Predictors of neutrophil extracellular traps markers in type 2 diabetes mellitus: associations with a prothrombotic state and hypofibrinolysis.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with a hypercoagulable state and increased neutrophil extracellular traps formation (NETosis). We investigated predictors of NETosis and cell death markers in circulating blood and their association with a prothrombotic state in T2DM. METHODS: In a cross-sectional study involving 113 T2DM patients aged 63.7 ± 8.2 years, we investigated citrullinated histone H3 (H3Cit), cell-free deoxyribonucleic acid (cfDNA), myeloperoxidase, neutrophil elastase, and inflammation markers, along with thrombin generation (TG), plasma clot lysis time (CLT), clot permeability (Ks) and fibrinolysis inhibitors. RESULTS: On multivariate logistic regression analysis adjusted for age and gender, predictors of high H3Cit (≥ 7.36 ng/mL, upper quartile) were: glycated hemoglobin (HbA1c) ≥ 7.0% and interleukin-6. Interleukin-6 was also found to be a predictor of high cfDNA (≥ 2.84 µg/mL, upper quartile) along with glucose. Citrullinated histone H3 and cfDNA correlated positively with CLT and inversely with Ks, while TG associated solely with cfDNA. These associations were not seen with myeloperoxidase and neutrophil elastase. Patients with previous myocardial infarction (n = 21, 18.6%) had higher H3Cit (+108%, p < 0.001) and cfDNA (+45%, p = 0.022). On multivariable analysis adjusted for potential confounders, H3Cit and cfDNA, along with plasminogen activator inhibitor-1 and concomitant cardiovascular disease, were predictors of CLT. Citrullinated histone H3 alone was a predictor of Ks and only cfDNA was a predictor of peak thrombin generated. CONCLUSIONS: In T2DM, NETosis detectable in circulating blood is associated with inflammatory state and a prothrombotic state, especially hypofibrinolysis.

Quantitative Analysis of Human Red Blood Cell Proteome.

Red blood cells (RBCs) are the most abundant cell type in the human body. RBCs and, in particular, their plasma membrane composition have been extensively studied for many years. During the past decade proteomics studies have extended our knowledge on RBC composition; however, these studies did not provide quantitative insights. Here we report a large-scale proteomics investigation of RBCs and their "white ghost" membrane fraction. Samples were processed using the multienzyme digestion filter-aided sample preparation (MED-FASP) and analyzed using Q-Exactive mass spectrometer. Protein abundances were computed using the total protein approach (TPA). The validation of the data with stable isotope-labeled peptide-based protein quantification followed. Our in-depth analysis resulted in the identification of 2650 proteins, of which 1890 occurred at more than 100 copies per cell. We quantified 41 membrane transporter proteins spanning an abundance range of five orders of magnitude. Some of these, including the drug transporter ABCA7 and choline transporters SLC44A1 and SLC44A2, have not previously been identified in RBC membranes. Comparison of protein copy numbers assessed by proteomics showed a good correlation with literature data; however, abundances of several proteins were not consistent with the classical references. Because we validated our findings by a targeted analysis using labeled standards, our data suggest that some older reference data from a variety of biochemical approaches are inaccurate. Our study provides the first "in-depth" quantitative analysis of the RBC proteome and will promote future studies of erythrocyte structure, functions, and disease.

Interaction of glycated and acetylated human α2-antiplasmin with fibrin clots.

: In type 2 diabetes mellitus (T2DM), increased α2-antiplasmin incorporation in fibrin and impaired fibrinolysis have been reported. Acetylsalicylic acid (ASA), used in cardiovascular prevention, modulates fibrinolysis and exerts weaker therapeutic effect in this disease. We investigated how glycation and acetylation of α2-antiplasmin affects its interaction with fibrin. Using surface plasmon resonance, we analyzed fibrin binding by α2-antiplasmin incubated with no ß-D-glucose or ASA (control); incubated with ß-D-glucose (5, 10, 50 mmol/l); (3) incubated with 1.6 mmol/l acetylsalicylic acid (ASA) and (4) incubated with 1.6 mmol/l ASA and 50 mmol/l ß-D-glucose. Incubation with glucose decreased affinity of α2-antiplasmin for fibrin compared with control α2-antiplasmin in a glucose concentration-depending manner. α2-Antiplasmin incubation with ASA did not affect its affinity to fibrin. α2-Antiplasmin incubation with ASA and glucose resulted in 4.2-fold increased affinity to fibrin compared with α2-antiplasmin incubated with 50 mmol/l glucose (P < 0.001). In conclusion, α2-antiplasmin incubation with glucose at concentrations encountered in T2DM is associated with decreased binding affinity of α2-antiplasmin to fibrin. ASA alone does not affect the binding affinity of α2-antiplasmin to fibrin, but partly reverses the effect introduced by the incubation with 50 mmol/l glucose. This study suggests new mechanisms involved in regulating fibrinolysis efficiency in hyperglycemia.

Plasma Protein Oxidation as a Determinant of Impaired Fibrinolysis in Type 2 Diabetes.

