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Background Deficiency of antithrombin increases risk of venous thromboembolism. We hypothesized that antithrombin deficiency affects fibrin clot structure and function. Methods We evaluated 148 patients (age: 38 [32–50] years; 70% women) with genetically confirmed antithrombin deficiency and 50 healthy controls. Fibrin clot permeability (Ks) and clot lysis time (CLT) along with thrombin generation capacity were assessed before and after antithrombin activity normalization in vitro. Results Antithrombin-deficient patients had lower antithrombin activity (−39%) and antigen levels (−23%) compared with controls (both p Conclusions Our study suggests that enhanced thrombin generation and prothrombotic plasma fibrin clot phenotype can contribute to increased risk of thrombosis in patients with antithrombin deficiency.

Introduction: Neutrophil-activating peptide 2 (NAP-2, CXCL7), a platelet-derived neutrophil chemoattractant, is involved in inflammation. We investigated associations between NAP-2 levels, neutrophil extracellular traps (NETs) formation, and fibrin clot properties in atrial fibrillation (AF). Materials and Methods: We recruited 237 consecutive patients with AF (mean age, 68±11 years; median CHA2DS2VASc score of 3 [2-4]) and 30 apparently healthy controls. Plasma NAP-2 concentrations were measured, along with plasma fibrin clot permeability (Ks) and clot lysis time (CLT), thrombin generation, citrullinated histone H3 (citH3), as a marker of NETs formation, and 3-nitrotyrosine reflecting oxidative stress. Results: NAP-2 levels were 89% higher in AF patients than in controls (626 [448-796] vs. 331 [226-430] ng/ml; p2DS2-VASc score, or the AF manifestation. Patients with NAP-2 in the top quartile (>796 ng/ml) were characterized by higher neutrophil count (+31.7%), fibrinogen (+20.8%), citH3 (+86%), and 3-nitrotyrosine (+111%) levels, along with 20.2% reduced Ks and 8.4% prolonged CLT as compared to the remaining subjects (all ps. Conclusions: Elevated NAP-2, associated with increased oxidative stress, has been identified as a novel modulator of prothrombotic plasma fibrin clot properties in patients with AF.

Venous thromboembolism is associated with formation of denser fibrin clots resistant to lysis. We investigated whether prothrombotic plasma clot properties are associated with the severity of acute pulmonary embolism (PE). We enrolled 126 normotensive acute PE patients (aged 58 ± 14 years) and 25 age- and sex-matched healthy controls. Plasma fibrin clot permeability (Ks), clot lysis time (CLT), endogenous thrombin potential (ETP), plasminogen activator inhibitor-1 (PAI-1), and citrullinated histone H3 (citH3) were evaluated on admission. PE patients compared to controls had 370% higher citH3 levels, 41% higher ETP, 16.5% reduced Ks, and 25.6% prolonged CLT. Patients with intermediate-high (n = 29) and intermediate-low (n = 77) PE mortality risk had reduced Ks and prolonged CLT, increased PAI-1 and ETP as compared to low-risk PE (n = 20) patients. Prolonged CLT was predicted by PAI-1 and citH3, while low Ks by C-reactive protein. During a 12-month follow-up 9 (7.1%) patients who had 24% higher ETP, 45% higher citH3 levels, and 18% prolonged CLT at baseline died. High ETP combined with elevated citH3 levels and prolonged CLT was associated with eightfold increased risk of PE-related death. Prothrombotic fibrin clot properties and enhanced neutrophil extracellular traps formation are associated with higher early mortality risk in acute PE patients, which suggests a prognostic role of these biomarkers.

OBJECTIVES The Fontan procedure is the treatment of choice in congenital cardiac malformations defined as the single ventricle. Fontan patients are at high risk of thromboembolism, but the exact mechanism of this is poorly understood. The aim of this study was to evaluate an involvement of thrombin generations and microparticles (MPs) in prothrombotic state in adults with Fontan circulation. METHODS This study included hospitalized patients after Fontan procedure and healthy volunteers. We assessed laboratory tests including thrombin generation by calibrated automated thrombography in three variants [platelet-poor plasma (impact of coagulation factors), platelet-rich plasma (PRP) (influence of platelets) and related with MPs]. The technique allows for a comprehensive evaluation of the coagulation system. RESULTS The study groups consisted of 81 adult Fontan patients [41 females (50.6%); median age 22 interquartile range [20–27] years] and 54 control subjects. In patients with Fontan circulation, higher values of endogenous thrombin potential and peak values were observed for both platelet-poor plasma (+17% and +33%) and MPs (+29% and 41%) compared to controls (all P 0.05). CONCLUSIONS This study for the first time showed that thrombin generation associated with MPs may be an important contributor to the prothrombotic state in the Fontan population.

