Fibrinopeptide A induces C-reactive protein expression through the ROS-ERK1/2/p38-NF-κB signal pathway in the human umbilical vascular endothelial cells.

Atherosclerosis is a chronic inflammatory disease of the arterial wall. Inflammation causes endothelial injury and dysfunction, which is an initial step of atherosclerosis. Fibrinopeptide A (FPA) is a biomarker of the activation of the coagulation system, and a high concentration of FPA in the blood occurs in patients with ischemic cardiocerebrovascular diseases. The present research observed that FPA stimulated the generation of C-reactive protein (CRP), IL-1ß, and IL-6 in human umbilical vascular endothelial cells (HUVECs); and anti-IL-1 ß and anti-IL-6 neutralizing antibodies did not alter FPA-induced CRP expression in HUVECs. The subchronic administration of FPA into rats increased the plasma FPA and CRP levels. Further studies showed that FPA stimulated superoxide anion generation, activated ERK1/2 and p38, promoted nuclear factor κB (NF-κB) nuclear translocation, and raised the NF-κB level in the nuclei of HUVECs. Antioxidant N-acetylcysteine (NAC), complex II inhibitor thenoyltrifluoroacetone (TTFA), and NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited FPA-stimulated generation of superoxide anion, and NAC reduced FPA-induced expressions of the phosphorylated ERK1/2 and p38. NAC, TTFA, DPI, inhibitors of ERK1/2, p38, and NF-κB all downregulated FPA-induced CRP expression. These results indicate that FPA induces CRP expression in HUVECs via the ROS-ERK1/2/p38-NF-κB signal pathway. Moreover, this is the first report that FPA produces a proinflammatory effect on the vascular endothelial cells.

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-1ß and IL-6 in VSMCs, and anti-IL-1ß 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.

Fibrinopeptide A-induced blood-feeding arrest in the yellow fever mosquito Aedes aegypti.

Female mosquitoes engage in blood feeding from their hosts to facilitate egg maturation but cease feeding once a sufficient blood meal has been acquired. Abdominal distention has been proposed as a contributing factor; however, it has also been suggested that there are chemical controls. In this study, we focus on negative chemical regulators of blood feeding, particularly those present in the host blood. Serum derived from animal blood inhibits the feeding of ATP, a phagostimulant of blood feeding in Aedes aegypti. Fibrinopeptide A (FPA), a 16-amino acid peptide cleaved from fibrinogen during blood coagulation, serves as an inhibitory factor in the serum. Our findings suggest that blood-feeding arrest in female mosquitoes is triggered by the detection of FPA in the host blood, which increases as blood coagulation proceeds in the mosquito's midgut, highlighting the role of host-derived substances as negative regulators of mosquito behavior.

Serum fibrinopeptide A is increased in patients with acute coronary syndrome.

OBJECTIVE: Acute coronary syndrome (ACS) is one of the leading causes of mortality, globally. Atherosclerosis is an underlying factor in ACS process and coagulative cascade is activated secondary to atherosclerotic plaque rupture. Fibrinopeptide A (FPA) takes an active role in thrombus formation and is an indicator of coagulative process. We aimed to evaluate serum FPA level in patients with ACS. METHODS: Patients diagnosed with ACS and chronic coronary syndrome (CCS), with non-obstructive coronary artery disease as a control group, were included in the study. Blood samples and demographic data of all patients were obtained at admission. Obtained data were compared between ACS and control groups. RESULTS: The study consisted of 107 patients with ACS and 69 patients with CCS. ACS group was older (p<0.001) with male preponderance (p<0.001), more likely to had hypertension (p<0.001), and had a higher smoking rate (p<0.001). Serum FPA level was highest in the ST elevated myocardial infarction group (p<0.001). FPA>3.38 ng/mL predicted ACS with 89.7% sensitivity and 78% specificity (AUC: 0.825, 95% CI 0.745-0.905; p<0.001). CONCLUSION: Serum FPA may be used for the differential diagnosis of ACS. In addition, patients with increased FPA may be considered to be given more aggressive antithrombotic medication.

A Circulating Risk Score, Based on Combined Expression of Exo-miR-130a-3p and Fibrinopeptide A, as Predictive Biomarker of Relapse in Resectable Non-Small Cell Lung Cancer Patients.

