Differences in the mechanism of blood clot formation and nanostructure in infants and children compared with adults

Introduction: Infants and children have a lower incidence of thrombosis compared with adults. Yet, the mechanism of blood clot formation and structure in infants and children, as the end product of coagulation, has not been studied. This study aimed to establish differences in the mechanism of thrombin generation, fibrin clot formation and response to thrombolysis in infants and children compared with adults. Materials and methods: We studied thrombin generation, fibrin clot formation, structure and fibrinolysis in healthy infants, children and adults. Results: Younger populations had a decreased potential to generate thrombin, at a slower velocity compared with adults, correlating positively with age. Clot formation at venous shear rate was decreased in infants and children compared with adults, with increased time for fibrin formation, decreased fibrin formation velocity, resulting in decreased tendency for fibrin formation in younger populations. These differences were less pronounced at arterial shear rate. Studies of the fibrin clot structure in paediatric age groups showed a significantly larger pore size compared with adults, suggestive of a clot that is less resistant to fibrinolysis. The presence of tissue plasminogen activator (tPA) resulted in a significant decrease in the pore size of infants and children, but not in adults. Conclusions: This is the first study to suggest that the mechanism of blood clot formation and nanostructure, as well as response to thrombolytic therapy is different in infants and children compared with adults

Remote Ischemic Preconditioning (RIPC) Modifies the Plasma Proteome in Children Undergoing Repair of Tetralogy of Fallot: A Randomized Controlled Trial

<div><p>Background</p><p>Remote ischemic preconditioning (RIPC) has been applied in paediatric cardiac surgery. We have demonstrated that RIPC induces a proteomic response in plasma of healthy volunteers. We tested the hypothesis that RIPC modifies the proteomic response in children undergoing Tetralogy of Fallot (TOF) repair.</p><p>Methods and Results</p><p>Children (n=40) were randomized to RIPC and control groups. Blood was sampled at baseline, after cardiopulmonary bypass (CPB) and 6, 12 and 24h post-CPB. Plasma was analysed by liquid chromatography mass spectrometry (LC-MS) in an untargeted approach. Peptides demonstrating differential expression (p<0.01) were subjected to tandem LC-MS/MS and protein identification. Corresponding proteins were identified using the NCBI protein database. There was no difference in age (7.3±3.5vs6.8±3.6 months)(p=0.89), weight (7.7±1.8vs7.5±1.9 kg)(p=0.71), CPB time (104±7vs94±7 min)(p=0.98) or aortic cross-clamp time (83±22vs75±20 min)(p=0.36). No peptides were differentially expressed at baseline or immediately after CPB. There were 48 peptides with higher expression in the RIPC group 6h post-CPB. This was no longer evident at 12 or 24h, with one peptide down-regulated in the RIPC group. The proteins identified were: inter-alpha globulin inhibitor (42.0±11.8 vs 820.8±181.1, p=0.006), fibrinogen preproprotein (59.3±11.2 vs 1192.6±278.3, p=0.007), complement-C3 precursor (391.2±160.9 vs 5385.1±689.4, p=0.0005), complement C4B (151.5±17.8 vs 4587.8±799.2, p=0.003), apolipoprotein B100 (53.4±8.3 vs 1364.5±278.2, p=0.005) and urinary proteinase inhibitor (358.6±74.9 vs 5758.1±1343.1, p=0.009). These proteins are involved in metabolism, haemostasis, immunity and inflammation.</p><p>Conclusions</p><p>We provided the first comprehensive analysis of RIPC-induced proteomic changes in children undergoing surgery. The proteomic changes peak 6h post-CPB and return to baseline within 24h of surgery.</p><p>Trial Registration</p><p>ACTR.org.au <a href="http://www.anzctr.org.au/trial_view.aspx/ACTRN12610000496011" target="_blank">ACTRN12610000496011</a></p></div

Integrated peak area of the peptides in the 5 pooled samples in each group.

<p>Expression of each peptide is depicted as a line with the lines in red each representing the significantly higher expressed peptides in the RIPC group compared to the control group. There were 48 up-regulated peptides in the RIPC group compared to the control group. The histogram on the left indicates the false discovery rate.</p

Complement C3 concentration for the control and RIPC groups at the time points in the study.

<p>Individual samples were assessed in this ELISA. There was significantly higher levels of Complement C3 in the RIPC group at 6 h after CPB (p<0.05).</p

CONSORT diagram describing flow of the participants though the enrolment, allocation, follow up and analysis phases of the trial.

<p>CONSORT diagram describing flow of the participants though the enrolment, allocation, follow up and analysis phases of the trial.</p

Protein data for the 6 proteins that were up-regulated in RIPC group as compared to the control group at 6 hours after CPB.

<p>Protein data for the 6 proteins that were up-regulated in RIPC group as compared to the control group at 6 hours after CPB.</p

Time points of the sample collection in panel A.

<p>Pooling of the plasma samples at each time point in panel <b>B.</b> Plasma from all 20 patients of one group was pooled into 5 samples (each representing plasma of 4 patients).</p

Venn diagram detailing the number of differentially expressed peptides in RIPC as compared to control samples at each time point.

<p><b>A.</b> Baseline. <b>B.</b> End CPB. <b>C.</b> 6 hours post CPB sample. <b>D.</b> 12 hours post CPB. <b>E.</b> 24 h post CPB. <b>*—</b>down-regulated, †<b>—</b>up-regulated, ‡<b>—</b>significantly differentially expressed.</p

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

Abstract 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