Plasma fibrin clot properties and cardiovascular mortality in patients with type 2 diabetes: a long-term follow-up study.

BACKGROUND: Patients with type 2 diabetes mellitus (T2DM) are at high risk of cardiovascular mortality, but the mechanisms behind this remain unclear. Prothrombotic fibrin clot properties have been shown in T2DM and cardiovascular disease. We hypothesized that formation of denser clots, which are resistant to fibrinolysis, has a negative impact on cardiovascular mortality in T2DM. METHODS: We studied 133 T2DM patients aged 43-83 years. Plasma fibrin clot turbidity, permeation, compaction, and efficiency of clot lysis using 3 assays including the determination of maximum concentration (D-Dmax) and rate of increase in D-dimer concentration (D-Drate) released during tissue plasminogen activator-induced degradation, were evaluated at the time of enrollment, along with thrombin generation and fibrinolytic proteins. During a median follow-up period of 72 months, cardiovascular mortality was recorded. RESULTS: Cardiovascular deaths (n = 16, 12%) occurred more frequently in patients with increased D-Dmax (> 4.26 mg/l, hazard ratio [HR] 5.43, 95% confidence interval [CI] 1.99-14.79), or decreased D-Drate (< 0.07 mg/l/min, HR 2.97, 95% CI 1.07-8.23), or increased peak thrombin (> 283.5 nM, HR 5.65, 95% CI 2.07-15.51). These predictors had an even more potent impact on cardiovascular mortality in patients with prior cardiovascular disease (64.7%) and with corresponding risks as follows: HR 6.18, 95% CI 2.02-18.96; HR 8.98, 95% CI 2.99-26.96; and HR 5.35, 95% CI 1.62-17.72, respectively. Other investigated fibrin variables and fibrinolytic proteins did not associate with cardiovascular mortality. In multivariable analysis, cardiovascular mortality was predicted by D-Dmax > 4.26 mg/l, age > 65 years, prior cardiovascular disease, and C-reactive protein > 3 mg/l. CONCLUSIONS: This study is the first to show that formation of denser fibrin clots resistant to fibrinolysis could be a risk factor for long-term cardiovascular mortality in T2DM.

Identification of glycated and acetylated lysine residues in human α2-antiplasmin.

BACKGROUND: The post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues. AIM: We sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin. METHODS: We analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of ß-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM ß-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer. RESULTS: Eleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with ß-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with ß-d-glucose or ASA. Incubation with concomitant ASA and ß-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM ß-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM ß-d-glucose was associated the absence of acetylation at that site. CONCLUSION: Human α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use.

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.

[Determinants of the elevated factor VIII activity in patients following venous thromboembolism]. / Czynniki determinujace wysoka aktywnosc czynnika VIII u pacjentów po przebytej zylnej chorobie zakrzepowo-zatorowej.

INTRODUCTION: Activity of factor VIII (FVIII) increased above 150% of reference range predisposes to venous thromboembolism (VTE). The aim of this study was to identify predictors of increased FVIII activity in patients following VTE. MATERIALS AND METHODS: 241 (38% men) patients presented due to objectively documented VTE episode at least 3 months ago were included in this study. FVIII activity was measured using a clotting assay on the analyzer BCS XP. RESULTS: Among 241 patients with VTE, activity of FVIII above 150% (FVIII ≥ 150%) was observed in 96 (40%). These patients were older (p = 0.035) and their concentrations of fibrinogen and C-reactive protein (CRP) were higher by 12% and 88% (p < 0.001), respectively, compared with other patients. There was a positive correlation between FVIII and fibrinogen (r = 0.34; p < 0.001), FVIII and CRP (r = 0.30; p < 0.001). Type of treatment, time from the VTE episode and type of VTE were not associated with FVIII. Twenty patients (8%) had activity of FVIII increased above 200% (FVIII > 200%) and this group was also older (p = 0.015), more patients in that group had obesity (p = 0.015), idiopathic VTE (p = 0.043), less of them had positive family history (p = 0.010) and they were characterized by fibrinogen and CRP increased by 28% (p < 0.001) and 102% (p = 0.004), respectively, compared with patients with FVIII between 150-200%. Independent predictors of FVIII ≥ 150% were: fibrinogen (p < 0.001), bilirubin (p = 0.002), hemoglobin (p = 0.016), glucose (p = 0.040), CRP (p = 0.023), total homocysteine (p = 0.032). Fibrinogen was the only independent predictor of FVIII > 200% (p = 0.016). CONCLUSIONS: The activity of FVIII in patients after VTE episode is influenced by age, concentration of fibrinogen, bilirubin, hemoglobin, glucose, CRP and homocysteine. Our results suggest the role of environmental factors, mainly inflammatory response in maintaining elevated FVIII activity following VTE.