The effect of epicardial left atrial appendage (LAA) occlusion therapy on lipid and glucose metabolism in atrial fibrillation (AF) patients over the long-term follow-up are unclear.In a single-center prospective observational study, 60 patients with longstanding persistent AF with cardiovascular risk factors had undergone an epicardial exclusion procedure. Anthropometric parameters and glucose, glycated hemoglobin (HbA1c), insulin, leptin, adiponectin, free fatty acids, beta-hydroxybutyrate, and total cholesterol levels were evaluated on fasting at baseline before the procedure and compared with levels at 24 h, 7 days, 1, 3, 6, and 24 months follow the procedure.The mean age of the patients was 67.5 ± 8.1. Insulin levels significantly increased at 7 days, 1, 3, 6, 12, and 24 months follow-up. The leptin levels showed a significant increase in 6, 12, and 24 months when compared to baseline. Whereas the adiponectin levels showed a significant decrease at 3, 6, 12, and 24 months when compared to baseline levels. In patients with the epicardial procedure, when compared to baseline, glucose, glycated hemoglobin, total cholesterol, and beta-hydroxybutyrate levels did not show any significant changes at baseline and 24 months follow-up.The epicardial exclusion ligation in AF patients was associated with significant changes in insulin, leptin, and adiponectin over long follow-up.

Recent data suggest that epicardial left atrial appendage closure (LAAC) is associated with several short-term neurohormonal effects. However, the long-term effects are currently unknown.To investigate the effects of percutaneous epicardial left atial appendage (LAA) exclusion using LARIAT on neurohormonal profiles at long-term follow-up.In a prospective single centre study, 60 patients with long-standing, persistent atrial fibrillation (AF) LARIAT were treated. The major hormones of the adrenergic system, renin-angiotensin-aldosterone system (RAAS), and natriuretic peptides were assessed before the intervention and at regular intervals during the following two years.In patients with epicardial LAAC, atrial natriuretic peptide (ANP) levels were significantly increased from baseline at 24 h and decreased at 7 days, 1 month, and 3 months, while remaining unchanged at 12 and 24 months. Noradrenaline levels were significantly lower at 24 h, 7 days, 1 month, 6 months, 12 months, and 24 months, while epinephrine levels decreased significantly at 1 month, 6 months, 12 months, and 24 months. Plasma renin activity significantly decreased at 7 days, 1 month, 6 months, 12 months, and 24 months, while aldosterone levels significantly decreased at 6 months, 12 months, and 24 months. Endothelin-1 and vasopressin showed a significant increase and decrease, respectively, at 24 h, 7 days, 1 month, 6 months, 12 months, and 24 months. There was also a significant decrease in systolic and diastolic blood pressure at 3 months, 6 months, 1 year, and 2 years after the intervention.Epicardial LAAC in AF patients is associated with persistent neurohormonal changes favouring blood pressure reduction.

Enhanced oxidative stress occurs in atrial fibrillation (AF), however its impact on the efficacy and safety of anticoagulation is unknown. We sought to evaluate whether 8-isoprostaglandin F2 (8-isoprostane) levels are associated with clinical outcomes in anticoagulated AF patients.In a study involving 243 AF patients (median age 69 years), we measured serum 8-isoprostane, along with prothrombotic markers, including plasma fibrin clot permeability, clot lysis time (CLT), endogenous thrombin potential (ETP), von Willebrand factor (VWF), and fibrinolytic proteins. Ischemic cerebrovascular events, major bleeding, and death were recorded during a median follow-up of 53 months while on anticoagulation, largely on non-vitamin K antagonist oral anticoagulants (NOACs).Increased 8-isoprostane levels were observed in women, in patients with arterial hypertension, and those with paroxysmal or persistent AF. Patients with 8-isoprostane levels ≥559 pg/mL (the top quartile) compared with those with 8-isoprostane250 pg/mL (the bottom quartile) had higher fibrinogen, lower VWF, higher plasminogen activator inhibitor 1, along with lower fibrin clot permeability with no difference in CHAIncreased 8-isoprostane levels partly through altered fibrin clot structure are associated with thromboembolic events despite anticoagulant therapy in AF patients.

Changes in coagulation and fibrinolysis have been reported in women undergoing controlled ovarian hyperstimulation (COH) supporting a potential hypercoagulable condition related to this treatment. This study aimed at evaluating the changes in fibrin clot properties and thrombin generation induced by two different COH protocols: long with gonadotropin-releasing hormone agonist (GnRH-a) and GnRH antagonist (GnRH-ant). Primary outcomes included determination of plasma fibrin clot properties, including clot permeability (K

