Lesley G. Mitchell

Anticoagulant effects of heparin in neonatal plasma.

ABSTRACT: Available data on the anticoagulant effects of heparin in neonatal plasma are scarce and conflicting: relative to adult plasma, neonatal plasma has been reported to show both resistance as well as sensitivity to heparin. We explored this apparent paradox by comparing how well heparin accelerated inhibition of exogenous thrombin and prevented thrombin generation in defibrinated neonatal and adult plasmas. Using amidolytic assays, we determined the effects of heparin on 7) the neutralization of exogenous human a-thrombin and on 2) the formation of endogenous thrombin activity after contact activation and recalcification. Neonatal plasma proved resistant to heparin (0.05 U/ mL) during inhibition of added thrombin (15 NIH U/mL). Inhibition of thrombin in heparinized neonatal plasma became as efficient as in adult plasma only after raising the AT III activity to normal adult values. However, de novo generation of thrombin activity was very susceptible to inhibition by heparin, even in neonatal plasmas with physiologically low AT HI levels. Peak thrombin activity generated in neonatal plasma in the absence of heparin was 50% or less of peak adult activity, and this already reduced ability of neonatal plasma to generate thrombin activity upon prothrombin activation was further decreased by heparin (0.05-0.2 U/mL). We conclude that due to the neonatal AT III deficiency, added thrombin is inactivated less effectively by heparin in neonatal than in normal adult plasma. Yet, the generation of thrombin activity is impaired in neonatal plasma and easily suppressed by heparin. We speculate that newborn infants may be resistant to heparin therapy during overt thrombotic disease, when neutralization of abnormal thrombin activity is the therapeutic goal. In contrast, lower plasma heparin levels may be required to prevent the formation of thrombin activity in newborn infants than in adult patients.

Significant association with location of central venous line placement and risk of venous thrombosis in children

Venous thromboembolic events (VTE) in children are frequently associated with central venous lines (CVL). Identifying risk factors related to CVL management could potentially minimize CVL-related thrombotic complications. The objectives of the study were to assess whether CVL location, type, size, and duration of placement are associated with the incidence of VTE in children. The study was a prospective, multicentre cohort study in a general pediatric population requiring CVL. Data on CVL characteristics were documented prospectively using standardized case report forms. Outcome assessments were by i) clinical monitoring for symptomatic VTE which were confirmed by appropriate objective test, or ii) screening by venography at study exit. Among 158 children, 21 (13%) hadVTE. The incidence of VTE was increased with femoral CVL (32%) and subclavian CVL (27%) compared to brachial CVL (12%) and jugular CVL (8%; p = 0.01). The incidence of VTE was independent of CVL type (peripherally inserted central catheters, untunneled CVL, tunneled exteriorized CVL, subcutaneous ports; p = 0.90), and CVL size (CVL diameter, p = 0.42; number of CVL lumen, p = 0.58). The incidence of VTE did not increase with duration of CVL placement: 0-5 days (17% VTE), 6-20 days (19%), 21-35 days (10%), and 36-50 days (11%, p = 0.68). The incidence of CVL-relatedVTE may be reduced by preferred placement of CVL in brachial or jugular veins. The choice of CVL type and size does not significantly influence the risk of VTE. Short-term CVL are associated with a similar risk of VTE as longer-term CVL.

The effect of 1 month of therapy with midodrine, octreotide-LAR and albumin in refractory ascites: a pilot study

Background: The pathogenesis of refractory ascites (RA) is linked to splanchnic vasodilation. We hypothesized that a combination of midodrine, octreotide long‐acting release (LAR) and albumin would result in increased natriuresis, better control of ascites and an improvement in renal function in patients with RA±Type 2 hepatorenal syndrome.

Assessing the anticoagulant effect of dabigatran in children: An in vitro study

OBJECTIVES Age-related changes in the hemostatic system result in variation in response to anticoagulants and coagulation assays over childhood. This study used in vitro methods to determine i) optimum coagulation assays for dabigatran in children and ii) anticoagulant effect of dabigatran across pediatric age groups. MATERIALS AND METHODS Pooled plasma samples from healthy children aged 0 to <1, 1 to <5, 5 to <10, 10 to <17 years and adults were spiked with increasing concentrations of dabigatran and the effect was assessed in five coagulation assays. The samples were also assessed for overall hemostasis potential using a fibrin clot formation and lysis assay. RESULTS In all five coagulation assays, there were no differences in responses to dabigatran over all pediatric age groups. The international normalized ratio was the least sensitive measure. Activated partial thromboplastin time showed moderate sensitivity and a nonlinear response curve. Thrombin time (TT), dilute TT (dTT) and ecarin clotting time were linearly correlated with dabigatran concentrations; however, the ecarin time and TT were overly sensitive. In the overall hemostasis potential assay, increasing dabigatran concentrations delayed the initiation of clot formation and reduced the time to 50% clot lysis. The responses to initiation of clot formation and clot lysis were consistent across all pediatric groups and comparable to responses in adults. CONCLUSION The dTT is the most suitable assay for measuring dabigatran concentrations in children. Fibrin clot generation and lysis assay responses to dabigatran in children over all ages were consistent and comparable to those of adults.

