Fibrinopeptide A Research Articles

Issue Information

Front cover: Female mosquitoes suck blood to mature their eggs. It has been reported that fi brinopeptide A (FPA), which is cleaved from fi brinogen α chain during blood coagulation, makes mosquitoes feel “full” and stop sucking blood (Sakuma et al. (2024) Cell Reports 43: 114354, DOI: 10.1016/j.celrep.2024.114354). The mother in this painting is trying to use a mosquito net imbued with the ability of FPA to override the child's nightmare of a bloodsucking mosquito with the image of a satiated mosquito. In addition to fi brinogen and FPA, the mother's kimono shows schematic images of fi brin that has just begun to coagulate. Designed by TRAIS Co., Ltd. (Kobe, Japan). image

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.

Enhanced in vivo and ex vivo thrombin generation after lower-leg trauma, but not after knee arthroscopy

BackgroundThere is room for improvement of prevention of venous thromboembolism (VTE) after lower-leg cast application or knee arthroscopy. Information about the mechanism of clot formation in these patients may be useful to identify new prophylaxis targets. We aimed to study the effect of 1) lower-leg injury and 2) knee arthroscopy on thrombin generation.MethodsA cross-sectional study was conducted using plasma samples of POT-(K)CAST trials to measure ex vivo thrombin generation (Calibrated Automated Thrombography [CAT]) and plasma levels of prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin (TAT), fibrinopeptide A (FPA). Plasma was obtained shortly after lower-leg trauma or before and after (< 4 h) knee arthroscopy. Participants were randomly selected from those who did not develop VTE. For aim 1, samples of 88 patients with lower-leg injury were compared with 89 control samples (i.e., preoperative samples of arthroscopy patients). Linear regression was used to obtain mean differences (or ratios if ln-retransformed because of skewedness) adjusted for age, sex, body mass index, comorbidities. For aim 2, pre- and postoperative samples of 85 arthroscopy patients were compared, for which mean changes were obtained.ResultsIn patients with lower-leg injury (aim 1), endogenous thrombin potential, thrombin peak, velocity index, FPA and TAT were increased as compared with controls. In arthroscopy patients (aim 2), pre- and postoperative levels were similar for all parameters.ConclusionLower-leg trauma increases thrombin generation both ex vivo and in vivo, in contrast to knee arthroscopy. This may imply that the pathogenesis of VTE is different in both situations.

Nanopore Discrimination of Coagulation Biomarker Derivatives and Characterization of a Post-Translational Modification.

One of the most important health challenges is the early and ongoing detection of disease for prevention, as well as personalized treatment management. Development of new sensitive analytical point-of-care tests are, therefore, necessary for direct biomarker detection from biofluids as critical tools to address the healthcare needs of an aging global population. Coagulation disorders associated with stroke, heart attack, or cancer are defined by an increased level of the fibrinopeptide A (FPA) biomarker, among others. This biomarker exists in more than one form: it can be post-translationally modified with a phosphate and also cleaved to form shorter peptides. Current assays are long and have difficulties in discriminating between these derivatives; hence, this is an underutilized biomarker for routine clinical practice. We use nanopore sensing to identify FPA, the phosphorylated FPA, and two derivatives. Each of these peptides is characterized by unique electrical signals for both dwell time and blockade level. We also show that the phosphorylated form of FPA can adopt two different conformations, each of which have different values for each electrical parameter. We were able to use these parameters to discriminate these peptides from a mix, thereby opening the way for the potential development of new point-of-care tests.

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.

Simple SummaryTo date, the five-year survival rate of early stages of non-small cell lung cancer (NSCLC) is still disappointing and reliable prognostic factors are mandatory. Here, we performed in-depth high-throughput analyses of plasma circulating markers, including exosomal microRNAs and peptidome to identify a prognostic score. 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 relapse patients. Notably, a stepwise algorithm selected Exo-miR-130a-3p and the greatest FpA (2–16) to build a prognostic score, where high-risk patients had 18 months of median disease-free survival. Overexpression of miR-130a-3p cells led to a deregulation of pathways such as angiogenesis as well as the coagulation and metalloprotease, which might be linked to FpA reduction. The risk score integrating circulating markers may help clinicians predict early-stage NSCLC patients who are more likely to relapse after surgery.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.

