Differential sialic acid content in adult and neonatal fibrinogen mediates differences in clot polymerization dynamics

Neonates possess a molecular variant of fibrinogen, known as fetal fibrinogen, characterized by increased sialic acid, a greater negative charge, and decreased activity compared to adults. Despite these differences, adult fibrinogen is used for treatment of bleeding in neonates, with mixed efficacy. In order to determine safe and efficacious bleeding protocols for neonates, more information on neonatal fibrin clot formation and the influence of sialic acid on these processes is needed. Here, we examine the influence of sialic acid on neonatal fibrin polymerization. We hypothesized that the increased sialic acid content of neonatal fibrinogen promotes fibrin B:b knob hole interactions and consequently influences the structure and function of the neonatal fibrin matrix. We explored this hypothesis through analysis of structural properties and knob:hole polymerization dynamics of normal and desialylated neonatal fibrin networks and compare to those formed with adult fibrinogen. We then characterized normal neonatal fibrin knob:hole interactions by forming neonatal and adult clots with either thrombin or snake-venom thrombin like enzymes (SVTLEs) that preferentially cleave fibrinopeptide A or B. We determined that sialic acid content of neonatal fibrinogen is a key determinant of resulting clot properties. Experiments analyzing knob:hole dynamics indicated typical neonatal fibrin clots are formed with the release of more fibrinopeptide B and less fibrinopeptide A than adults. After the removal of sialic acid, fibrinopeptide release was roughly equivalent between adults and neonates indicating the influence of sialic acid on fibrin neonatal fibrin polymerization mechanisms. These results could inform future studies developing neonatal specific treatments of bleeding.

Association Between Hemostatic Profile and Migraine: A Mendelian Randomization Analysis

Objective:To assess support for a causal relationship between hemostatic measures and migraine susceptibility using genetic instrumental analysis.Methods:Two-sample Mendelian randomization (MR) instrumental leveraging available genome-wide association study (GWAS) summary statistics was applied to hemostatic measures as potential causal for migraine and its subtypes, migraine with aura (MA) and migraine without aura (MO). Twelve blood-based measures of hemostasis were examined, including plasma level or activity of eight hemostatic factors and two fibrinopeptides together with two hemostasis clinical tests.Results:There were significant instrumental effects between increased coagulation factor VIII activity (FVIII, OR [95% CI]=1.05[1.03, 1.08]/SD, P=6.08×10-05), von Willebrand factor level (VWF, 1.05[1.03, 1.08]/SD, P=2.25×10-06), and phosphorylated fibrinopeptide A level (1.13[1.07, 1.19]/SD, P=5.44×10-06) with migraine susceptibility. When extended to migraine subtypes, FVIII, VWF, and phosphorylated fibrinopeptide A showed slightly stronger effects with MA than overall migraine. Fibrinogen level was inversely linked with MA (0.76[0.64, 0.91]/SD, P=2.32×10-03) but not overall migraine. None of the hemostatic factors was linked with MO. In sensitivity analysis, effects for fibrinogen and phosphorylated fibrinopeptide A were robust, while independent effects of FVIII and VWF could not be distinguished, and FVIIII associations were potentially affected by pleiotropy at the ABO locus. Causal effects from migraine to the hemostatic measures were not supported in reverse MR. However, MA was not included due to lack of instruments.Conclusions:The findings support potential causality of increased FVIII, VWF, and phosphorylated fibrinopeptide A, and decreased fibrinogen in migraine susceptibility, especially for MA, potentially revealing etiologic relationships between hemostasis and migraine.

Covid-19-Associated Coagulopathy: Biomarkers of Thrombin Generation and Fibrinolysis Leading the Outcome

