in real-world experience using a global multicenter dataset. METHODS:We queried the TriNetX global electronic health record-derived real-world data research platform for patients <18 years of age who underwent cardiac surgery for congenital heart disease with a diagnosis of VTE within 1 year of surgery. Data on patient and VTE characteristics, antithrombotic therapies, and recurrent thromboembolism were descriptively analyzed. RESULTS:Of 24 879 children, 1475 (5.9%) developed an acute incident VTE within 1 year of surgery. Lower extremity deep venous thrombosis (n = 999, 67.7%) was the most common VTE type, and the Glenn procedure was the most common surgery type (n = 432, 29.3%). Unfractionated heparin was utilized for acute (<7 days after diagnosis) and subacute (≥7 days to 3 months after diagnosis) VTE treatment in 1022 (69.3%) and 895 (60.7%) patients, respectively. The 1-year rate of recurrent thromboembolism was high (n = 372, 25%), most of which were lower extremity deep venous thrombosis (n = 305, 81.9%). CONCLUSION:Approximately 6% of children undergoing cardiac surgery develop VTE within 1 year. The 1-year risk of thromboembolism recurrence is 25%. Prospective multicenter studies are essential to identify factors associated with the occurrence and recurrence of VTE in children undergoing cardiac surgery. This research will provide valuable insights for future interventional trials focused on preventing incident and recurrent VTE in this vulnerable population.

The anticoagulant proteins antithrombin, protein C, protein S, and tissue factor pathway inhibitor are physiological regulators of the blood coagulation pathway and have complex roles in causing or modifying the severity of bleeding and clotting disorders. The anticoagulant activity, as well as alternative physiological functions, of these proteins also impact many other diseases beyond bleeding and clotting disorders. We searched PubMed for articles published between 2018 and early 2025 that pertained to alternative functions of these physiological anticoagulants in clinical disease and selected therapeutic applications in the following categories: cancer, cardiovascular disease, novel therapeutics, pregnancy health, inflammation and infection, blood-brain barrier protection, stroke, novel developments in thrombosis, and endothelial and platelet interactions. This review highlights recently identified physiological implications of antithrombin, protein C, protein S, and tissue factor pathway inhibitor functions that present an exciting therapeutic avenue for a diverse array of human diseases.

Rationale: Variations in antibiotic treatment for bacterial tracheostomy-associated infections (TARI; e.g., pneumonia, tracheitis) have been observed with no clear benefit in clinical outcomes. A mechanistic understanding of airway and systemic physiologic response to antibiotic therapy and infection resolution may guide treatment decision making. An important first step is to characterize cytokine signaling and inflammatory signatures across multiple specimen types in children hospitalized for TARI. Methods: We performed a prospective proof-of-concept study in children <17 years of age hospitalized for TARI and treated with an antibiotic course. Blood, tracheal aspirate (TA), and urine samples were collected within 48 hours of hospital admission and 24 hours before discharge. Specimens were processed according to Olink® recommendations. The Olink Target 48 Cytokine Panel targeting 45 protein biomarkers of pathways related to cytokine signaling and inflammatory processes was used. Normalized protein expression values were uploaded into the Mass Dynamics platform for data interpretation. Results were analyzed using principle component analysis (PCA) and volcano plots to identify unique protein signatures present across groups. False discovery rate threshold was set at 0.05. Results: Forty samples from eight patients representing 12 hospitalizations were analyzed, consisting of 11 plasma, 24 TA, and five urine samples. PCA clustered plasma samples separately from TA and urine (Fig. 1A). Compared to plasma, TA specimens had a significantly higher abundance of pro-inflammatory IL-33, EGF, IL-1β, IL-6, CXCL8, IL-7, and OSM and a significantly lower abundance of LTA, CXCL12, FLT3LG, CCL11, IL-27, TSLP, CCL13, CCL19, and IL-17C (Fig. 1B). Compared to urine, TA specimens had a significantly higher abundance for 21 targets, with IL-1β, CSF3, CSCL8, and OSM comprising those with the largest difference (Fig. 1C). Compared to urine, plasma had a significantly higher abundance for 29 targets, with CCL19, MMP1, MMP12, CXCL11, and CSF3 comprising the largest difference, and a significantly lower abundance of EGF and OLR1 (Fig. 1D). Compared to admission samples, discharge samples had a decreased abundance of TNF, IFN-γ, IL-10, CCL8, and IL-17C in TA (Fig. 1E) and an increased abundance of TGFA, IL-7, CCL4, FLT3LG, and CCL11 in plasma (Fig. 1F), although these differences failed to reach statistical significance. Conclusions: Higher abundance of proinflammatory cytokines were observed in TA and plasma samples. Tracheal aspirate specimens showed a potentially meaningful decrease and plasma specimens showed an increase in inflammatory signatures from admission to discharge. This proof-of-concept study suggests inflammatory signatures may be objective markers of treatment response among TARI patients.

Immunoglobulin E (IgE) is a group of antibodies involved with allergic sensitisation. Levels of total IgE change with age and tend to be higher in individuals with allergic diseases than in those without. To improve the clinical utility of total IgE, this study aimed to establish continuous paediatric reference intervals (RIs) for healthy individuals. The study participants consisted of healthy term neonates and children aged 30 days to 18 years. Venous blood samples were collected, and total serum IgE concentrations were measured by performing a fluoroenzyme immunoassay. Fractional polynomial regression modelling and quantile regression were applied in the statistical analysis to generate continuous RIs. A total of 441 samples were analysed for total serum IgE, including 12 neonate samples and 429 paediatric samples from birth to 18 years of age, with males representing 51.5% (n=227) of the samples. For individuals without allergic conditions (n=305), the RI formulae for 2.5th percentile and 97.5th percentile were 2.00 and 37.29+101.95∗(age/10), respectively. For individuals with allergic conditions (n=136), the RI formulae for 2.5th percentile and 97.5th percentile were 1.50+0.30∗(age/10) and 1751.73+14.82∗(age/10), respectively. This study established continuous paediatric RIs for total serum IgE in otherwise healthy term neonates and children with and without allergic conditions. The continuous RIs generated from this study showed the age-based dynamic changes and allergy-specific differences for total serum IgE.