First Probable Case of Subcutaneous Infection Due to Truncatella angustata: a New Fungal Pathogen of Humans?

Truncatella angustata is a coelomycetous fungus, typically associated with vascular plants as either an endophyte or a pathogen. This organism has not previously been implicated in human disease. This report describes a case of T. angustata subcutaneous infection in an immunocompetent patient. A conclusive diagnosis was achieved through partial sequencing of ribosomal DNA (rDNA) cluster. The patient was successfully treated with voriconazole followed by itraconazole.

Warfarin Metabolites in Patients Following Cardiac Valve Implantation: A Contribution of Clinical and Genetic Factors.

INTRODUCTION: Warfarin, a racemic mixture of S- and R-enantiomers, is the cornerstone of therapy in patients following cardiac valve replacement. S-warfarin is metabolized to 7-S-hydroxywarfarin by the cytochrome P450 isoform 2C9 encoded by CYP2C9 gene. R-warfarin is metabolized by multiple cytochromes P450. We sought to assess the impact of clinical and genetic factors on circulating warfarin metabolites following valve implantation. MATERIAL AND METHODS: Venous blood was collected from 120 patients after 3 months since elective mitral and/or aortic valve replacement. Plasma S-warfarin, R-warfarin, S-7-hydroxywarfarin, and R-7-hydroxywarfarin were determined using high-performance liquid chromatography. The S-7-hydroxywarfarin/S-warfarin and S-warfarin/R-warfarin (S/R) ratios, along with warfarin sensitivity index (WSI), defined as INR/S-warfarin ratio, were calculated. Vitamin K epoxide reductase complex subunit 1 (VKORC1) c.-1639A, CYP2C9*3 and CYP2C9*2 alleles were determined using real-time polymerase chain reaction. RESULTS: The S-warfarin was higher in former smokers (p = 0.047) and the VKORC1 c.-1639A allele carriers (p < 0.0001). The S-7-hydroxywarfarin was lower in carriers of the VKORC1 c.-1639A allele (p = 0.0005) and CYP2C9*3 (p = 0.047). The S-7-hydroxywarfarin/S-warfarin ratio was lower in the carriers of CYP2C9*3 (p = 0.008), but not in those with VKORC1 -c.1639A allele. The S/R ratio was higher in patients with hypertension (p = 0.01). The independent predictors of elevated S/R ratio defined as the upper quartile were diabetes (p = 0.045), CYP2C9*3 (p < 0.0001) and CYP2C9*2 (p = 0.0002). The independent predictors of elevated WSI were current smoking (p = 0.049), implantation of mechanical valve (p = 0.006) and VKORC1c.-1639A allele (p = 0.007). CONCLUSION: We conclude that not only genetic, but also several clinical factors affect warfarin metabolites in patients following cardiac valve implantation.

Glycation and acetylation sites on fibrinogen in plasma fibrin clot of patients with type 2 diabetes: Effects of low-dose acetylsalicylic acid.