Objectives: thromboembolic complications are a major cause of morbidity and mortality following Fontan (FO) surgery. It is also well established that altered FO circulation results in systemic complications, including liver and endothelium damage. We sought to evaluate whether dysfunctions of these sources of hemostatic factors may result in changes of fibrin clot properties. Methods: a permeation coefficient (Ks) and clot lysis time (CLT) were assessed in 66 FO patients, aged 23.0 years [IQR 19.3–27.0], and 59 controls, aged 24.0 years [IQR 19.0–29.0]. Ks was determined using a pressure-driven system. CLT value was measured according to assay described by Pieters et al. Endothelium and liver-derived hemostatic factors along with liver function parameters were evaluated. The median time between FO operation and investigation was 20.5 years [IQR 16.3–22.0]. Results: FO patients had lower Ks (p = 0.005) and prolonged CLT (p < 0.001) compared to that of controls. Ks correlated with CLT (r = −0.28), FVIII (r = −0.30), FIX (r = −0.38), fibrinogen (r = −0.41), ALT (r = −0.25), AST (r = −0.26), GGTP (r = −0.27) and vWF antigen (r = −0.30), (all p < 0.05). CLT correlated with the time between FO operation and investigation (r = 0.29) and FIX (r = 0.25), (all p < 0.05). After adjustment for potential cofounders, TAFI antigen and GGTP were independent predictors of reduced Ks (OR 1.041 per 1% increase, 95% CI 1.009–1.081, p = 0.011 and OR 1.025 per 1 U/L increase, 95% CI 1.005–1.053, p = 0.033, respectively). Protein C and LDL cholesterol predicted prolonged CLT (OR 1.078 per 1% increase, 95% CI 1.027–1.153, p = 0.001 and OR 6.360 per 1 μmol/L increase, 95% CI 1.492–39.894, p = 0.011, respectively). Whereas elevated tPA was associated with lower risk of prolonged CLT (OR 0.550 per 1 ng/mL, 95% CI 0.314–0.854, p = 0.004). GGTP correlated positively with time between FO surgery and investigation (r = 0.25, p = 0.045) and patients with abnormal elevated GGTP activity (n = 28, 42.4%) had decreased Ks, compared to that of the others (5.9 × 10−9 cm2 vs. 6.8 × 10−9 cm2, p = 0.042). Conclusion: our study shows that cellular liver damage and endothelial injury were associated with prothrombotic clot phenotype reflected by Ks and CLT.

Neutrophil extracellular traps (NETs) formation contributes to thrombosis but its role in atrial fibrillation (AF) is poorly explored. We investigated whether increased circulating NETs markers in relation to a hypercoagulable state can predispose to ischemic stroke in anticoagulated AF patients during long-term follow-up.In this cohort study 243 AF patients (median age 69 years) were assessed. Serum levels of citrullinated histone H3 (H3cit), myeloperoxidase (MPO), and peptidylarginine deiminase 4 (PAD4), along with plasma fibrin clot permeability (KIschemic cerebrovascular events were observed in 20 patients (1.9%/year) who had at baseline higher H3cit, MPO, and PAD4 levels, all positively associated with CLT. Increased thrombin generation correlated positively with H3cit and PAD4, while KEnhanced NETs formation related to prothrombotic markers is associated with increased risk of stroke/TIA in AF patients, suggesting a prognostic value of NETosis in AF.

BACKGROUND Prothrombotic fibrin clot properties, including increased clot density, are in part genetically determined. We investigated whether fibrinogen alpha-chain gene (FGA) c.991A>G (rs6050), fibrinogen beta chain gene (FGB) -455G>A (rs1800790) and factor XIII gene (F13) c.103G>T (rs5985) polymorphisms affect plasma fibrin clot properties in patients with acute pulmonary embolism (PE). METHODS As many as 126 normotensive patients with PE, free of cancer, were genotyped by TaqMan assay. Fibrin clot permeability (Ks ), clot lysis time (CLT) and endogenous thrombin potential (ETP) were assessed on admission. RESULTS The minor allele frequencies were as follows: FGA rs6050 (n = 62, 0.31), FGB rs1800790 (n = 40, 0.17) and F13 rs5985 (n = 49, 0.23). There were no differences related to any of the polymorphisms with regard to demographic, clinical and laboratory data, except for fibrinogen concentration, which was higher in carriers of F13 rs5985 polymorphism (p = .024), and PE combined with deep-vein thrombosis, which was less prevalent in FGB rs1800790 polymorphism carriers (p = .004). Carriers of FGB rs1800790 A allele and F13 rs5985 T allele had lower Ks , prolonged CLT and higher ETP compared with major homozygotes (all p