Correlation and mechanism between cardiac magnetic resonance imaging and oral streptococcus count in patients with primary microvascular angina pectoris.

Although primary microvascular angina (PMVA) can be diagnosed clinically, the etiology and pathophysiology of PMVA remain unclear. The effects of conventional clinical medications (aspirin, statins, and nitrates) are unsatisfactory, and PMVA can lead to serious cardiovascular events. The present study was designed to analyze the correlation between the load perfusion cardiovascular magnetic resonance imaging (CMR) results and the Streptococcus sanguinis(S sanguinis) count and the correlations between the S sanguinis count in oral cavity subgingival plaque and changes in the plasma levels of platelet alpha-granule membrane glycoprotein 140 (GMP-140), fibrinopeptide A (FPA), von Willebrand factor (vWF), and homocysteine (Hcy) in patients with PMVA after increased anti-infective treatment of the oral cavity. This study also discusses the pathogenesis of PMVA from this perspective. The differences in the S sanguinis count in oral cavity subgingival plaque and oral health status between healthy people and PMVA patients will be compared, and the correlation between the oral cavity health status and disease in PMVA patients will be analyzed. The present randomized controlled trial with a parallel control group will be conducted in 68 PMVA patients diagnosed by the in-patient cardiology department. The selected patients will be randomly divided into 2 groups, one receiving routine drug treatment and the other a combination of anti-infective treatments. The normal control group will comprise 30 healthy people with no infectious oral cavity disease matched by age and sex. We will conduct CMR, and the presence of S sanguinis in subgingival plaques will be used to determine the bacterial count in PMVA patients. Blood samples will also be collected to determine the levels of GMP-140, FPA, vWF, and Hcy. S sanguinis in the subgingival plaque of PMVA patients will be further analyzed after increasing the oral cavity anti-infective treatment; the resulting changes and their correlations with changes in GMP-140, FPA, vWF, and Hcy levels will be assessed. Additionally, the differences in the S sanguinis count and the oral cavity health status of oral cavity dental plaque between healthy people and PMVA patients will be determined, and the correlation between the oral cavity conditions and PMVA will be analyzed. The relationship between the perfusion CMR results and the oral cavity S sanguinis count of PMVA patients, and the potential pathogenesis, will be explored. We will use the SPSS19.0 statistical software package to analyze the data. The measurements will be expressed as means±standard deviation. Student t test will be used for intergroup comparisons, a relative number description will be used for the count data, and the chi-square test will be used for intergroup comparisons. Multivariate logistic regression will be performed to identify associations. A P value < .05 will be considered significant. In this study, the correlation between the perfusion CMR results and the S sanguinis count in oral cavity subgingival plaque of PMVA patients will be analyzed. Changes in the levels of GMP-140, FPA, vWF, and Hcy of PMVA patients after receiving increased oral cavity anti-infective treatment will be explored, and the difference in the S sanguinis count in oral cavity subgingival plaque and the oral cavity health status between healthy people and PMVA patients will be compared. Chinese Clinical Trial Registry, (http://www.chictr.org.cn/showprojen.aspx?proj=45091).

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

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. 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. 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. 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.