Background: Coronavirus Disease 2019 (COVID-19)-associated coagulopathy is characterized by a prothrombotic state not yet comprehensively studied. We investigated the coagulation pattern of patients with COVID-19 acute respiratory distress syndrome (ARDS), comparing patients who survived to those who did not. Methods: In this prospective cohort study on 20 COVID-19 ARDS patients, the following biomarkers were measured: thrombin generation (prothrombin fragment 1 + 2 (PF 1 + 2)), fibrinolysis activation (tissue plasminogen activator (tPA)) and inhibition (plasminogen activator inhibitor 2 (PAI-2)), fibrin synthesis (fibrinopeptide A) and fibrinolysis magnitude (plasmin–antiplasmin complex (PAP) and D-dimers). Measurements were done upon intensive care unit (ICU) admission and after 10–14 days. Results: There was increased thrombin generation; modest or null release of t-PA; and increased levels of PAI-2, fibrinopeptide A, PAP and D-dimers. At baseline, nonsurvivors had a significantly (p = 0.014) higher PAI-2/PAP ratio than survivors (109, interquartile range (IQR) 18.1–216, vs. 8.7, IQR 2.9–12.6). At follow-up, thrombin generation was significantly (p = 0.025) reduced in survivors (PF 1 + 2 from 396 pg/mL, IQR 185–585 to 237 pg/mL, IQR 120–393), whereas it increased in nonsurvivors. Fibrinolysis inhibition at follow-up remained stable in survivors and increased in nonsurvivors, leading to a significant (p = 0.026) difference in PAI-2 levels (161 pg/mL, IQR 50–334, vs. 1088 pg/mL, IQR 177–1565). Conclusion: Severe patterns of COVID-19 ARDS are characterized by a thrombin burst and the consequent coagulation activation. Mechanisms of fibrinolysis regulation appear unbalanced toward fibrinolysis inhibition. This pattern ameliorates in survivors, whereas it worsens in nonsurvivors.

Covid-19-associated coagulopathy (CoAC): thrombin burst and insufficient fibrinolysis leading to bad outcome.

Background: COVID-19 associated coagulopathy is characterized by a pro-thrombotic state. However, the nature of this pattern has not been comprehensively studied. We investigated the coagulation pattern of patients with COVID-19 acute respiratory distress syndrome (ARDS) comparing survivors to not survivors. Methods: Prospective cohort study conducted in the Intensive Care Unit (ICU) of a University Hospital . Twenty COVID-19 ARDS patients received measurements of markers of thrombin generation (prothrombin fragment 1+2, PF 1+2); fibrinolysis activation (tissue plasminogen activator, tPA) and inhibition (plasminogen activator inhibitor-2, PAI-2); fibrin synthesis (fibrinopeptide A) and fibrinolysis magnitude (plasmin-antiplasmin complex, PAP, and D-dimers). Measurements were done at the ICU admission and after 10-14 days. Results: The general pattern showed an increased thrombin generation, modest or null release of t-PA, and increased levels of PAI-2, Fibrinopeptide A, PAP and D-dimers. At baseline, non survivors had a significantly (P=0.014) higher PAI-2/PAP ratio than survivors (109, interquartile range [IQR] 18.1-216, vs. 8.7, IQR 2.9-12.6). At follow-up, thrombin generation was significantly (P=0.025) reduced in survivors (PF 1+2 from 396 pg/mL, IQR 185-585 to 237 pg/mL, IQR 120-393), whereas it increased in non-survivors. Fibrinolysis inhibition at follow-up remained stable in survivors, and increased in non-survivors, leading to a significant (P=0.026) difference in PAI-2 levels (161 pg/mL, IQR 50-334, vs. 1,088 pg/mL, IQR 177-1,565). Conclusions: Severe patterns of COVID-19 infection (ARDS) are characterized by a thrombin burst, triggered by the release of IL-6 and other cytokines, and the consequent release of Tissue Factor. Mechanisms of fibrinolysis regulation appear unbalanced toward fibrinolysis inhibition. In survivors, this pattern ameliorates, whereas in non-survivors it worsens, leading to the environment for clinically relevant thrombi generation, that was found in 58% of non-surviving patients. Trial registration: clinicaltrials.gov (NCT04441502).

Fibrinopeptide-A and fibrinopeptide-B may help to D-dimer as early diagnosis markers for acute mesenteric ischemia

Background The aim of this study was to investigate the importance of fibrinopeptide-A and fibrinopeptide-B, which occur during the formation of D-dimer, the most commonly used laboratory parameter, in the early diagnosis of acute mesenteric ischemia (AMI). Materials and methods This experimental study was performed in 30 male pathogen-free Wistar albino rats. The experimental animals were divided into 3 equal groups: Control group (n = 10), Sham group (n = 10) and Ischemia group (n = 10). Blood samples were taken 0, 1, 3, and 6 h after the simulation of mesenteric ischemia. Results Fibrinopeptide-A and fibrinopeptide-B levels increased significantly in the first 6 h in the ischemic group, similar to the increase in D-dimer levels. The statistical change between 0, 1, 3 and 6 h was more significant for fibrinopeptide-A and fibrinopeptide-B. Conclusion Fibrinopeptide-A and fibrinopeptide-B may be markers that can be used for early diagnosis of mesenteric ischemia, early diagnosis is highly important for decreasing mortality and morbidity.