Acetylsalicylic acid (ASA) and type 2 diabetes mellitus (T2DM) affect fibrin clot properties through fibrinogen acetylation or glycation. We aimed to identify glycation and acetylation sites on fibrinogen in plasma fibrin clot of T2DM patients with respect to effects of ASA and fibrin clot properties. In fibrin clots generated from plasma of 9 T2DM patients, we performed mass-spectrometric analysis of Nε-fructosyl-(FL), Nε-carboxyethyl-(CEL) and Nε-carboxymethyl-lysine (CML), and acetylation sites, before and after one-month administration of 75 mg/d ASA confirmed with determination of thromboxane B2 concentration (TXB2), along with clot permeability and lysis time, and thrombin generation. In the proteomic analysis, 216 proteins were identified. Among 10 glycation sites identified in α, 10 in ß and 6 in γ fibrinogen chain, there were 17 FL, 5 CEL and 4 CML sites. Some of glycation sites in fibrinogen were previously reported to be involved in cross-linking by factor XIII (αK-208, αK-448 and αK-539) and plasmin cleavage (αK-81). There were 7 acetylation sites in α and ß chains, and none in fibrinogen γ chain. Two acetylation sites were identical with FL sites (αK-195 and ß-247), while one with CML site (ßK-353). In 7 patients with low post-ASA TXB2, intensity of acetylation, as well as clot properties were unaffected by ASA. This study identifies glycation and acetylation sites on fibrinogen in plasma fibrin clot of T2DM and supports the view that low-dose ASA does not increase fibrinogen acetylation in T2DM. Our findings suggest that glycation may block sites previously identified to be acetylated in vitro.

Plasma fibrin clot proteomics in patients with acute pulmonary embolism: Association with clot properties.

It has been reported that 476 proteins can be detected in plasma fibrin clots from patients with venous thromboembolism. Plasma fibrin clots proteomic composition in relation to their properties has not been studied in acute pulmonary embolism (PE). Clots generated from plasma of 20 PE patients and 20 healthy controls were assessed using mass spectrometry, clot permeability (Ks), and clot lysis time (CLT). The proteomic composition of plasma fibrin clots from acute PE patients differed from that of control subjects in regard to 198 clot-bound proteins. In the acute PE group, we observed increased clot-bound fibrinogen, apolipoprotein B-100, platelet glycoprotein Ib, lipopolysaccharide-binding protein, and histones H3 + 4 and reduced fibronectin, α2-antiplasmin, α2-macroglobulin, factor (F)XIII, histidine-rich glycoprotein, antithrombin, von Willebrand Factor, plasminogen, and prothrombin. Among PE patients, low Ks (≤3.83 × 10-9 cm2) was associated with increased clot-bound C-reactive protein, kininogen-1, protein S, ß-2-microglobulin, and thromboxane-A synthase when compared with patients having Ks > 3.83 × 10-9 cm2. Ks correlated inversely with FIX and FV, thrombin-activatable fibrinolysis inhibitor, complement C1s, C7, C8, and apolipoprotein A-I. The specific protein composition in plasma fibrin clots from acute PE patients is associated with denser clot formation. Several proteins unrelated to the coagulation system can modulate fibrin phenotype in acute thrombotic states. SIGNIFICANCE: Our study significantly advances the field of thrombosis and hemostasis. The plasma fibrin clot proteomics findings fill the gap of knowledge about the presence and the role of other proteins to the plasma fibrin clot in the acute phase of pulmonary embolism, aside fibrinogen, which is the main component of fibrin. The reported methodology, which involves the sample preparation using Multienzyme Digestion-Filter Aided Sample Preparation (MED FASP), data acquisition with the Quadrupole-Orbitrap mass spectrometer, and data analysis using the advanced tools such as MaxQuant, Total Protein Approach and Perseus, allows to gain not only the qualitative, but also the quantitative insights into the microworld of proteins entangled among the fibrin network. By comparing the clots formed from plasma of patients with acute pulmonary embolism with the clots from healthy control, we provide the specific protein composition associated with unfavorable clot properties observed in this disease. Moreover, our findings emphasize that several proteins unrelated to the coagulation system, can modulate fibrin phenotype in acute thrombotic states.

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.