Introduction Atrial fibrillation (AF), a risk factor for stroke and systemic thromboembolism, is associated with unfavorable fibrin clot properties and increased thrombus formation in peripheral blood. The left atrial appendage (LAA) is known to be the primary site of thrombus formation. Aim We investigated the relative differences in plasma fibrin clot features including plasma fibrin clot permeability (Ks) and clot lysis time (CLT) between the right atrium (RA), right ventricle (RV), left atrium (LA), left ventricle (LV), LAA, and peripheral blood. Methods Sixteen patients with nonvalvular AF who stopped oral anticoagulant therapy at least 2 days before a LARIAT procedure participated in a single-center prospective study. We measured fibrinogen and plasminogen levels along with Ks, CLT, and endogenous thrombin potential (ETP) during the LARIAT procedure in blood obtained from the right femoral vein, RA, RV, LA, LV and LAA. Results LAA clot porosity was reduced by 16.2% compared to peripheral blood (p = 0.026), also after adjustment for fibrinogen levels (p = 0.038). Ks was similar for the RA, RV, LA, LV, and LAA (all p > 0.05). We found 14.7% prolonged CLT for clots prepared from blood samples obtained from the LAA compared to those prepared from peripheral blood, but no differences between the RA, RV, LA and LV (all p > 0.05) were found. There were no significant differences in other parameters, including ETP, between heart chambers. Conclusions Patients with AF are characterised by a local prothrombotic state as reflected by formation of compact fibrin clots in the LAA compared to peripheral blood, which may contribute to LAA thrombus formation and device-related thrombi.

Large amounts of clot-bound lipoproteins were reported in proteomic analysis of plasma clot but their impact on fibrin clot properties is unknown. We investigated a contribution of lipid profile and apolipoproteins (apo) to the prothrombotic plasma fibrin clot phenotype in patients with aortic stenosis (AS).In 138 patients with isolated severe AS, we determined serum apoA-I, A-II, B, C-II, C-III, E, oxidized low-density lipoprotein (OxLDL) and lipoprotein(a) concentrations. Plasma fibrin clot permeability (Ks), maximal absorbance (MaxAbsAfter adjustment for confounding factors, including statin use, only low-density lipoprotein cholesterol (LDL-C) and apoB levels were inversely associated with Ks. Triglycerides, apoC-II, and C-III were associated with MaxAbsApolipoproteins and OxLDL contribute to prothrombotic fibrin clot phenotype in severe AS. Moreover, apolipoproteins better than serum lipids predicted hypofibrinolysis, which provides additional argument for a role of elevated lipoproteins in the prothrombotic state.

BACKGROUND Post-pulmonary embolism (PE) syndrome occurs in up to 50% of PE patients. The pathophysiology of this syndrome is obscure. OBJECTIVE We investigated whether enhanced oxidative stress and prothrombotic state may be involved in post-PE syndrome. METHODS We studied 101 normotensive noncancer PE patients (aged 56.5 ± 13.9 years) on admission, after 5-7 days and after a 3-month anticoagulation, mostly with rivaroxaban. A marker of oxidative stress, 8-isoprostane, endogenous thrombin potential, fibrinolysis proteins, clot lysis time (CLT) and fibrin clot permeability (Ks ), along with PE biomarkers, were determined. RESULTS Patients who developed the post-PE syndrome (n = 31, 30.7%) had at baseline 77.6% higher N-terminal brain natriuretic propeptide and 46.8% higher growth differentiation factor 15, along with 14.1% longer CLT associated with 34.4% higher plasminogen activator inhibitor-1 as compared to subjects without post-PE syndrome (all P

Background Aortic stenosis (AS) prevalence is estimated to reach 4.5 million cases worldwide by the year 2030. AS is a progressive disease without a pharmacological treatment. In the current review we aimed to investigate novel therapeutic approaches for non-surgical AS treatment, at least in patients with mild-to-moderate AS. Materials and methods The most recent and relevant papers concerned with novel molecular pathways that have potential as therapeutic targets in AS were selected from searches of PubMed and Web of Science up to February 2021. Results Growing evidence indicates that therapies using proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, simvastatin/ezetimibe combination, cholesteryl ester transfer protein inhibitors or antisense oligonucleotides targeting apolipoprotein(a) reduce the risk of AS progression. It has been shown that enhanced valvular lipid oxidation may drive AS development by leading to the activation of valvular interstitial cells (VICs), the most abundant valvular cells having a major contribution to valve calcification. Since VICs are able to release proinflammatory cytokines, clotting factors, and proteins involved in calcification, strategies targeting these cells activation seem promising as therapeutic interventions. Recently non-vitamin K antagonist oral anticoagulants (NOACs) have been shown to inhibit activation of VICs. Conclusion Several novel molecular pathways of AS development have been identified over the past few years. Therapies using PCSK9 inhibitors, simvastatin/ezetimibe combination, lipoprotein(a)-lowering therapy are highly promising candidates as therapeutics in the prevention of mild AS progression, while preclinical studies show that NOACs may inhibit valvular inflammation and coagulation activation and slower the rate of AS progression.