Fibrinogen has a rapid turnover in the healthy newborn lamb.

ABSTRACT: The half-lives for coagulation factors in the healthy newborn infant are not known and may be different than for the adult. We measured the half-life for fetal sheep fibrinogen and compared it to the half-life of adult sheep fibrinogen. Fibrinogen was purified from adult and fetal sheep plasma and radiolabeled with either 125I or 131I. The half-lives for these fibrinogens were determined in the adult sheep and newborn lamb. In addition, the fetal and adult sheep fibrinogens were compared by reptilase time, thrombin clotting time, sialic acid content, and the behavior of the N-glycans derived from these fibrinogens on the immobilized lectin, Sepharose-concanavalin A. Finally, the in vivo response of coinjected radiolabeled fibrinogens to increasing doses of infused thrombin was determined. The fetal sheep fibrinogen differed from the adult as indicated by a prolonged reptilase time and an increased sialic acid content (fetal: 10–11 residues/340 Kd versus adult: 8–9 residues/340 Kd). The latter was also reflected in differing chromotographic profiles for the N-glycans on Sepharose-concanavalin A. The half-lives for both the adult and fetal fibrinogen were significantly more rapid in the newborn lamb (fetal: 47 ± 2.0 h; adult: 46 ± 2.4 h, mean ± SEM) than in the adult (fetal: 116 ± 6.5 h; adult: 121 ± 6.9 h). Finally, the adult and fetal sheep fibrinogen responded to thrombin in an identical fashion in the intact animal. In summary, both adult and fetal fibrinogen have faster half-lives in the lamb compared to the adult, despite a higher sialic acid content for the fetal fibrinogen. We speculate that the more rapid turnover of coagulation proteins in the newborn may be of significance when planning replacement therapy for congenital or acquired coagulopathies.

Proteolytic cleavage of carboxypeptidase N markedly increases its antifibrinolytic activity

Summary.  Background: Carboxypeptidase N (CPN) is a constitutively active basic carboxypeptidase sharing specificity with activated thrombin‐activable fibrinolysis inhibitor (TAFIa). Generally, CPN is regarded as being non‐antifibrinolytic. However, this assumption has not been thoroughly investigated, particularly with respect to long‐term antifibrinolysis. In addition, a recent report has shown that plasmin cleavage increases the catalytic activity of CPN. Therefore, we investigated the antifibrinolytic properties of CPN and plasmin‐cleaved CPN (CPNc). Methods: CPN was incubated with plasmin for various periods of time and the prolongation of clot lysis at various concentrations of CPN/CPNc mixture was investigated in TAFI‐depleted plasma. CPN cleavage was analyzed by electrophoresis and catalytic activity was determined by monitoring cleavage of the small substrate, FA‐Ala‐Lys. Results: CPN exhibited antifibrinolytic properties in plasma clot lysis assays when present at supraphysiological concentrations. Depletion of CPN from plasma decreased the lysis time of clots formed from minimally diluted plasma at low tissue‐type plasminogen activator (t‐PA) concentrations. Plasmin cleavage of CPN markedly increased the antifibrinolytic properties. CPN and CPNc prolonged lysis in a non‐saturable, dose‐dependent, and t‐PA‐dependent manner. At sufficient concentration, CPN and CPNc prolonged lysis at least forty‐fivefold. CPNc was 700% more antifibrinolytic than CPN but only 7% more active toward FA‐Ala‐Lys. The active site inhibitor GEMSA eliminated the antifibrinolytic effects of CPN and CPNc. Antifibrinolytic activity correlated with cleavage of active and/or regulatory subunits, presumably generating heterodimeric CPNc. Conclusions: Limited proteolysis of CPN by plasmin generates an enzyme with greatly increased antifibrinolytic properties. We speculate that (patho)physiological proteolysis of CPN may generate a long‐term antifibrinolytic enzyme.

Thromboprophylaxis of central venous lines in children with cancer: The first steps taken on the long road ahead

Approximately one third of children with malignancy and central venous lines develop catheter‐related thrombosis, with the reported sequelae including death, pulmonary embolism, chylothoraces, superior vena cava syndrome and post‐thrombotic syndrome. These complications prompt the design and completion of randomized, controlled trials to determine a safe and efficacious therapy to prevent these thromboses. The authors Ruud et al. have presented a well‐designed, randomized, controlled trial which demonstrates the lack of utility of using low‐dose warfarin to prevent catheter‐related thrombosis in children with central lines and acute lymphoblastic leukaemia. The difficulties with warfarin in children are again demonstrated in this study.