OBJECTIVE: We investigated clinical and laboratory determinants of plasma protein oxidation and its associations with clot fibrinolysis in type 2 diabetes patients. MATERIALS AND METHODS: Our cross-sectional study consisted of 246 type 2 diabetic patients, 143 (58%) with concomitant cardiovascular disease (CVD), including 41 (17%) with previous myocardial infarction (MI). We measured total protein carbonylation (PC), thiobarbituric acid reactive substances (TBARS) and total antioxidant capacity (TAC) along with clot lysis time (CLT) and clot permeation (Ks ), fibrinogen, plasminogen, α-2-antiplasmin, plasminogen activator inhibitor-1 (PAI-1), thrombin activatable fibrinolysis inhibitor (TAFI) and thrombomodulin. RESULTS: Total PC correlated positively, while TAC correlated inversely with glycated haemoglobin and diabetes duration (all p < 0.05). Diabetic patients with CVD had higher total PC, TBARS and lower TAC compared with the remainder (all p < 0.001). Among correlations of total PC with Ks , PAI-1, thrombomodulin and TAFI, the strongest was with CLT (r = 0.687, all p < 0.01). High total PC, defined as ≥ 3.45 nmol/mg, was predicted by time since diabetes diagnosis ≥ 5 years (odds ratio [OR]: 3.0, 95% confidence interval [CI]: 1.36-6.63) and previous MI (OR: 11.31, 95% CI: 4.37-29.32). After adjustment for potential confounders, total PC accounted for 34.9% of the total variance in CLT. Total PC at a cut-off of 3.44 nmol/mg showed high discriminatory power for identifying patients with prolonged CLT (area under the curve: 0.845, 95% CI: 0.792-0.898, p < 0.001). CONCLUSION: Elevated plasma PC, largely driven by a long history of diabetes and concomitant CVD, is an important determinant of hypofibrinolysis in type 2 diabetes.

Polish regional differences in patient knowledge on atrial fibrillation and its management as well as in patterns of oral anticoagulant prescription.

BACKGROUND The Jessa Atrial Fibrillation Knowledge Questionnaire (JAKQ) was successfully used to assess knowledge gaps in patients with atrial fibrillation (AF). AIMS To evaluate the regional differences among Polish patients in their awareness of AF diagnosis and oral anticoagulation use. METHODS A total of 1583 patients with AF at a median (IQR) age of 72 (66-79) years completed the JAKQ in 3 cardiology centers (center I, Kraków; center II, Torun; center III, Kielce) from January 2017 to June 2018. The final analysis included 1525 patients, 32.9% were on vitamin K antagonists (VKAs) and 67.1% on non-VKA oral anticoagulants (NOACs), that is, rivaroxaban and dabigatran (28.9% each), and apixaban (9.3%). RESULTS The mean (SD) score on the JAKQ was 55.5% (18.4%) with better results among patients on VKAs compared with NOACs (58% [18.3%] vs 54.3% [18.4%]; P = 0.0002) with time from AF diagnosis more than 12 months (57.4% [17.5%] vs 50% [19.9%]; P <0.0001). There was a significant difference in the knowledge scores between the 3 centers (I, 59.5%; II, 48.5%; III, 54.3%; P <0.0001). In all centers the number of correct answers correlated inversely with patient's age (r = -0.20; P <0.0001). NOACs were more frequently used in center III. The percentage of correct responses was lower in patients on reduced NOAC doses (35.4% of patients on NOACs), compared with the full-dose NOAC groups in center I (56.9% vs 62.5%; P = 0.012) and II (48.1% vs 56.2%; P = 0.003). CONCLUSIONS Patients from a high-volume academic center showed better knowledge than their peers from district hospitals. There are large regional differences in prescription patterns of oral anticoagulants, including the preferred NOAC.

Differences in knowledge among patients with atrial fibrillation receiving non-vitamin K antagonist oral anticoagulants and vitamin K antagonists.

BACKGROUND: Non-vitamin K antagonist oral anticoagulants (NOACs) are increasingly used for stroke prevention in patients with atrial fibrillation (AF) worldwide. Few articles have compared current understanding of AF patients about the disease and anticoagulant therapy in relation to the medications used. AIM: We sought to compare the knowledge of AF and anticoagulation between AF patients treated with NOACs and those on vitamin K antagonists (VKAs). METHODS: We used the Jessa AF Knowledge Questionnaire (JAKQ), developed and validated in Belgium. Patients were re-cruited at a tertiary centre in Kraków, Poland. RESULTS: A total of 479 AF patients completed the JAKQ. Patients on NOACs (n = 276, 57.6%) compared with those on VKAs (n = 175, 36.5%) did not differ regarding demographic and clinical variables. The mean score of the JAKQ was very similar in the NOAC and VKA group (60.7 ± 17.0% vs. 61.6 ± 17.1%; p = 0.4, respectively). The differences in the proportion of correct responses referred to three questions. Consequences of AF, such as blood clots and cerebral infarction, were more obvious for patients on NOACs compared with those on VKAs (81.5% vs. 70.9%; p = 0.01). The patients on NOACs (78.7% vs. 67.6%; p = 0.009) more frequently considered consulting a physician for advice concerning anticoagulant treatment before surgery, while fewer patients on NOACs were aware of the need to take their medication even if they did not feel AF (76.1% vs. 89.7%; p = 0.0004). Only 25.9% of the VKA patients and 49.3% of the NOAC users knew what to do if they missed a dose of the anticoagulant. CONCLUSIONS: The knowledge of arrhythmia and anticoagulation is better regarding the safety issues among subjects on NOACs compared with those on VKAs. Irrespective of the type of oral anticoagulation therapy, education of AF patients should be improved.