Sex-specific alteration to α2-antiplasmin incorporation in patients with type 2 diabetes.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with hypofibrinolysis and increased factor XIII-mediated α2-antiplasmin incorporation into the fibrin clot. It is unclear whether there are sex-related differences in α2-antiplasmin incorporation in relation to impaired clot lysis in T2DM. AIM: We investigated α2-antiplasmin incorporation into fibrin clots as a determinant of clot lysability in patients of both sexes with T2DM. METHODS: In a group of 113 T2DM patients, 54 (47.8%) of which were women, we investigated α2-antiplasmin incorporation using an in-house sandwich enzyme-linked immunoassay and plasma clot lysis by turbidimetry, along with fibrinogen and thrombin generation using calibrated automated thrombogram and factor XIII activity. RESULTS: Female patients had 15.2% greater α2-antiplasmin incorporation into the fibrin clot (p = 0.008) and slightly higher plasma α2-antiplasmin concentration (p = 0.005) along with 8.4% longer time to 50% lysis (Lys50MA, p = 0.012) compared with men. Female patients had enhanced thrombin generation represented by shorter lag phase (p = 0.042), shorter time to peak (p = 0.033), and higher endogenous thrombin potential (p = 0.003) compared with men, while factor XIII activity was comparable between sexes (p = 0.085). On multivariate regression, patient sex and glycated hemoglobin (HbA1c) level were the predictors of α2-antiplasmin incorporation in the entire patient group, while α2-antiplasmin incorporation was associated with Lys50MA, as were fibrinogen, male sex and body-mass index. CONCLUSIONS: This study suggests that a more compromised fibrinolysis in diabetic women when compared with men could be in part mediated by increased α2-antiplasmin incorporation into the fibrin.

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.

Bleeding predictors in patients following venous thromboembolism treated with vitamin K antagonists: Association with increased number of single nucleotide polymorphisms.

INTRODUCTION: Single nucleotide polymorphisms (SNP) in genes encoding proteins involved in metabolism and action of vitamin K antagonists (VKA) affect anticoagulation stability. We investigated how those polymorphisms influence bleeding rates in patients following venous thromboembolism (VTE). MATERIALS AND METHODS: In 324 patients following unprovoked VTE, 143 (44%) on warfarin and 181 (56%) on acenocoumarol, we recorded bleeds within the preceding 24 months. We assessed eight SNP, including those in cytochrome P450 isoform 2C9 (CYP2C9) and isoform 4F2 (CYP4F2), vitamin K epoxide reductase complex subunit 1 (VKORC1), gamma-glutamyl carboxylase (GGCX), apolipoprotein E (APOE) and multidrug resistance gene 1 (MDR1). RESULTS: Within 48 months before enrolment, bleeding events occurred in 80 (25%) patients, including 14 (4%) major bleeds. Patients with bleeds had 16.2% lower median time in therapeutic range (TTR) and were more often carriers of CYP2C9*3 variant (26 [33%] vs. 19 [8%], p < 0.001) compared with the remainder. Bleeding occurred more frequently in patients with ≥4 SNP compared with the remainder (27 [34%] vs. 47 [19%], p = 0.009) with no intergroup differences of TTR. Number of SNP was one of the predictors of any bleeding. The regression model for major bleeding including factors such as CYP2C9*3 c. 1075 C, VKORC1 c. -1639 A and APOE c. 388 C showed good predictive ability (area under the curve - 0.79). CONCLUSIONS: In VTE patients on the maintenance treatment with VKA, bleeding episodes are associated with CYP2C9 gene variations and increased number of SNP of genes involved in the action and metabolism of VKA.

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.

Prediction of unstable anticoagulation with acenocoumarol versus warfarin in atrial fibrillation.