Coagulopathy in Patients with Plasmacellular Dyscrasia

Disorders of fibrinogen (Fg) are usually the result of genetic mutations that hesitate in reduced levels of protein (hypofibrinogenemia) or in an abnormal molecule (dysfibrinogenemia). However, plasma factors or microenvironment can determine an acquired defect: reduced concentration or altered function. For example, antibodies may bind fibrinogen and/or fibrin by interfering with the polymerization and inhibiting the coagulation.Our goal is to identify the cause of dysfibrinogenemia in a man of 65 years, diagnosed because of a discrepancy between values of immunological Fg and values of coagulative Fg; despite a reduced concentration of functional fibrinogen and an elongation of the thrombin time (TT), activated partial thromboplastin time (aPTT) and of the prothrombin time (PT) we could not put the diagnosis of hereditary deficit, because of normal values of coagulation tests performed by the patient about 12 months before.The identification of a band on plasma immunofixation (western blot), referable to the light chain k, near to the band of fibrinogen, led us to investigate for a possible plasmacellular dyscrasia. Bone marrow plasma cells percentage was within normal range (4-5%). However the cytofluorimetric assay showed a k monoclonality in 80% of the patient's bone marrow plasma cells.FLC-MGUS (monoclonal gammopatia of uncertain significance with the presence of monoclonal protein consisting of only light chain) diagnosis was made.Treatment with dexametasone resulted in an almost complete correction of hemocoagulation parameters (with the only exception of TT elongation).Despite serious altered coagulation tests, patient have never had haemorrhagic manifestations and/or thrombotic diseases.This case is particularly unusual because the inhibitory effect on fibrinogen molecules was induced by a single light chain rather than by a complete antibody molecule. MGUS diagnosis was made on plasma immunofixation: no clonality was identified on serum immunofixation maybe because k chains were binded to fibrinogen molecules, absent in serum assay. In agreement with this hypothesis, after steroid treatment, FLC k (free light chain) values rose as if k light chains were released from fibrinogen molecules.No coagulation tests correction was observed after PNP (pooled normal plasma) addition, (1:1 ratio) or after addition of Fg (obtained from PNP) to patient's plasma: patient's k light chain could inhibit Fg in normal plasma pool as well. Purified Fg from patient plasma was added to purified Fg from PNP: by increasing the patient's Fg share in relation to normal Fg share, we observed an initial decline in functional Fg value until 5:1 ratio was reached. No further functional Fg decline was observed increasing Fg share in dilution: functional fg reached a plateau as if saturation of the interfering capacity of light chains was achieved .There were no differences between coagulation test at 4°C or at 37°C so we could rule out an interaction between k light chain and glucose part of fibrinogen molecule. No alterations in platelet aggregation test were observed, so an interaction in the fibrinogen's binding site for platelets IIb/IIIa glycoprotein, was excluded. As no hemorrhagic events were reported by the patient, an interaction in the fibrinogen's binding site for fXIII (coagulation factor XIII) or a FpA (fibrinopeptide A) impaired release were unlikely. However reptilase time test wasn't available at our laboratoty to assess FpA release.It is known that pathological immunoglobulins (Ig) multiple myeloma (MM) patients interfere with coagulation test, so we screened 20 MM patients control group for dysfibrinogenemia, like patient's one. In 4 MM patients we found TT elongation but normal functional Fg values. In plasma immunofixation light chain bands didn't run at the same level of fibrinogen molecules.We could rule out an interference between Fg and monoclonal light chain in MM patients: we speculated that pathological Ig interefered with fibrin polimerization. However only a comparison between functional and immunological fibrinogen tests should surely exclude dysfibrinogenemia in these patients, despite normal functional fibrinogen values.A specific test to assay interaction between monoclonal light chain (obtained from patient's plasma) and Fg from PNP will be necessary to surely prove dysfibrinogenemia related to monoclonal FLCs. DisclosuresNo relevant conflicts of interest to declare.

Combination of polycarboxybetaine coating and factor XII inhibitor reduces clot formation while preserving normal tissue coagulation during extracorporeal life support