Aims/hypothesis Type 2 diabetes has been demonstrated to predispose to aortic valve calcification. We investigated whether type 2 diabetes concomitant to aortic stenosis (AS) enhances valvular inflammation and coagulation activation via upregulated expression of NF-κB, with subsequent increased expression of bone morphogenetic protein 2 (BMP-2). Methods In this case–control study, 50 individuals with severe isolated AS and concomitant type 2 diabetes were compared with a control group of 100 individuals without diabetes. The median (IQR) duration of diabetes since diagnosis was 11 (7–18) years, and 36 (72%) individuals had HbA1c ≥48 mmol/mol (≥6.5%). Stenotic aortic valves obtained during valve replacement surgery served for in loco NF-κB, BMP-2, prothrombin (FII) and active factor X (FXa) immunostaining. In vitro cultures of valve interstitial cells (VICs), isolated from obtained valves were used for mechanistic experiments and PCR investigations. Results Diabetic compared with non-diabetic individuals displayed enhanced valvular expression of NF-κB, BMP-2, FII and FXa (all p ≤ 0.001). Moreover, the expression of NF-κB and BMP-2 positively correlated with amounts of valvular FII and FXa. Only in diabetic participants, valvular NF-κB expression was strongly associated with serum levels of HbA1c, and moderately with fructosamine. Of importance, in diabetic participants, valvular expression of NF-κB correlated with aortic valve area (AVA) and maximal transvalvular pressure gradient. In vitro experiments conducted using VIC cultures revealed that glucose (11 mmol/l) upregulated expression of both NF-κB and BMP-2 (p N-acetyl-l-cysteine), the expression of NF-κB and BMP-2 was significantly suppressed. A comparable effect was observed for VICs cultured with glucose in combination with NF-κB inhibitor (BAY 11–7082), suggesting that high doses of glucose activate oxidative stress leading to proinflammatory actions in VICs. Analysis of mRNA expression in VICs confirmed these findings; glucose caused a 6.9-fold increase in expression of RELA (NF-κB p65 subunit), with the ROS and NF-κB inhibitor reducing the raised expression of RELA by 1.8- and 3.2-fold, respectively. Conclusions/interpretation Type 2 diabetes enhances in loco inflammation and coagulation activation within stenotic valve leaflets. Increased valvular expression of NF-κB in diabetic individuals is associated not only with serum HbA1c and fructosamine levels but also with AVA and transvalvular gradient, indicating that strict long-term glycaemic control is needed in AS patients with concomitant type 2 diabetes. This study suggests that maintaining these variables within the normal range may slow the rate of AS progression. Graphical abstract

Coronary artery bypass grafting (CABG) done on-pump may cause a significant blood loss. Low fibrinogen is associated with perioperative bleeding. The influence of cardiopulmonary bypass on fibrin clot properties is poorly investigated. We studied 55 patients with isolated coronary artery disease on aspirin undergoing on-pump CABG with tranexamic acid. Fibrinogen levels, fibrinolytic capacity expressed as clot lysis time (CLT), thrombin generation potential and platelet count were assessed before and after the surgery (prior to admission to the intensive care unit). A postoperative drop in haemoglobin (-30% from baseline), haematocrit (-31% from baseline) and platelet count (-42% from baseline) was observed (all, P < 0.0001). Postoperative fibrinogen level was lower by 57%, compared with preoperative value (1.5 [1.3-1.8] vs. 3.5 [2.8-3.9] g/l, P < 0.0001). Postoperative CLT was longer by 48 min, compared with preoperative (182 [170-218] vs. 134 [122-165] min, P < 0.0001). Thrombin generation was impaired postoperatively: both lag time and time to peak thrombin were prolonged by 44 and 45%, respectively, whereas endogenous thrombin potential and peak thrombin generation decreased by 45 and 78%, respectively (all P < 0.0001). Median postoperative drainage at 12 h was 400 [290-570] ml. Predictors of blood loss at 12 h identified in multivariable linear regression model adjusted for sex and preoperative fibrinogen level were: BMI (b = -23.4, P = 0.048) and postoperative CLT (b = -2.4, P = 0.042). Despite decreased fibrinogen levels after on-pump CABG with tranexamic acid, fibrin clot susceptibility to lysis is impaired, as reflected by prolonged CLT. Postoperative CLT is associated with mediastinal drainage at 12 h.

Aortic stenosis (AS) is a progressive disease, with no pharmacological treatment. The prevalence of diabetes mellitus (DM) among AS patients is higher than in the general population. DM significantly increases the risk of AS development and the rate of its progression from mild to severe. However, a mechanism of the interaction between AS and DM is not fully understood. Limited data regarding the influence of hyperglycemia on valvular calcification are available, while understanding the cross-talk between them is pivotal to design an effective therapeutic approach to prevent or at least retard AS development and/or progression in DM patients. Analysis of aortic stenotic valves revealed that increased accumulation of advanced glycoxidation end products (AGEs) was associated with enhanced valvular oxidative stress, inflammation, expression of coagulation factors and markers of calcification. Moreover, AGEs valvular expression correlated with AS severity. Interestingly, in diabetic AS patients valvular inflammation correlated only with long-term glycemic control parameters, i.e. glycated hemoglobin and fructosamine but not with serum glucose levels. It has been demonstrated that transcatheter aortic valve replacement (TAVI) is beneficial for AS patients also with concomitant DM and safer as compared to surgical aortic valve replacement (SAVR). Moreover, new antidiabetic drugs, such as glucagon-like peptide-1 receptor antagonists agents and sodium-glucose cotransporter-2, inhibitors targeting an inhibition of AGEs-mediated oxidative, have been proposed to reduce the risk of AS development in DM patients. The aim of this review was to comprehensively discuss the impact of DM on AS and its potential therapeutic implications.

