Changes in von Willebrand Factor Multimers, Concentration, and Function During Pediatric Extracorporeal Membrane Oxygenation.

OBJECTIVES: To investigate changes in von Willebrand factor (VWF) concentration, function, and multimers during pediatric extracorporeal membrane oxygenation (ECMO) and determine whether routine monitoring of VWF during ECMO would be useful in predicting bleeding. DESIGN: Prospective observational study of pediatric ECMO patients from April 2017 to May 2019. SETTING: The PICU in a large, tertiary referral pediatric ECMO center. PATIENTS: Twenty-five neonates and children (< 18 yr) supported by venoarterial ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Arterial blood samples were collected within 24 hours pre-ECMO, daily for the first 5 days of ECMO, every second day until decannulation, and 24 hours post-ECMO. The STA R Max analyzer was used to measure VWF antigen (VWF:Ag) and ristocetin cofactor (VWF:RCo) activity. VWF collagen binding (VWF:CB) was measured using an enzyme-linked immunosorbent assay. VWF multimers were measured using the semi-automated Hydragel 11 VWF Multimer assay. Corresponding clinical data for each patient was also recorded. A total of 25 venoarterial ECMO patients were recruited (median age, 73 d; interquartile range [IQR], 3 d to 1 yr). The median ECMO duration was 4 days (IQR, 3-8 d) and 15 patients had at least one major bleed during ECMO. The percentage of high molecular weight multimers (HMWM) decreased and intermediate molecular weight multimers increased while patients were on ECMO, irrespective of a bleeding status. VWF:Ag increased and the VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios decreased while patients were on ECMO compared with the baseline pre-ECMO samples and healthy children. CONCLUSIONS: Neonates and children on ECMO exhibited a loss of HMWM and lower VWF:CB/VWF:Ag and VWF:RCo/VWF:Ag ratios compared with healthy children, irrespective of major bleeding occurring. Therefore, monitoring VWF during ECMO would not be useful in predicting bleeding in these patients and changes to other hemostatic factors should be investigated to further understand bleeding during ECMO.

Platelet Phenotype and Function Changes With Increasing Duration of Extracorporeal Membrane Oxygenation.

OBJECTIVES: To investigate platelet pathophysiology associated with pediatric extracorporeal membrane oxygenation (ECMO). DESIGN: Prospective observational study of neonatal and pediatric ECMO patients from September 1, 2016, to December 31, 2019. SETTING: The PICU in a large tertiary referral pediatric ECMO center. PATIENTS: Eighty-seven neonates and children (< 18 yr) supported by ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Arterial blood samples were collected on days 1, 2, and 5 of ECMO and were analyzed by whole blood flow cytometry. Corresponding clinical data for each patient was also recorded. A total of 87 patients were recruited (median age, 65 d; interquartile range [IQR], 7 d to 4 yr). The median duration of ECMO was 5 days (IQR, 3-8 d) with a median length of stay in PICU and hospital of 18 days (IQR, 10-29 d) and 35 days (IQR, 19-75 d), respectively. Forty-two patients (48%) had at least one major bleed according to a priori determined definitions, and 12 patients (14%) had at least one thrombotic event during ECMO. Platelet fibrinogen receptor expression decreased (median fluorescence intensity [MFI], 29,256 vs 26,544; p = 0.0005), while von Willebrand Factor expression increased (MFI: 7,620 vs 8,829; p = 0.0459) from day 2 to day 5 of ECMO. Platelet response to agonist, Thrombin Receptor Activator Peptide 6, also decreased from day 2 to day 5 of ECMO, as measured by binding with anti-P-selectin, PAC-1 (binds activated GPIIb/IIIa), and anti-CD63 monoclonal antibodies (P-selectin area under the curve [AUC]: 63.46 vs 42.82, respectively, p = 0.0022; PAC-1 AUC: 93.75 vs 74.46, p = 0.0191; CD63 AUC: 55.69 vs 41.76, p = 0.0020). CONCLUSIONS: The loss of platelet response over time may contribute to bleeding during ECMO. These novel insights may be useful in understanding mechanisms of bleeding in pediatric ECMO and monitoring platelet markers clinically could allow for prediction or early detection of bleeding and thrombosis.