Blood contact with high surface area medical devices, such as dialysis and extracorporeal life support (ECLS), induces rapid surface coagulation. Systemic anticoagulation, such as heparin, is thus necessary to slow clot formation, but some patients suffer from bleeding complications. Both problems might be reduced by 1) replacing heparin anticoagulation with artificial surface inhibition of the protein adsorption that initiates coagulation and 2) selective inhibition of the intrinsic branch of the coagulation cascade. This approach was evaluated by comparing clot formation and bleeding times during short-term ECLS using zwitterionic polycarboxybetaine (PCB) surface coatings combined with either a potent, selective, bicyclic peptide inhibitor of activated Factor XII (FXII900) or standard heparin anticoagulation. Rabbits underwent venovenous ECLS with small sham oxygenators for 60 min using three means of anticoagulation (n = 4 ea): (1) PCB coating + FXII900 infusion, (2) PCB coating + heparin infusion with an activated clotting time of 220–300s, and (3) heparin infusion alone. Sham oxygenator blood clot weights in the PCB + FXII900 and PCB + heparin groups were 4% and 25% of that in the heparin group (p < 10−6 and p < 10−5), respectively. At the same time, the bleeding time remained normal in the PCB + FXII900 group (2.4 ± 0.2 min) but increased to 4.8 ± 0.5 and 5.1 ± 0.7 min in the PCB + heparin and heparin alone groups (p < 10−4 and 0.01). Sham oxygenator blood flow resistance was significantly lower in the PCB + FXII900 and PCB + heparin groups than in the heparin only group (p < 10−6 and 10−5). These results were confirmed by gross and scanning electron microscopy (SEM) images and fibrinopeptide A (FPA) concentrations. Thus, the combined use of PCB coating and FXII900 markedly reduced sham oxygenator coagulation and tissue bleeding times versus the clinical standard of heparin anticoagulation and is a promising anticoagulation method for clinical ECLS.

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

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. 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. 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). 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.

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

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. 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. 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). 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.

Mathematical Models for the Prediction of Coagulation Activity in Patients with Paroxysmal Atrial Fibrillation

Our previous studies showed activation of coagulation in the early hours of the clinical manifestation of paroxysmal atrial fibrillation (PAF). Plasma coagulation activity of factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, vWF, tissue factor levels, FVIII, vWF, prothrombin fragment 1+2(F1 + 2) and fibrinopeptide A (FPA) were significantly increased as early as the first twenty-four hours of the disease. The results suggest that there is a correlation between the studied parameters and development of the disease. Aim: To search for a statistical model that predicts coagulation activity in PAF patients. Materials and methods: Coagulation parameters were examined in 51 PAF patients (26 males, 25 females; mean age 59.84 ±1.60 years, onset of PAF episode &lt; 24h prior to hospitalization). Controls included 52 individuals (26 males, 26 females; mean age 59.50 ± 1.46 years) with no prior anamnestic or ECG AF data, corresponding to patients in sex, age, BMI and comorbidities. A linear regression model was used to predict coagulation activity in PAF. Regression models showed good correlation between the duration of arrhythmia and six of the fourteen coagulation parameters studied: F1+2 (r = 0.83, p &lt;0.001), FPA (r = 0.84, p &lt;0.001), FVIII levels (r = 0.85, p &lt;0.001) as well as activity of FII (r = 0.83, p &lt;0.001), FVIII (r = 0.83, p &lt;0.001) and FXII (r = 0.78, p &lt;0.001). Changes in F1+2 plasma levels were most sensitive to PAF duration, where the contribution of duration to the values of the indicator is the greatest (b = 15.31). Conclusion: Linear regression analysis allowed us to create models with a high correlation coefficient for predicting the values of F1+2, FPA, FVIII levels, as well as activity of FII, FVIII and FXII in PAF patients. These models could allow for quantification of the procoagulatory process and thrombotic potential of the disease.