BACKGROUND: The SAMe-TT2R2 (sex female, age, medical history, treatment, tobacco use, race) score was developed in patients with atrial fibrillation (AF) on warfarin. The present study aimed to 1) compare the anticoagulation quality and management of AF patients treated with warfarin with those on acenocoumarol and 2) optimize the SAMe-TT2R2 score to detect AF patients at high risk of unstable anticoagulation with acenocoumarol and warfarin. METHODS: In a single-center retrospective study, 320 patients with AF, including 15 (5%) after valve replacement, aged 40-82 (median 70) years, including 203 (63%) receiving acenocoumarol and 117 (37%) treated with warfarin, were studied. The SAMe-TT2R2 score was modified based on the candidate factors retrieved from univariate regression and assessed using the receiver operating curves (ROC). RESULTS: A median SAMe-TT2R2 score was 2 (1-3). Proportions of patients with ≥ 2 points and 0-1 points in the SAMe-TT2R2 score who had the time in therapeutic range (TTR) ≤ 70% were similar (61 [67%] vs. 63 [56%], p = 0.11). A modified score, involving medical history (myocardial infarction [MI] and chronic obstructive pulmonary disease [COPD], 1 point), statin treatment (1 point) and tobacco use (2 points) had a higher area under the curve (AUC) in patients on acenocoumarol compared to SAMe- TT2R2 (0.66; 95% confidence interval 0.58-0.73 vs. 0.56; 0.48-0.64, p = 0.042); ≥ 1 point indicated TTR > 70% with a sensitivity and specificity of 61% and 63%, respectively. CONCLUSIONS: The SAMe-TT2R2 score is less effective in predicting unstable anticoagulation with acenocoumarol versus warfarin. Adding statin use and highlighting COPD and previous MI increases a predictive value of this score for acenocoumarol.

Interplay between elevated cellular fibronectin and plasma fibrin clot properties in type 2 diabetes.

Type 2 diabetes is associated with faster formation of poorly lysable, denser fibrin clots and elevated cellular fibronectin (cFn), a marker of vascular injury. We investigated whether cFn affects clot properties in type 2 diabetes. In 200 consecutive patients with type 2 diabetes and 100 control subjects matched for age and sex, we determined plasma cFn along with clot formation and degradation using turbidimetric and permeability assays. Diabetic patients had elevated cFn (median, 3.99 [interquartile range, 2.87-4.81] µg/ml]), increased clot density (MaxAbsC) and prolonged lysis time (LysT) compared with those without type 2 diabetes (all p<0.01). Diabetic patients with documented cardiovascular disease (CVD, n=127, 63.5 %) had increased cFn (4.53 [3.68-4.95] µg/ml), decreased clot permeability (Ks) and increased MaxAbsC compared with those without CVD (all p<0.001). Diabetic patients with cFn in the top quartile (>4.81 µg/ml) were two times more likely to have CVD compared with those in the lowest quartile (odds ratio 1.80, 95 % confidence interval 1.41-2.46, p<0.001). No differences in cFn were observed in relation to microvascular complications. After adjustment for potential confounders, cFn accounted for 10.2 % of variance in Ks, 18.2 % of variance in clot density and 10.2 % of variance in AUC in diabetic patients. This study shows that elevated cFn is associated with unfavourably modified clot properties in type 2 diabetes, especially with concomitant CVD, which indicates novel links between vascular injury and prothrombotic alterations in diabetes. Coagulation, cellular fibronectin, type 2 diabetes, cardiovascular disease.

Heavy menstrual bleeding in women treated with rivaroxaban and vitamin K antagonists and the risk of recurrent venous thromboembolism.

Anticoagulants increase the risk of heavy menstrual bleeding (HMB). We sought to investigate the incidence, predictors and management of HMB in women on rivaroxaban compared to those on vitamin K antagonists (VKA). We addressed the issue as to whether HMB is associated with VTE recurrences. We performed a single-center prospective study in menstruating women aged 18-55years treated with rivaroxaban or VKA≥3months since the index VTE episode. Seventy six women on rivaroxaban and 45 patients on VKA were included. Patients on rivaroxaban more commonly reported HMB compared with those on VKA (31 [41%] vs. 8 [18%], p=0.009). Women treated with rivaroxaban more frequently needed interventions to reduce menstruation compared with those on VKA (29 [38%] vs. 6 [13%], p=0.004). During the median follow-up time of 13months, there were 8 (11%) recurrent VTE on rivaroxaban and 3 (7%) on VKA (p=0.5). Rivaroxaban treatment predisposed to HMB (odds ratio [OR] 3.2, 95% [confidence interval] CI 1.4-8.2, p=0.007) and the interruption of anticoagulant treatment for 2-3days (OR 3.2, 95% CI 1.1-11.6, p=0.033). HMB during the rivaroxaban treatment predisposed to recurrent VTE (OR 5.3, 95% CI 1.1-33.3, p=0.038). In menstruating women following VTE, rivaroxaban is associated with a two-fold higher risk of HMB compared with VKA. HMB predisposes to recurrent VTE episode, most likely due to the short interruptions of anticoagulation.