Integrating endogenous peptides analysis and protease mapping for identification of potential serum biomarkers in gastric adenocarcinoma.

e15564 Background: Gastric cancer (GC) is the third cause of cancer death in the world. In Brazil 21.000 new cases had been diagnosed in 2018 and the mortality rate is associated with late diagnosis that contributed to poor prognosis. The objective was to study endogenous peptides in serum of patients with GC. Methods: Fifteen serum samples of patients with diagnosis of gastric adenocarcinoma (TNM stage I-IV) and 15 controls were included. Endogenous peptides were extracted and pooled of random mode in 5 biological replicates (n = 3 samples) for each group. The mixture peptides was submitted to nLC-MS/MS analysis and peptide sequences identified were used to protease mapping. Results: A total of 191 peptides (≥ 7 amino acids) were identified corresponding to 36 proteins involved mainly on metabolic and immune system processes, signal transduction, platelet and neutrophils degranulation. Peptidome-based protease mapping identified 59 proteases (29 serine-, 19 metallo-, 8 cisteine- and 3 aspartic-), in which Prothrombin, Plasminogen, MMP14, MMP7 and MMP3 proteases yielded most of peptides. Twenty sequences derived from Fibrinogen A, Fibrinogen B, Complement C3 (C3f), Apolipoprotein A-I (N-terminal), Prothrombin (C-terminal of Activation peptide fragment 2; N-terminal of Thrombin light chain) and Coagulation factor XIII A (N-terminal) were significantly different (p ≤ 0.5). Between these peptides, 14 were up regulated and six downregulated in GC patients. The phosphopeptide 20ADSpGEGDFLAEGGGVR35 (Fibrinopeptide A) was significantly increased (p< 0.03) in GC serum samples, while the non-phosphorylated Fibrinopeptide A was decreased (p<0.04), suggesting a higher phosphorylation rate in this protein in GC patients. Further, the peptide 31QGVNDNEEGFFSAR44 (Fibrinopeptide B) was downregulated (p<0.04) in GC serum samples; in counterpart, the Fibrinopeptide B derived peptides NDNEEGFF (p<0.0007) and QGVNDNEEGFFS (p<0.0013) was up regulated, suggesting a higher proteolysis in GC serum samples. Conclusions: The investigation of protease/substrate activity relationship may be described such as a novel panorama for discovery of serum biomarkers.

Fibrinopeptide A Induces Expression of C-Reactive Protein via the ROS-ERK1/2/ P38-NF-κB Signal Pathway in Vascular Smooth Muscle Cells

Background/Aims: Atherosclerosis is a chronic inflammatory disease in the artery walls. Fibrinopeptide A (FPA) is a biomarker of the activation of coagulation system, and a high concentration of FPA in blood occurs in patients with ischemic heart disease etc. However, there exist few studies on the pathological effects of FPA in cardiovascular system. Therefore, the present study examined the effect of FPA on CRP expression in VSMCs and the molecular mechanisms. Methods: mRNA and protein expression was identified by quantitative real-time PCR and Western blot, respectively. Reactive oxygen species (ROS) and the immunofluorescence staining were observed by a fluorescence microscope. Plasma FPA and CRP level was determined by ELISA. Results: FPA induced the expressions of CRP, IL-1β and IL-6 in VSMCs, and anti-IL-1β and anti-IL-6 neutralizing antibodies partially reduced FPA-induced CRP expression in VSMCs. The subchronic administration of FPA to rats increased FPA level in plasma and CRP expression in the aortic artery walls. The further studies showed that FPA promoted superoxide anion generation in VSMCs. Antioxidant NAC antagonized FPA-stimulated superoxide anion generation and inhibited FPA-induced CRP expression in VSMCs. FPA activated ERK1/2 and p38 phosphorylation, and PD98059 and SB203580 reduced FPA-induced CRP expression. Moreover, NAC inhibited the activation of ERK1/2 and p38. In addition, FPA enhanced NF-κB level in the nuclei of VSMCs, and PDTC reduced FPA-induced expression of CRP. Conclusions: FPA induces CRP expression in VSMCs via ROS-ERK1/2/p38-NF-κB signal pathway. This finding for the first time provides an experimental evidence for pro-inflammatory effect of FPA.