Whole blood flow cytometry protocol for the assessment of platelet phenotype, function, and cellular interactions.

Platelets are a key component of the hemostatic system and their roles in inflammation via interactions with leukocytes have also gained attention in recent years. Changes in platelet phenotype and function can cause bleeding and/or thrombosis and, as such, monitoring platelet-specific changes is crucial to assessing hemostasis in the clinical setting. Currently, available platelet function tests such as platelet aggregometry and thromboelastography require a large volume of blood, which is a major limitation for the pediatric population. Whole blood flow cytometric analysis of platelets is increasingly utilized in recent years, primarily due to the sensitivity of this method, but also because it only requires a small amount of blood with minimal sample manipulation. We have developed a whole blood flow cytometry methodological approach that enables the assessment of platelet phenotype, function, and their interactions with monocytes and neutrophils.

Guidelines for panel design, optimization, and performance of whole blood multi-color flow cytometry of platelet surface markers.

Flow cytometry is a valuable tool in determining the phenotype and function of platelets accurately. The emergence of platelet flow cytometry in recent years provides an attractive alternative to other platelet analytical techniques, with advantages such as requiring small volumes and being highly sensitive to minimal changes in receptor function and expression. Here we present a methodical approach encompassing the stages in the development and optimization of platelet flow cytometry panels based on our extensive experience in this area.

Factors XI and XII in extracorporeal membrane oxygenation: longitudinal profile in children.

Background: Extracorporeal membrane oxygenation (ECMO) is used in children with cardiopulmonary failure. While the majority of ECMO centers use unfractionated heparin, other anticoagulants, including factor XI and factor XII inhibitors are emerging, which may prove suitable for ECMO patients. However, before these anticoagulants can be applied in these patients, baseline data of FXI and FXII changes need to be acquired. Objectives: This study aimed to describe the longitudinal profile of FXI and FXII antigenic levels and function before, during, and after ECMO in children. Methods: This is a prospective observational study in neonatal and pediatric patients with ECMO (<18 years). All patients with venoarterial ECMO and with sufficient plasma volume collected before ECMO, on day 1 and day 3, and 24 hours postdecannulation were included. Antigenic levels and functional activity of FXI and FXII were determined in these samples. Longitudinal profiles of these values were created using a linear mixed model. Results: Sixteen patients were included in this study. Mean FXI and FXII antigenic levels (U/mL) changed from 7.9 and 53.2 before ECMO to 6.0 and 34.5 on day 3 and they recovered to 8.8 and 39.4, respectively, after stopping ECMO. Function (%) of FXI and FXII decreased from 59.1 and 59.0 to 49.0 and 50.7 on day 3 and recovered to 66.0 and 54.4, respectively. Conclusion: This study provides the first insights into changes of the contact pathway in children undergoing ECMO. FXI and FXII antigen and function change during ECMO. Results from this study can be used as starting point for future contact pathway anticoagulant studies in pediatric patients with ECMO.

Platelet Phenotype and Function in the Setting of Pediatric Extracorporeal Membrane Oxygenation (ECMO): A Systematic Review.

