Protocol for the Investigation of Plasma and Whole Blood Clot Property of Fibrin Fiber Thickness Using Scanning Electron Microscopy.

Blood clot formation represents a key component of the coagulation process for preventing excessive hemorrhage. The structural characteristics of blood clots are associated with their strength and susceptibility to fibrinolysis. Scanning electron microscopy is a technique that allows for state-of-the-art image capture of blood clots, providing visualization of topography, fibrin thickness, fibrin network density, and blood cell involvement and morphology. In this chapter, we provide a detailed protocol for characterization of plasma and whole blood clot structure using SEM, covering the spectrum from blood collection, in vitro clot formation, sample preparation for SEM, imaging, and image analysis, specifically focusing on the measurement of fibrin fiber thickness.

Platelet function in neonates and children.

Platelets are major regulators of haemostasis and coagulation. The primary role of platelets in coagulation is to form a stable clot and stop bleeding. Studies of platelet phenotype and function in neonates and children have been restricted by the large volumes required for many common platelet function tests such as platelet aggregometry. Developmental changes in platelets have not been as well described as developmental changes in plasma coagulation proteins, and overall, platelet phenotype and function in neonates and children has been understudied when compared to adults. Recent developments in more sensitive platelet function testing methods requiring smaller blood volumes such as flow cytometry has enabled recent studies to further investigate platelet phenotype and function in neonates and children. In this review we will provide an overview of recent advances from the past five years in platelets in the context of developmental haemostasis, as well as the role of platelets in neonatal paediatric disease.

Blood Collection Processing and Handling for Plasma and Serum Proteomics.

The plasma and serum proteome has enormous potential as a tool for understanding the health of a number of physiological systems. Despite this potential, the use of plasma and serum proteomics clinically and for research is limited, and there are no strict guidelines on how samples should be collected and prepared for proteomic analysis. Given the sensitivity of proteomic analysis, there are a number of pre-analytical variables that should be considered and determined prior to undertaking proteomics-based methodologies.In this chapter, we provide an example of a blood processing protocol and highlight major considerations for pre-analytical variables involving the collection, processing, and handling of blood samples for plasma and serum proteomics. We provide comprehensive notes on aspects of the protocol that must be considered before commencing sample collections for a proteomic study as well as a thorough checklist to be used when designing new proteomic studies.

Proteomic Applications and Considerations: From Research to Patient Care.

Despite technological advancements in the field of proteomics, the rate at which serum and plasma biomarkers identified using proteomic approaches are translated into clinical use remains extremely low. In this chapter, we describe recent technological advancements and analytical strategies in proteomic methods. We also describe the progress of proteomic blood-based biomarkers to date and discuss what the future of proteomics might entail with the use of multi-omic approaches and implementing machine learning on large proteomic datasets. Lastly, we provide several key considerations for biomarker studies, ranging from sample type to the use of reference samples, in order to achieve progress from bench to bedside, ultimately improving patient diagnosis, disease, and/or therapeutic monitoring and care.

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.

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.

Fibrin clot characteristics and anticoagulant response in a SARS-CoV-2-infected endothelial model.

Coronavirus disease 2019 (COVID-19) patients have increased thrombosis risk. With increasing age, there is an increase in COVID-19 severity. Additionally, adults with a history of vasculopathy have the highest thrombotic risk in COVID-19. The mechanisms of these clinical differences in risk remain unclear. Human umbilical vein endothelial cells (HUVECs) were infected with SARS-CoV-2, influenza A/Singapore/6/86 (H1N1) or mock-infected prior to incubation with plasma from healthy children, healthy adults or vasculopathic adults. Fibrin on surface of cells was observed using scanning electron microscopy, and fibrin characteristics were quantified. This experiment was repeated in the presence of bivalirudin, defibrotide, low-molecular-weight-heparin (LMWH) and unfractionated heparin (UFH). Fibrin formed on SARS-CoV-2 infected HUVECs was densely packed and contained more fibrin compared to mock-infected cells. Fibrin generated from child plasma was the thicker than fibrin generated in vasculopathic adult plasma (p = 0.0165). Clot formation was inhibited by LMWH (0.5 U/ml) and UFH (0.1-0.7 U/ml). We show that in the context of the SARS-CoV-2 infection on an endothelial culture, plasma from vasculopathic adults produces fibrin clots with thinner fibrin, indicating that the plasma coagulation system may play a role in determining the thrombotic outcome of SARS-CoV-2 infection. Heparinoid anticoagulants were most effective at preventing clot formation.

Pathophysiological pathway differences in children who present with COVID-19 ARDS compared to COVID -19 induced MIS-C.

COVID-19 has infected more than 275 million worldwide (at the beginning of 2022). Children appear less susceptible to COVID-19 and present with milder symptoms. Cases of children with COVID-19 developing clinical features of Kawasaki-disease have been described. Here we utilise Mass Spectrometry proteomics to determine the plasma proteins expressed in healthy children pre-pandemic, children with multisystem inflammatory syndrome (MIS-C) and children with COVID-19 induced ARDS. Pathway analyses were performed to determine the affected pathways. 76 proteins are differentially expressed across the groups, with 85 and 52 proteins specific to MIS-C and COVID-19 ARDS, respectively. Complement and coagulation activation are implicated in these clinical phenotypes, however there was significant contribution of FcGR and BCR activation in MIS-C and scavenging of haem and retinoid metabolism in COVID-19 ARDS. We show global proteomic differences in MIS-C and COVID-ARDS, although both show complement and coagulation dysregulation. The results contribute to our understanding of MIS-C and COVID-19 ARDS in children.