Age at cancer diagnosis, non-O blood group and asparaginase therapy are independently associated with deep venous thrombosis in pediatric oncology patients: A risk model

INTRODUCTION Pediatric oncology patients are at increased risk for deep venous thrombosis (DVT). Determining the sub-population of children at increased DVT risk is critical for optimum clinical management. Therefore, the aim of the current study was to identify clinical risk factors for DVT which are easily identifiable at cancer diagnosis. MATERIALS AND METHODS A Canadian multicenter case control study in survivors of childhood cancer. Survivors who had DVT (Cases) while being treated for pediatric cancer where matched by center with a minimum of two survivors who did not experience DVT (Controls). Clinical information including age at diagnosis, type of cancer and chemotherapy were collected. Genotyping of blood group was done by single nucleotide polymorphisms analysis. RESULTS 218 subjects were recruited at 4 Canadian pediatric centers. Multivariable analysis demonstrated 3 significant variables (reported as Odds Ratio (OR), (95% CI), p value): age at diagnosis p<0.001, non-O blood group OR 2.6 (1.3-5.2) p=0.005 and asparaginase treatment OR 2.4 (1.2-4.8) p=0.011. In order to optimise clinical utility, we reanalysed the study data with age at diagnosis categorised into four subgroups 0-≤2years, >2-≤7years, >7≤10years, >10years. A significant association with DVT were seen in children 0-≤2years (OR 3.1 (1.1-8.3) p=0.026) and >10years (OR 3.8, 1.7-8.5 p=0.001). Significant associations with DVT remained for non-O blood group, OR 2.2 (1.2-4.4) p=0.016 and asparaginase treatment, OR 2.1 (1.1-4.0) p=0.027. The value for the clinical risk model receiver operating characteristics curve was 0.67. CONCLUSIONS We have shown 3 independent risk factors for DVT in childhood cancer.

Phase IIa study of dabigatran etexilate in children with venous thrombosis: pharmacokinetics, safety, and tolerability

Essentials Dabigatran etexilate may provide a new treatment option for pediatric venous thromboembolism. Children aged 1 to < 12 years were given dabigatran etexilate in an open‐label, single‐arm study. The pharmacokinetic–pharmacodynamic relationship was similar to that seen in adult patients. There were no serious adverse events, bleeding events or recurrent venous thromboembolism.

Design and Methods of the Pan-Canadian Applying Biomarkers to Minimize Long-Term Effects of Childhood/Adolescent Cancer Treatment (ABLE) Nephrotoxicity Study

Background: Childhood cancer survivors experience adverse drug events leading to lifelong health issues. The Applying Biomarkers to Minimize Long-Term Effects of Childhood/Adolescent Cancer Treatment (ABLE) team was established to validate and apply biomarkers of cancer treatment effects, with a goal of identifying children at high risk of developing cancer treatment complications associated with thrombosis, graft-versus-host disease, hearing loss, and kidney damage. Cisplatin is a chemotherapy well known to cause acute and chronic nephrotoxicity. Data on biomarkers of acute kidney injury (AKI) and late renal outcomes in children treated with cisplatin are limited. Objective: To describe the design and methods of the pan-Canadian ABLE Nephrotoxicity study, which aims to evaluate urine biomarkers (neutrophil gelatinase–associated lipocalin [NGAL] and kidney injury molecule-1 [KIM-1]) for AKI diagnosis, and determine whether they predict risk of long-term renal outcomes (chronic kidney disease [CKD], hypertension). Design: This is a 3-year observational prospective cohort study. Setting: The study includes 12 Canadian pediatric oncology centers. Patients: The target recruitment goal is 150 patients aged less than 18 years receiving cisplatin. Exclusion criteria: Patients with an estimated glomerular filtration rate (eGFR) <30 mL/min/1.73 m2 or a pre-existing renal transplantation at baseline. Measurements: Serum creatinine (SCr), urine NGAL, and KIM-1 are measured during cisplatin infusion episodes (pre-infusion, immediate post-infusion, discharge sampling). At follow-up visits, eGFR, microalbuminuria, and blood pressure are measured and outcomes are collected. Methods: Outcomes: AKI is defined as per SCr criteria of the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines. CKD is defined as eGFR <90 mL/min/1.73m2 or albumin-to-creatinine ratio≥3mg/mmol. Hypertension is defined as per guidelines. Procedure: Patients are recruited before their first or second cisplatin cycle. Participants are evaluated during 2 cisplatin infusion episodes (AKI biomarker validation) and at 3, 12, and 36 months post-cisplatin treatment (late outcomes). Limitations: The study has a relatively moderate sample size and short follow-up duration. There is potential for variability in data collection since multiple sites are involved. Conclusions: ABLE will provide a national platform to study biomarkers of late cancer treatment complications. The Nephrotoxicity study is a novel study of AKI biomarkers in children treated with cisplatin that will greatly inform on late cisplatin renal outcomes and follow-up needs.