Background: Despite increasing technical improvement and extracorporeal membrane oxygenation (ECMO)-related knowledge over the past three decades, morbidity and mortality associated with bleeding and clotting complications remain high in pediatric patients undergoing ECMO. Platelets, a key element of the coagulation system, have been proposed to be the main cause of coagulopathy in the setting of ECMO. This systematic review aims to summarize and discuss the existing knowledge of platelet phenotype and function in the pediatric ECMO population. Methods: A systematic review was conducted for the Embase, Medline, and PubMed databases following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Results: The detailed study selection process yielded a total of 765 studies and only 3 studies that fulfilled the selection criteria were included in this review. Techniques used to assess platelet function in the three existing studies included platelet aggregometry, flow cytometry, and thromboelastography-platelet mapping. The finding that is common to the three studies is reduced platelet function in pediatric patients during ECMO either compared to before the initiation of ECMO or in non-survivors compared to survivors. Two studies demonstrated reduced platelet aggregation that are irreversible by platelet transfusion during ECMO. Two studies reported bleeding events and mortality in children on ECMO and none of the studies investigated thrombotic events. Conclusions: This systematic review demonstrates the extremely limited information available for platelet phenotype and function in the pediatric ECMO population. Evidence from the existing literature suggests reduced platelet aggregation and increased platelet activation in children during ECMO. However, this needs to be interpreted with care due to the limitations associated with the techniques used for platelet function testing. Furthermore, the association between platelet dysfunction and clinical outcomes in the pediatric ECMO population remains elusive. Multiple research gaps have been identified when it comes to the knowledge of platelet phenotype and function of children on ECMO, highlighting the need for robust, well-designed studies in this setting.

Glycyrrhizic acid prevents high calorie diet-induced metabolic aberrations despite the suppression of peroxisome proliferator-activated receptor γ expression.

OBJECTIVE: To investigate the effects of glycyrrhizic acid supplementation on glucose and lipid metabolism in rodents consuming a high-fat, high-sucrose diet. METHODS: Twenty-four male, 8-week old Sprague Dawley rats with an initial weight of 160 to 200 g were randomised into three groups (n = 6 for each group): groups A (standard rat chow), B (high-fat, high-sucrose diet), and C (high-fat, high-sucrose diet + 100 mg/kg/d of glycyrrhizic acid via oral administration). The rats were treated accordingly for 4 wk. Glycaemic parameters, lipid profile, stress hormones, and adiponectin levels were measured after the treatment. Relative gene expressions of peroxisome proliferator-activated receptor α and γ, lipoprotein lipase as well as gluconeogenic enzymatic activities in different tissues were also determined. RESULTS: Consumption of high-fat, high-sucrose diet triggered hyperglycaemia, insulin resistance, and dyslipidemia, which were effectively attenuated by supplementation with glycyrrhizic acid. Glycyrrhizic acid supplementation also effectively reduced circulating adrenaline, alleviated gluconeogenic enzymes overactivity, and promoted the upregulation of lipoprotein lipase expression in the cardiomyocytes and skeletal muscles. A high calorie diet also triggered hypoadiponectinaemia and suppression of peroxisome proliferator-activated receptor γ expression, which did not improve with glycyrrhizic acid treatment. CONCLUSION: Supplementation with glycyrrhizic acid could alleviate high calorie diet-induced glucose and lipid metabolic dysregulations by reducing circulatory stress hormones, normalizing gluconeogenic enzyme activities, and elevating muscular lipid uptake. The beneficial effects of these bioactivities outweighed the adverse effects caused by diet-induced repression of peroxisome proliferator-activated receptor γ expression, resulting in the maintenance of lipid and glucose homeostasis.

Modulation of lipid metabolism in glycyrrhizic acid-treated rats fed on a high-calorie diet and exposed to short or long-term stress.