Context.— Direct oral anticoagulants (DOACs) may cause false negative results of antithrombin (AT) deficiency screening. Objective.— To evaluate the impact of DOAC-Stop, an agent reversing in vitro effects of DOACs, on AT testing in anticoagulated patients. Design.— We assessed 130 venous thromboembolism patients aged 46.7 ± 13.5 years. Blood samples were collected 2 to 27 hours after DOAC intake from 49 patients on rivaroxaban, 54 on apixaban, and 27 on dabigatran. Antithrombin activity was assessed using the activated factor X (FXa)–based and the activated factor II (FIIa)–based method twice, before and after DOAC-Stop treatment, together with plasma DOAC levels using coagulometric assays. Results.— The use of DOAC-Stop did not influence AT activity measured using the FIIa-based assay, whereas there was a marked decrease in AT activity determined using the FXa-based assay (ΔAT = 16.9%; 95% CI, 12.9%–19.1%). The AT-FIIa assay revealed decreased AT level ( Conclusions.— Application of DOAC-Stop enables reliable evaluation of AT deficiency screening in patients taking rivaroxaban or apixaban and tested using the FXa-based method.

Research question Is there a difference in fibrin clot phenotype in women with endometriosis before and after ovarian stimulation? Design Prospective study including 73 infertile women in two age-matched groups: (i) with confirmed endometriosis (n = 29); (ii) without endometriosis (n = 44). Assessments of plasma fibrin clot permeability (Ks), efficiency of fibrinolysis using clot lysis time (CLT), along with thrombin generation (prothrombin fragments 1+2 [F1+2] and endogenous thrombin potential [ETP]) and fibrinolysis inhibitors were performed together with clinical pregnancy rate. Results Endometriosis was associated with increased thrombin generation, reflected by both higher F1+2 (+96.1%, P = 0.005) and ETP (+14.2%, P = 0.014) along with unfavourably altered fibrin clot properties represented by lower Ks (–31%, P Conclusions Endometriosis is associated with the prothrombotic fibrin clot phenotype and increased thrombin generation. Ovarian stimulation favourably alters fibrin clot properties and leads to comparable pregnancy outcomes to those in women without endometriosis.

Background Prothrombotic fibrin clot properties are associated with higher early mortality risk in acute pulmonary embolism (PE) patients. It is unknown whether different types of PE are associated with particular clot characteristics. Methods We assessed 126 normotensive, noncancer acute PE patients (median age: 59 [48–70] years; 52.4% males), who were categorized into central versus peripheral PE with or without concomitant deep vein thrombosis (DVT). Plasma fibrin clot permeability (K s), clot lysis time (CLT), thrombin generation, platelet-derived markers, and fibrinolytic parameters were measured on admission. Plasma fibrin clot morphology was assessed by scanning electron microscopy (SEM). Results Patients with central PE (n = 76; 60.3%) compared with peripheral PE (n = 50; 39.7%) had 17.8% higher K s and 14.3% shortened CLT (both p Conclusion Our findings suggest that looser fibrin networks composed of thicker fibers with increased susceptibility to lysis characterize patients with central PE, suggesting that fibrin clot phenotype affects the size of thrombi occluding the pulmonary arteries, highlighting the role of fibrin structures in thrombus formation and stability.

OBJECTIVES Low von Willebrand factor (VWF) increases the risk of bleeding. The objective was to assess the influence of VWF on bleeding after valvular surgery. METHODS We studied 82 consecutive patients in median age of 65.5 years with severe isolated aortic stenosis (AS, n = 62) or mitral stenosis (MS, n = 20), undergoing heart valve surgery in extracorporeal circulation. Preoperatively, we assessed VWF antigen (VWF:Ag) and activity (VWF:RCo), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), and fibrinolysis inhibitors. RESULTS Compared with AS, MS patients were more frequently female (80 vs. 55%, p = 0.045) with atrial fibrillation (AF) (80 vs. 8%, p