To date, the 5-year overall survival rate of 60% for early-stage non-small cell lung cancer (NSCLC) is still unsatisfactory. Therefore, reliable prognostic factors are needed. Growing evidence shows that cancer progression may depend on an interconnection between cancer cells and the surrounding tumor microenvironment; hence, circulating molecules may represent promising markers of cancer recurrence. In order to identify a prognostic score, we performed in-depth high-throughput analyses of plasma circulating markers, including exosomal microRNAs (Exo-miR) and peptides, in 67 radically resected NSCLCs. The miRnome profile selected the Exo-miR-130a-3p as the most overexpressed in relapsed patients. Peptidome analysis identified four progressively more degraded forms of fibrinopeptide A (FpA), which were depleted in progressing patients. Notably, stepwise Cox regression analysis selected Exo-miR-130a-3p and the greatest FpA (2-16) to build a score predictive of recurrence, where high-risk patients had 18 months of median disease-free survival. Moreover, in vitro transfections showed that higher levels of miR-130a-3p lead to a deregulation of pathways involved in metastasis and angiogenesis, including the coagulation process and metalloprotease increase which might be linked to FpA reduction. In conclusion, by integrating circulating markers, the identified risk score may help clinicians predict early-stage NSCLC patients who are more likely to relapse after primary surgery.

A novel variant fibrinogen, AαE11del, demonstrating the importance of AαE11 residue in thrombin binding.

INTRODUCTION: We identified a novel heterozygous AαE11del variant in a patient with congenital dysfibrinogenemia. This mutation is located in fibrinopeptide A (FpA). We analyzed the effect of AαE11del on the catalyzation of thrombin and batroxobin and simulated the stability of the complex structure between the FpA fragment (AαG6-V20) peptide and thrombin. MATERIALS AND METHODS: We performed fibrin polymerization and examined the kinetics of FpA release catalyzed by thrombin and batroxobin using purified plasma fibrinogen. To clarify the association between the AαE11 residue and thrombin, we calculated binding free energy using molecular dynamics simulation trajectories. RESULTS: Increasing the thrombin concentration improved release of FpA from the patient's fibrinogen to approximately 90%, compared to the previous 50% of that of normal fibrinogen. Fibrin polymerization of variant fibrinogen also improved. In addition, greater impairment of variant FpA release from the patient's fibrinogen was observed with thrombin than with batroxobin. Moreover, the calculated binding free energy showed that the FpA fragment-thrombin complex became unstable due to the missing AαE11 residue. CONCLUSIONS: Our findings indicate that the AαE11 residue is involved in FpA release in thrombin catalyzation more than in batroxobin catalyzation, and that the AαE11 residue stabilizes FpA fragment-thrombin complex formation.

Paroxysmal Atrial Fibrillation: An Independent Risk Factor for Prothrombotic Conditions.

OBJECTIVE: It remains unclear whether atrial fibrillation (AF) alone determines systemic changes in hemocoagulation. Our aim was to examine the prothrombin fragment F1+2 and fibrinopeptide A (FPA) as early markers of coagulation activity still in the first twenty-four hours of paroxysmal AF (PAF) and to correlate them with the arrhythmia onset. METHODS: 51 non-anticoagulated patients (26 men, 25 women, aged 59.84±1.6 years) and 52 controls (26 men, 26 women, aged 59.50±1.46 years) were sequentially selected. F1+2 and FPA plasma levels were measured by enzyme-linked immunoassays. RESULTS: F1+2 was significantly higher in patients (292.61pmol/L±14.03pmol/L vs 183.40pmol/L±8.38pmol/L; p<0.001). FPA was also substantially higher (4.47ng/mL±0.25 ng/mL vs 3.09ng/mL±0.15ng/mL, p<0.001). Among the potential predictors for these deviations: age, gender, BMI, PAF duration and CHA2DS2-VASc score, it was established that higher F1+2 and FPA plasma levels were independently associated only with PAF duration (p<0.05). Moreover, longer episodes were associated with higher values of F1+2 (Adjusted R2 = 0.68) and FPA (Adjusted R2 = 0.70). CONCLUSIONS: Increased coagulation activity was present still in the first twenty-four hours of PAF clinical presentation. The disease itself was associated with increasing hypercoagulability over time, suggesting its importance as an independent risk factor for thromboembolic events.

Markers of Fibrinolysis in Indian Patients with Isolated Head Trauma.