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.

Proteomics in Thrombosis and Hemostasis.

Proteomics, the simultaneous study of all proteins in a given cell, tissue or organism, is an innovative approach used to identify novel markers for diagnosis, prognosis and the pathophysiological mechanisms associated with diseases. Proteomic methodologies have been used in a variety of contexts such as investigating changes in protein abundance that may occur with disease presence, the response to therapeutic treatments as well as the impacts of age on the plasma proteome.Over the last decade, significant technological advancements in proteomic techniques have resulted in an increase in the use of proteomics in thrombosis and hemostasis research, particularly in order to identify relevant and novel clinical markers associated with bleeding and thrombosis. This mini-review explores the use of proteomics in the setting of thrombosis and hemostasis from 2010-2020, across five main domains (platelets, blood clot composition, stroke, venous thromboembolism, and therapeutics), as well as provides insights into key considerations for conducting proteomic studies.

Advances and Utility of the Human Plasma Proteome.

The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods. We discuss examples where plasma proteomics contributed valuable insights into SARS-CoV-2 infections, aging, and hemostasis and the opportunities offered by combining proteomics with genetic data. As a contribution to the Human Proteome Organization (HUPO) Human Plasma Proteome Project (HPPP), we present the Human Plasma PeptideAtlas build 2021-07 that comprises 4395 canonical and 1482 additional nonredundant human proteins detected in 240 MS-based experiments. In addition, we report the new Human Extracellular Vesicle PeptideAtlas 2021-06, which comprises five studies and 2757 canonical proteins detected in extracellular vesicles circulating in blood, of which 74% (2047) are in common with the plasma PeptideAtlas. Our overview summarizes the recent advances, impactful applications, and ongoing challenges for translating plasma proteomics into utility for precision medicine.

The use of proteomics for blood biomarker research in premature infants: a scoping review.

Over the last decade, the use of proteomics in the setting of prematurity has increased and has enabled researchers to successfully identify biomarkers for an array of associated morbidities. The objective of this scoping review was to identify the existing literature, as well as any knowledge gaps related to proteomic biomarker discoveries in the setting of prematurity. A scoping review was conducted using PubMed, Embase and Medline databases following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines. The study selection process yielded a total of 700 records, of which 13 studies were included in this review. Most studies used a tandem Mass Spectrometry (MS/MS) proteomics approach to identify key biomarkers. The corresponding studies identified proteins associated with retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD), necrotising enterocolitis (NEC), late onset sepsis (LOS) and gestational age. This scoping review demonstrates the limited use of proteomics to identify biomarkers associated with severe complications of prematurity. Further research is warranted to identify biomarkers of other important morbidities associated with prematurity, such as intraventricular haemorrhage (IVH) and cerebral palsy, and to investigate the mechanisms associated with these outcomes.

Investigating potential protein markers of cardiovascular disease in children with type 1 diabetes mellitus.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a metabolic disease characterized by dysglycaemia. Cardiovascular disease (CVD) is a major complication among T1DM patients and the leading cause of mortality later in life. METHODS: The study subjects consisted of T1DM children with poor glycemic control (HbA1c > 7.5%) and healthy age and gender matched controls. Venous blood samples were collected and tested by utilizing a novel immunoassay panel with 96 protein biomarkers. Data were analyzed using non-linear regression analysis and the expression of biomarkers was compared between T1DM and healthy control groups using an unpaired student's t-test. Dynamic principal component analysis (PCA) was operated based on the differentially expressed proteins. RESULTS: Ten T1DM children and 10 healthy controls were analyzed. Twelve CVD markers show significant differential expression between T1DM patients and healthy controls (p < 0.05). Dynamic PCA clustering based on differentially expressed proteins demonstrated an obvious clustering between the two populations. CONCLUSIONS: This preliminary study reveals the feasibility of utilizing a novel immunoassay panel to investigate potential biomarkers for predicting incipient CVD in children with T1DM. In future, longitudinal studies are required to track the relationships between measurements of the selected protein markers and the development of CVD in T1DM patients.

Age-specific differences in the in vitro anticoagulant effect of Bivalirudin in healthy neonates and children compared to adults.

Bivalirudin is a reversible direct thrombin inhibitor that inhibits both bound and free thrombin and binds to the active (catalytic) and fibrinogen-binding sites of thrombin, with high affinity and specificity. Off-label use of bivalirudin in the paediatric population has increased, as an alternative to heparin, particularly in the setting of anticoagulation for patients undergoing coronary bypass surgery (CPB), extracorporeal life support (ECLS) and those on ventricular assist devices (VAD). This study aimed to determine the age-specific in vitro effect of bivalirudin in children compared to adults. Age-specific pools (neonates, ≤2 years, >2 to 5 years, 6 to 10 years, 11 to 17 years and Adults) were prepared using platelet poor plasma samples from 20 individuals per age group. Pooled plasma was spiked with increasing concentrations of Bivalirudin (from 0 g/mL to 10µg/mL), and thrombin inhibition was measured using standard coagulation assays. There was a significantly increased response to bivalirudin across all paediatric age groups as compared to adults. The age-specific difference in response to bivalirudin was specifically evident in neonates, where the potential to generate thrombin was decreased 2-fold compared to adults (p < 0.001). Our findings support the concept of age-specific pharmaco-dynamic responses to Bivalirudin and support the need for further ex vivo studies in hospitalised children to determine accurate clinical dosing recommendations.