Aortic stenosis (AS) has been associated with impaired fibrinolysis and increased oxidative stress. This study aimed to investigate whether oxidative stress could alter fibrin clot properties in AS. We studied 173 non-diabetic patients, aged 51&ndash, 79 years, with isolated AS. We measured plasma protein carbonylation (PC) and thiobarbituric acid reactive substances (TBARS), along with plasma clot permeability (Ks), thrombin generation, and fibrinolytic efficiency, which were evaluated by two assays: clot lysis time (CLT) and lysis time (Lys50). Coagulation factors and fibrinolytic proteins were also determined. Plasma PC showed an association with AS severity, reflected by the aortic valve area and the mean and maximum aortic gradients. Plasma PC was positively correlated with CLT, Lys50, plasminogen activator inhibitor-1 (PAI-1), and tissue factor (TF) antigens. TBARS were positively correlated with maximum aortic gradient, Lys50, and TF antigen. Regression analysis showed that PC predicted prolonged CLT (&gt, 104 min, odds ratio (OR) 6.41, 95% confidence interval (CI) 2.58&ndash, 17.83, p &lt, 0.001) and Lys50 (&gt, 565 s, OR 5.83, 95% CI 2.23&ndash, 15.21, p &lt, 0.001). Multivariate regression analysis showed that mean aortic gradient, PC, &alpha, 2-antiplasmin, PAI-1, and triglycerides were predictors of prolonged CLT, while PC, &alpha, 2-antiplasmin, and fibrinogen were predictors of Lys50. Our findings suggest that elevated oxidative stress contributes to impaired fibrinolysis in AS and is associated with AS severity.

BACKGROUND The objective of this study was to compare the impact of skeletonized versus pedicled left internal mammary artery (LIMA) harvesting on bleeding after coronary artery bypass grafting (CABG). METHODS In a randomized, single-blinded trial with a parallel group design and equal allocation, we randomly assigned 62 patients undergoing primary elective CABG in a tertiary cardiac center to skeletonized or pedicled LIMA dissection. Before surgery, all aspects of coagulation were assessed. Patients were blinded to LIMA dissection technique and monitored for cumulative drainage at 12 hours (primary outcome) as well as myocardial necrosis markers. RESULTS With recruitment complete, there were 31 patients in each group; all patients were analyzed. Median postoperative drainage was 395 mL at 12 hours in all patients; it was lower by 28% at 12 hours (P = .02) in patients with skeletonized LIMA (Cohen's d, 0.6; 95% confidence interval (CI), 0.09-1.11). Patients with a LIMA pedicle received more fresh-frozen plasma transfusions than did the skeletonized LIMA group (Median 3; interquartile range 3-5 versus median 3; interquartile range 3-3; P = .03). Study arms did not differ in blood coagulation. Left internal mammary artery skeletonization (odds ratio = 0.04; 95% CI, 0.003-0.44; P = .009) and higher body mass index (odds ratio = 0.63; 95% CI, 0.45-0.89; P = .008) decreased the odds of being in the top drainage quartile at 12 hours (≥550 mL). Creatine kinase was lower in skeletonized LIMA directly after surgery (median 218 U/L; interquartile range 175-310 U/L versus median 424 U/L; interquartile range 256-510 U/L; P < .001), at 6 hours (median 324 U/L; interquartile range 239-424 U/L versus median 529 U/L; interquartile range 374-707 U/L; P < .001), and 12 hours after surgery (median 351 U/L; interquartile range 277-552 U/L versus median 695 U/L; interquartile range 509-1067 U/L; P < .001). CONCLUSIONS Left internal mammary artery skeletonization results in lower mediastinal drainage after CABG than pedicled LIMA harvesting.

Background: Similarities between the pathobiology of aortic stenosis (AS) and atherosclerosis have led to the concept that pharmacological strategies effective in atherosclerosis might attenuate valvular inflammation. Objective: The objective of this study was to assess how angiotensin-converting enzyme inhibitors (ACEIs) might affect valvular expression of coagulation and inflammatory proteins in AS. Material/Methods: We studied 111 advanced AS patients (62 males, aged 63.3±11.2 years) undergoing valve replacement. Plasma levels, valvular expression and mRNA transcripts of tissue factor (TF), TF pathway inhibitor (TFPI), prothrombin, along with C-reactive protein (CRP) and interleukin-6 (IL-6) were evaluated. Results: TF-, TFPI-, CRP and prothrombin valvular expression was not related to demographics, concomitant diseases or plasma TF, free-TFPI or IL-6. ACEIs-treated patients (n=37), mainly due to hypertension (n=24, 65%), showed decreased areas for valvular TF (13.64±6.43 vs. 18.05±6.81%, p=0.03), TFPI (32.6±7.8 vs. 49.15±9.5%, p

Calcific aortic stenosis (CAS) is the most common cause of acquired valvular heart disease in adults with no available pharmacological treatment to inhibit the disease progression to date. This review provides an up-to-date overview of current knowledge of molecular mechanisms underlying CAS pathobiology and the related treatment pathways. Particular attention is paid to current randomized trials investigating medical treatment of CAS, including strategies based on lipid-lowering and antihypertensive therapies, phosphate and calcium metabolism, and novel therapeutic targets such as valvular oxidative stress, coagulation proteins, matrix metalloproteinases, and accumulation of advanced glycation end products.

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, β-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.