CONTEXT: Head injury causes disseminated intravascular coagulation as the most severe complication which is associated with high mortality. Elevated levels of markers of fibrinolysis such as D-dimer and fibrinopeptide A (FPA) have been correlated with poor outcome in these patients. AIM: The study aimed to correlate the levels of plasma fibrinogen, D-dimer, and FPA with outcome in patients with isolated head trauma. SETTINGS AND DESIGN: This cross-sectional descriptive study was conducted in the Departments of Pathology and Neurosurgery, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, on 100 patients admitted within 12 h of isolated head trauma. SUBJECTS AND METHODS: Plasma fibrinogen, D-dimer, and FPA were measured in 100 patients admitted within 12 h of isolated head trauma. While plasma fibrinogen and D-dimer were estimated in all patients, FPA was measured in 45 patients. STATISTICAL ANALYSIS: SPSS (20.2) software was used for mean, standard deviation, and median values of the quantitative parameters, and for all qualitative parameters, their frequencies were obtained. P < 0.05 was considered significant. RESULTS: Elevated D-dimer (>250 ng/ml) and FPA (>3 ng/ml) were observed in 64% and 91.1% patients, respectively. Both D-dimer and FPA were elevated in 66.6% of patients. Disseminated intravascular coagulation (DIC) score, calculated using standard criteria, was ≥5 in 28% of patients indicating overt DIC. Hypofibrinogenemia was observed in 48% of patients. D-dimer, FPA, and DIC score was significantly (P < 0.001) higher and plasma fibrinogen significantly (P < 0.001) lower in nonsurvivors as compared to survivors. Elevated D-dimer and FPA and low fibrinogen levels were seen in patients irrespective of severity of injury. CONCLUSIONS: Elevated D-dimer and FPA were frequently observed in patients with isolated head trauma. As these markers rise soon after injury and indicate poor outcome, their measurement will help identify patients who will benefit with additional therapy, thus reducing morbidity and mortality.

Human Antimicrobial Peptide Isolated From Triatoma infestans Haemolymph, Trypanosoma cruzi-Transmitting Vector.

The importance of antimicrobial peptides (AMPs) in relation to the survival of invertebrates is well known. The source and the mode of action on the insects' immune system of these molecules have been described from different perspectives. Insects produce their own AMPs as well as obtain these molecules from various sources, for example by absorption through the intestinal tract, as previously described for Boophilus microplus. Blood-sucking barber bug Triatoma infestans attracts social, economic and medical interest owing to its role in the transmission of Chagas disease. Despite new studies, descriptions of AMPs from this insect have remained elusive. Thus, the aims of this work were to characterize the antimicrobial potential of human fibrinopeptide A (FbPA) obtained from the T. infestans haemolymph and identify its natural source. Therefore, FbPA was isolated from the T. infestans haemolymph through liquid chromatography and identified by mass spectrometry. This peptide exhibited antimicrobial activity against Micrococcus luteus. Native FbPA from human blood and the synthetic FbPA also exhibited antimicrobial activity. The synthetic FbPA was conjugated with fluorescein isothiocyanate and offered to the insects. The haemolymph collected after 72 h exhibited fluorescence at the same wavelength as fluorescein isothiocyanate. Our experiments show that beyond intrinsic AMP production, T. infestans is able to co-opt molecules via internalization and may use them as AMPs for protection.

Coagulation and fibrinolysis in gastric cancer.

Coagulation is a highly conserved process occurring after an injury to a blood vessel and resulting in hemostasis. In the thrombus microenvironment, finely orchestrated events restore vessel integrity through platelet activation, adhesion, and aggregation (primary hemostasis), followed by the coagulation cascades, thrombin generation, and fibrin clot deposition (secondary hemostasis). Several studies on cancer have provided insight into dramatic changes to coagulation-related events (i.e., fibrin clot deposition, fibrinolysis) during tumor pathogenesis, progression, and metastasis, in addition to a tumor-driven systemic activation of hemostasis and thrombosis (Trousseau's syndrome). Diverse molecular and cellular effectors participate in the cross talk between hemostasis and tumors. Here, we focus on some aspects of the interconnection between cancer biology and hemostatic components, with particular attention to some key coagulation-related proteins (e.g., tissue factor, thrombin, fibrinogen, and D-dimers) in the particular case of gastric cancer (GC). Recent advances in deciphering the complex molecular link between GC and the coagulation system are described, showing their important roles in better management of patients affected by GC.

Fibrinogen α-chain-derived peptide is upregulated in hippocampus of rats exposed to acute morphine injection and spontaneous alternation testing.