Stress and high-calorie diets increase the risk of developing metabolic syndrome. Glycyrrhizic acid (GA) has been shown to improve dyslipidaemia in rats fed on a high-calorie diet. This study aimed to examine the effects of GA on lipid metabolism in rats exposed to short- or long-term stress and on a high-calorie diet. The parameters examined included serum lipid profiles, serum free fatty acids and fatty acid profiles in tissues, and expression of peroxisome proliferator-activated receptors (PPAR), lipoprotein lipase (LPL), elongases and desaturases. Within the 14- or 28-day exposure groups, neither stress nor GA affected the lipid profile and serum free fatty acids. Stress did not affect PPAR-α expression in both the 14- and 28-day exposure groups. However, GA-treated rats from the former group had increased PPAR-α expression only in the kidney while all other tissues from the latter group were unaffected. Stress increased PPAR-γ expression in the heart of the 28-day exposure group but its expression was unaffected in all tissues of the 14-day exposure group. GA elevated PPAR-γ expression in the kidney and the skeletal muscles. Neither stress nor GA affected LPL expressions in all tissues from the 14-day exposure group but its expressions were elevated in the QF of the stressed rats and heart of the GA-treated rats of the 28-day exposure group. As for the elongases and desaturases in the liver, stress down-regulated ELOVL5 in the long-term exposure group while up-regulated ELOVL6 in the short-term exposure group while hepatic desaturases were unaffected by stress. Neither elongase nor desaturase expressions in the liver were affected by GA. This research is the first report of GA on lipid metabolism under stress and high-calorie diet conditions and the results gives evidence for the role of GA in ameliorating MetS via site-specific regulation of lipid metabolism gene expressions and modification of fatty acids.

Glycyrrhizic acid can attenuate metabolic deviations caused by a high-sucrose diet without causing water retention in male Sprague-Dawley rats.

Glycyrrhizic acid (GA) ameliorates many components of the metabolic syndrome, but its potential therapeutic use is marred by edema caused by inhibition of renal 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2). We assessed whether 100 mg/kg per day GA administered orally could promote metabolic benefits without causing edema in rats fed on a high-sucrose diet. Groups of eight male rats were fed on one of three diets for 28 days: normal diet, a high-sucrose diet, or a high-sucrose diet supplemented with GA. Rats were then culled and renal 11ß-HSD2 activity, as well as serum sodium, potassium, angiotensin II and leptin levels were determined. Histological analyses were performed to assess changes in adipocyte size in visceral and subcutaneous depots, as well as hepatic and renal tissue morphology. This dosing paradigm of GA attenuated the increases in serum leptin levels and visceral, but not subcutaneous adipocyte size caused by the high-sucrose diet. Although GA decreased renal 11ß-HSD2 activity, it did not affect serum electrolyte or angiotensin II levels, indicating no onset of edema. Furthermore, there were no apparent morphological changes in the liver or kidney, indicating no toxicity. In conclusion, it is possible to reap metabolic benefits of GA without edema using the current dosage and treatment time.

Irregularities in glucose metabolism induced by stress and high-calorie diet can be attenuated by glycyrrhizic acid.

Stress and high-calorie diet increase the risk of developing metabolic syndrome. Glycyrrhizic acid (GA) has been shown to improve hyperglycaemia and dyslipidaemia under various physiological conditions. This study was aimed at examining the effects of stress and GA on glucose metabolism under short- or long-term stress. Forty-eight Sprague Dawley rats were divided into two groups with constant stress induced by light (300-400 lux) for either 14 days (short-term stress) or 28 days (long-term stress). Within each group, the rats were subdivided into three treatment groups i.e. Group A (control group): high-calorie diet (HCD) only; Group B: HCD + stress (14 or 28 days) and Group C: HCD + stress (14 or 28 days) + GA (100 mg/kg). The blood glucose concentrations of the rats exposed to 14-day stress were elevated significantly and GA lowered blood glucose concentration significantly in the 14-day exposure group. The 28-day exposure group adapted to stress as shown by the lower adrenaline level and gluconeogenic enzymes activities in most of the tissues than the 14-day exposure group. With regards to adrenaline and corticosterone, GA was found to increased adrenaline significantly in the short-term exposure group while lowering corticosterone in the long-term exposure group. GA-treated short- and long-term exposure groups had significant reduction in hexose-6-phosphate dehydrogenase activities in the visceral adipose tissues and quadriceps femoris respectively. The results may indicate the role of GA in improving blood glucose concentration in individuals exposed to short-term stress who are already on a high-calorie diet via selective action on gluconeogenic enzymes in different tissues.