Urinary fibrinopeptide-A as a predictive biomarker of exacerbation in asthma

BackgroundAsthma is a prevalent and potentially life-threatening disease associated with exacerbation and costly hospital admissions. The coagulation cascade is up-regulated in severe asthma and increased fibrinogenesis in the airway may precede exacerbation in moderate asthma. ObjectiveA longitudinal prospective study to test the hypothesis that levels of urinary fibrinopeptide A (FP-A), a marker of coagulation, increase prior to an exacerbation of asthma. Methods24 non-smoking participants with moderate to severe asthma were recruited and followed to exacerbation and to recovery for up to 8 weeks afterwards. Baseline measurements included spirometry, full blood count, atopic status and plasma markers of coagulation. Participants provided daily Peak Expiratory Flow (PEF) readings and three urine samples per week for analysis of FP-A, a specific marker of activation of coagulation. A novel method to concentrate urinary FP-A for immunodetection and quantification was developed. Participants were followed up until exacerbation, when baseline measurements were repeated, and monthly thereafter for 2 months or to recovery. Measurements and main findings17 participants exacerbated during the study. Significantly increased concentrations of plasma D-dimer (0.25(0.2–0.42) vs 0.21(0.12–0.29) μg FEU/ml, p = 0.02) were found at exacerbation. A peak in urinary FP-A concentration was detected on average 4.2 ± 2 days prior to exacerbation and was significantly (p < 0.05) higher than at exacerbation or 7 days later. Urinary FP-A concentrations correlated positively with time to recovery and negatively (p < 0.01) with IgE concentration. ConclusionFP-A is detectable in urine several days before the onset of an asthma exacerbation indicating disordered coagulation preceding asthma exacerbations.

Production of a correctly assembled fibrinogen using transgenic silkworms.

Fibrinogen from human blood is used as a main component of coagulants, including surgical tissue sealants. The development of a recombinant human fibrinogen (rFib) is anticipated to eliminate the risks of blood-borne infections. Here, we report the efficient production of rFib in a transgenic silkworm system. A silkworm line carrying cDNAs of the fibrinogen Aα and γ chains (Aα/γ-silkworm) produced Aα and γ chains in its cocoons, however, the Bβ chains were not detected from cocoons of another silkworm line carrying the cDNA of fibrinogen Bβ chains (Bβ-silkworm). A silkworm line for all three fibrinogen chains was generated by crossing Aα/γ-silkworms with Bβ-silkworms, which secreted Aα2Bβ2γ2 fibrinogen (rFib) into cocoons at high contents. The N-terminal amino acid sequences of the three rFib chains were identical to those of the corresponding chains of native fibrinogen (nFib). The N-glycan profile of the rFib comprised oligomannose-type (53%), complex-type (34%), and paucimannose-type (13%); neither high-mannose-type (six or more mannose residues) nor core-fucosylated glycans were observed. The coagulation activity of the rFib was evaluated for the amount of thrombin-released fibrinopeptide A (FpA) and the kinetics for turbidity increase (non-covalent network formation) in the solution. FpA release rates were equivalent between rFib and nFib; by contrast, the kinetics of the turbidity increase for rFib were accelerated nearly two-fold, for both the rate and maximum value, compared to those of nFib. These results demonstrate that the rFib produced in the transgenic silkworm system is comparable to nFib in both physical and coagulative properties. This rFib is a promising candidate component for safe hemostatic pharmaceuticals.

Application of Piezo-Based Measuring System for Evaluation of Nucleic Acid-Based Drugs Influencing the Coagulation.

During open-heart surgery, the status of hemostasis has to be constantly monitored to quickly and reliably detect bleeding or coagulation disorders. In this study, a novel optimized piezo-based measuring system (PIEZ) for rheological monitoring of hemostasis was established. The applicability of the PIEZ for the evaluation of nucleic acid-based drugs influencing coagulation was analyzed. Thrombin aptamers such as NU172 might be used during extracorporeal circulation (ECC) in combination with a reduced heparin concentration or for patients with heparin-induced thrombocytopenia (HIT). Therefore, the effect of the coagulation inhibiting thrombin aptamer NU172 and the abrogation by its complementary antidote sequence (AD) were investigated by this rheological PIEZ system. After the addition of different NU172 concentrations, the coagulation of fresh human blood was analyzed under static conditions and using an in vitro rotation model under dynamic conditions (simulating ECC). The clotting times (CTs) detected by PIEZ were compared to those obtained with a medical reference device, a ball coagulometer. Additionally, after the circulation of blood samples for 30 min at 37 °C, blood cell numbers, thrombin markers (thrombin-antithrombin III (TAT) and fibrinopeptide A (FPA)) and a platelet activation marker (β-thromboglobulin (β-TG)) were analyzed by enzyme-linked immunosorbent assays (ELISAs). The increase of NU172 concentration resulted in prolonged CTs, which were comparable between the reference ball coagulometer and the PIEZ, demonstrating the reliability of the new measuring system. Moreover, by looking at the slope of the linear regression of the viscous and elastic components, PIEZ also could provide information on the kinetics of the coagulation reaction. The shear viscosity at the end of the measurements (after 300 s) was indicative of clot firmness. Furthermore, the PIEZ was able to detect the abrogation of coagulation inhibition after the equimolar addition of NU172 aptamer´s AD. The obtained results showed that the established PIEZ is capable to dynamically measure the hemostasis status in whole blood and can be applied to analyze nucleic acid-based drugs influencing the coagulation.