Repeatedly administered antidepressant drugs modulate humoral and cellular immune response in mice through action on macrophages.

Depression is associated with an altered immune response, which could be normalized by antidepressant drugs. However, little is known about the influence of antidepressants on the peripheral immune response and function of macrophages in individuals not suffering from depression. Our studies were aimed at determining the influence of antidepressant drugs on the humoral and cellular immune response in mice. Mice were treated intraperitoneally with imipramine, fluoxetine, venlafaxine, or moclobemide and contact immunized with trinitrophenyl hapten followed by elicitation and measurement of contact sensitivity by ear swelling response. Peritoneal macrophages from drug-treated mice were either pulsed with sheep erythrocytes or conjugated with trinitrophenyl and transferred into naive recipients to induce humoral or contact sensitivity response, respectively. Secretion of reactive oxygen intermediates, nitric oxide, and cytokines by macrophages from drug-treated mice was assessed, respectively, in chemiluminometry, Griess-based colorimetry and enzyme-linked immunosorbent assay, and the expression of macrophage surface markers was analyzed cytometrically. Treatment of mice with fluoxetine, venlafaxine, and moclobemide results in suppression of humoral and cell-mediated immunity with a reduction of the release of macrophage proinflammatory mediators and the expression of antigen-presentation markers. In contrast, treatment with imipramine enhanced the humoral immune response and macrophage secretory activity but slightly suppressed active contact sensitivity. Our studies demonstrated that systemically delivered antidepressant drugs modulate the peripheral humoral and cell-mediated immune responses, mostly through their action on macrophages. Imipramine was rather proinflammatory, whereas other tested drugs expressed immunosuppressive potential. Current observations may be applied to new therapeutic strategies dedicated to various disorders associated with excessive inflammation.

Single-agent MOR208 salvage and maintenance therapy in a patient with refractory/relapsing diffuse large B-cell lymphoma: a case report.

BACKGROUND: Diffuse large B-cell lymphoma is the most common subtype of non-Hodgkin's lymphoma. Standard first-line treatment for this aggressive subtype comprises the anti-CD20 antibody rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone. If patients receiving such treatment have an early relapse, or their disease is initially refractory to such treatment, standard salvage regimens may not be effective. There is therefore a high unmet clinical need for new targeted agents that might improve the outcome for such patients. CD19 is a B-lymphocyte lineage-specific cell surface antigen that is expressed by most B-cell non-Hodgkin's lymphomas. MOR208 is an fragment-crystallizable engineered humanized monoclonal antibody with enhanced antitumor activity that targets CD19 and that may consequently have clinical utility in this setting. CASE PRESENTATION: We describe the case of a 33-year-old Caucasian man who presented with a 3-month history of general symptoms and who was admitted to our pulmonology ward with dyspnea due to pneumonia and severe anemia. A histopathological examination of an enlarged right suprasternal lymph node confirmed a diagnosis of T-cell/histiocyte-rich large B-cell lymphoma, an uncommon morphological variant of diffuse large B-cell lymphoma. Our patient had a complete response to first-line rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone, but had an early relapse 5 months after the end of treatment. After intensive salvage therapy consolidated with an autologous stem-cell transplant, our patient again had an early relapse and was subsequently enrolled in a phase IIa trial of single-agent MOR208. Following a scheduled 3 months of weekly treatment, a partial response was confirmed and MOR208 was continued as maintenance therapy, with administration every second week. Positron emission tomography-computed tomography confirmed a complete response 9 months later. This response is ongoing, with a duration of 24 months. MOR208 was well-tolerated by our patient and his quality of life and performance status remain high. No hospitalizations were required and our patient engaged in full-time work and physical activities. CONCLUSION: Third-line single-agent therapy with the CD19 antibody MOR208 was highly effective in this patient, despite a history of early relapse after standard first-line and second-line treatment regimens. These data provide support for future randomized studies of MOR208.