Background Treatment with non-vitamin K antagonist oral anticoagulants (NOACs) such as dabigatran (a direct thrombin inhibitor) or rivaroxaban (a direct inhibitor of factor [F] Xa) attenuates atherosclerotic plaque progression in hypercholesterolemic mice. Purpose: To evaluate the effect of NOACs application on the expression of coagulation proteins in loco within stenotic aortic valves and in valve interstitial cells (VICs) from patients with severe aortic stenosis (AS). Methods Primary cultures of VICs obtained from 90 patients undergoing aortic valve replacement were stimulated with TNF-α (50 ng/mL) and pre-treated with rivaroxaban (1 and 10 ng/mL) or dabigatran (25 and 250 ng/mL). The expression of coagulation proteins was analyzed by immunofluorescence. Cytokine levels were measured by ELISA. Results: FX, FXa, FVII, thrombin and PAR1/2 were present in loco within human aortic stenotic valves. Cultured VICs exhibited constant expression of FX, TF, PAR1/2. Exposure of VICs to TNF-α caused the upregulated expression of TF, PAR1/2 and induced expression of thrombin, FVII and FXa. FX was expressed by 80% of VICs, regardless of stimulation. Cultured VICs were able to synthesize metalloproteinases 1–3, IL-6, IL-32, IL-34, osteopontin and osteocalcin, the levels of which increased under TNF-α stimulation. NOACs added to culture inhibited coagulation factor and PAR1/2 expression. Moreover, NOACs down-regulated VIC-derived proteins responsible for valve calcification and extracellular matrix remodeling. Conclusions NOACs at therapeutic concentrations may inhibit the effects of FXa and thrombin at in vitro level. It might be speculated that long-term treatment with rivaroxaban or dabigatran could attenuate the progression of AS in humans.

Denser fibrin networks which are relatively resistant to lysis can predispose to post-thrombotic syndrome (PTS). Histidine-rich glycoprotein (HRG), a blood protein displaying antifibrinolytic properties, is present in fibrin clots. We investigated whether HRG may affect the risk of PTS in relation to alterations to fibrin characteristics. In venous thromboembolism (VTE) patients, we evaluated plasma HRG levels, plasma clot permeability, maximum absorbance, clot lysis time and maximum rate of increase in D-dimer levels released from clots after 3 months of the index event. We excluded patients with cancer and severe comorbidities. After 2 years of follow-up, 48 patients who developed PTS had 18.6% higher HRG at baseline. Baseline HRG positively correlated with clot lysis time, maximum absorbance, and thrombin-activatable fibrinolysis inhibitor (TAFI) activity but was inversely correlated with plasma clot permeability and maximum rate of increase in D-dimer levels released from clots. On multivariate regression model adjusted for age, fibrinogen and glucose, independent predictors of PTS were recurrent VTE, baseline HRG level, and TAFI activity. VTE recurred in 45 patients, including 30 patients with PTS, and this event showed no association with elevated HRG. Our findings suggest that increased HRG levels might contribute to the development of PTS, in part through prothrombotic fibrin clot properties.

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 β-D-glucose or ASA (control); incubated with β-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 β-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

Background High hemodynamic forces similar to those observed in aortic stenosis (AS) can trigger neutrophil extracellular traps (NETs) formation. Purpose An involvement of NETosis in the AS pathogenesis is unknown. Methods We enrolled 25 patients, median age 64.9 [range, 58–69] years with isolated severe AS (transvalvular pressure gradient, PGmean: 53.6±11.3 mm Hg, PGmax: 85.1±17.6 mmHg), after aortic valve replacement and 15 healthy sex/age-matched controls. Autopsy-derived aortic valves from 5 healthy donors served as negative control. Transverse valve sections were taken from the mid and commissural areas of the leaflet and cryosectioned by a Leica CM 1520 cryostat. Valvular expression of citrullinated histone H3 (citH3), together with myeloperoxidase (MPO), and neutrophil elastase (NE) as NETs biomarkers and macrophages (CD68) were evaluated by single- and double- immunostaining. Plasma concentrations of citH3 and interleukin 6 (IL-6) were also determined. Results All stenotic and healthy valves expressed citH3 in the leaflets' endothelial and sub-endothelial layers at the aortic side. The in loco expression of citH3-positive cells was higher in AS patients compared with controls (42.3±8.7% vs. 7.2±4.8%, p0.05). Double-staining revealed that within stenotic leaflets 28.3±8.4% of cells were citH3/MPO- and 25.2±7.1% citH3/NE-positive. None of control valves showed MPO or NE-positivity. Moreover, 6.6±1.9% of valvular cells (17.2±5.4% of citH3-positive cells) showed citH3/CD68 double-positivity and were identified as macrophages. Plasma levels of citH3 were 59% higher in AS patients then in controls (p Conclusions The presence of NETs in stenotic valves and association with AS severity might suggest novel mechanisms involved in the disease progression. This work was supported by the grant from the Polish National Science Center (DEC-2017/01/X/NZ5/02006 to R.K-T.). Acknowledgement/Funding This work was supported by the grant from the Polish National Science Center (DEC-2017/01/X/NZ5/02006 to R.K-T.).