Fibrinogen is a secreted glycoprotein that is synthesized in the liver, although recent in situ hybridization data support its expression in the brain. It is involved in blood clotting and is released in the brain upon injury. Here, we report changes in the extracellular levels of fibrinogen α-chain-derived peptides in the brain after injections of saline and morphine. More specifically, in order to assess hippocampus-related working memory, an approach pairing in vivo microdialysis with mass spectrometry was used to characterize extracellular peptide release from the hippocampus of rats in response to saline or morphine injection coupled with a spontaneous alternation task. Two fibrinopeptide A-related peptides derived from the fibrinogen α-chain-fibrinopeptide A (ADTGTTSEFIEAGGDIR) and a fibrinopeptide A-derived peptide (DTGTTSEFIEAGGDIR)-were shown to be consistently elevated in the hippocampal microdialysate. Fibrinopeptide A was significantly upregulated in rats exposed to morphine and spontaneous alternation testing compared with rats exposed to saline and spontaneous alternation testing (P < 0.001), morphine alone (P < 0.01), or saline alone (P < 0.01), respectively. The increase in fibrinopeptide A in rats subjected to morphine and a memory task suggests that a complex interaction between fibrinogen and morphine takes place in the hippocampus.

[A case of dysfibrinogenemia without hemorrhagic diathesis or thromboembolism linked to a new mutation p.H103N in fibrinogen γ chain]. / Un cas de dysfibrinogénémie sans complication hémorragique ni thrombotique lié à une nouvelle mutation p.H103N de la chaîne γ du fibrinogène.

This work describes a dysfibrinogenemia linked to a new mutation in the gene coding for fibrinogen γ chain. Dysfibrinogenemia was fortuitously discovered in a 9-year old boy consulting for symptoms suggesting meningitis. DNA was extracted from blood, the fibrinogen genes coding for Aα, Bß and γ chains were sequenced, and compared with consensus sequences. Apart from the patient, dysfibrinogenemia and the mutation p.H103N in the γ chain of fibrinogen with heterozygous status were found in his mother, without any symptom. This mutation is unknown in fibrinogen variant databases and seems to affect mostly fibrin polymerisation. The reporting of this new p.H103N mutation in the γ chain has a great interest for improving the knowledge of the fibrinogen gene and its expression. Even if no haemorrhage was observed in this case, the expression of this mutation impaired the function of the molecule, particularly polymerisation, and could induce bleeding during an important surgery.

Functional impact of oxidative posttranslational modifications on fibrinogen and fibrin clots.

Fibrinogen is a circulating multifunctional plasma protein vital for hemostasis. Activation of the coagulation cascade converts soluble fibrinogen to insoluble polymerized fibrin, which, along with platelets, forms the hemostatic clot. However, inappropriate formation of fibrin clots may result in arterial and venous thrombotic disorders that may progress to life-threatening adverse events. Often thrombotic disorders are associated with inflammation and the production of oxidants. Fibrinogen represents a potential target for oxidants, and several oxidative posttranslational modifications that influence fibrinogen structure and function have been associated with disease pathogenesis. Here, we review various oxidative modifications of fibrinogen and the consequences of these modifications on protein structure and the ability to form fibrin and how the resulting alterations affect fibrin architecture and viscoelastic and biochemical properties that may contribute to disease.

Hydrodynamic characterization of recombinant human fibrinogen species.

INTRODUCTION: Fibrinogen is a key component of the blood coagulation system and plays important, diverse roles in several relevant pathologies such as thrombosis, hemorrhage, and cancer. It is a large glycoprotein whose three-dimensional molecular structure is not fully known. Furthermore, circulating fibrinogen is highly heterogeneous, mainly due to proteolytic degradation and alternative mRNA processing. Recombinant production of human fibrinogen allows investigating the impact on the three-dimensional structure of specific changes in the primary structure. METHODS: We performed analytical ultracentrifugation analyses of a full-length recombinant human fibrinogen, its counterpart purified from human plasma, and a recombinant human fibrinogen with both Aα chains truncated at amino acid 251, thus missing their last 359 amino acid residues. RESULTS: We have accurately determined the translational diffusion and sedimentation coefficients (Dt(20,w)(0), s(20,w)(0)) of all three species. This was confirmed by derived molecular weights within 1% for the full length species, and 5% for the truncated species, as assessed by comparison with SDS-PAGE/Western blot analyses and primary structure data. No significant differences in the values of Dt(20,w)(0) and s(20,w)(0) were found between the recombinant and purified full length human fibrinogens, while slightly lower and higher values, respectively, resulted for the recombinant truncated human fibrinogen compared to a previously characterized purified human fibrinogen fragment X obtained by plasmin digestion. CONCLUSIONS: Full-length recombinant fibrinogen is less polydisperse but hydrodynamically indistinguishable from its counterpart purified from human plasma. Recombinant Aα251-truncated human fibrinogen instead behaves differently from fragment X, suggesting a role for the Bß residues 1-52 in inter-molecular interactions. Overall, these new hydrodynamic data will constitute a reliable benchmark against which models of fibrinogen species could be compared.