Citrullination of fibrinogen by peptidylarginine deiminase 2 impairs fibrin clot structure

BackgroundCitrullination is the post-translational conversion of arginine into citrulline in proteins. The reaction is catalyzed by peptidylarginine deiminase (PAD), of which five isoforms exist. Fibrinogen is a substrate for PAD2 and PAD4, and citrullinated fibrinogen (cFBG) has been detected in patients with inflammatory diseases. In purified systems, cFBG is known to inhibit the release of fibrinopeptide A (FPA) and B (FPB) and impairs fibrin polymerization. However, the effect of cFBG on fibrin structure and fibrinolysis in a plasma environment remains unclear. We hypothesized that citrullination of fibrinogen impairs fibrin properties. MethodsFibrinogen was citrullinated by recombinant PAD2 and PAD4. The impact of cFBG on fibrin structure was investigated by turbidity measurements in fibrinogen-deficient plasma spiked with cFBG or native fibrinogen. ResultsCitrullination of fibrinogen by PAD2 dose-dependently reduced the rate of fibrin polymerization, as well as the overall hemostasis potential of fibrin, the maximum velocity of fibrin formation, the fibrin mass/length ratio, and the lysis of fibrin clots. ConclusionCitrullination of fibrinogen by PAD2 affects not only fibrin polymerization but also fibrin fiber properties, indicating that the fibrin network formed in the presence of cFBG may influence hemostasis. Our results suggest that citrullination of fibrinogen alters the composition of fibrin fibers which may lead to a looser fibrin network that is more susceptible to fibrinolysis and thereby affecting the hemostatic balance.

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.

Histidine-rich Glycoprotein Could Be an Early Predictor of Vasospasm after Aneurysmal Subarachnoid Hemorrhage.

Cerebral vasospasm (CVS) is a major contributor to the high morbidity and mortality of aneurysmal subarachnoid hemorrhage (aSAH) patients. We measured histidine-rich glycoprotein (HRG), a new biomarker of aSAH, in cerebrospinal fluid (CSF) to investigate whether HRG might be an early predictor of CVS. A total of seven controls and 14 aSAH patients (8 males, 6 females aged 53.4±15.4 years) were enrolled, and serial CSF and serum samples were taken. We allocated these samples to three phases (T1-T3) and measured HRG, interleukin (IL)-6, fibrinopeptide A (FpA), and 8-hydroxy-2'-deoxyguanosine (8OHdG) in the CSF, and the HRG in serum. We also examined the release of HRG in rat blood incubated in artificial CSF. In contrast to the other biomarkers examined, the change in the CSF HRG concentration was significantly different between the nonspasm and spasm groups (p<0.01). The rat blood/CSF model revealed a time course similar to that of the human CSF samples in the non-spasm group. HRG thus appears to have the potential to become an early predictor of CVS. In addition, the interaction of HRG with IL-6, FpA, and 8OHdG may form the pathology of CVS.

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-1β, and IL-6 in human umbilical vascular endothelial cells (HUVECs); and anti-IL-1 β 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.

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.