Reduced fibrin clot permeability on admission and elevated E-selectin at 3 months as novel risk factors of residual pulmonary vascular obstruction in patients with acute pulmonary embolism.

BACKGROUND: Residual pulmonary vascular obstruction (RPVO) is common following pulmonary embolism (PE) but its association with fibrin clot properties is poorly understood. We investigated whether prothrombotic state and hypofibrinolysis markers can identify patients with RPVO. METHODS: In 79 normotensive noncancer patients (aged 56 ± 13.3 years) with acute PE, we determined fibrin clot permeability (Ks), clot lysis time (CLT), endogenous thrombin potential (ETP), fibrinolysis proteins, oxidative stress markers, and E-selectin on admission before initiation of anticoagulant therapy, after 5-7 days, and 3 months of anticoagulation. RPVO was diagnosed using computed tomography angiography 3-6 months since PE. RESULTS: Patients with RPVO (n = 23, 29.1%) had at baseline higher simplified Pulmonary Embolism Severity Index (sPESI) (P = 0.004), higher N-terminal brain natriuretic propeptide (P = 0.006) and higher D-dimer (P = 0.044). Patients with versus without RPVO had lower Ks (P < 0.001) and longer CLT (P < 0.05), both at baseline and 5-7 days since admission, but not at 3 months. Patients with RPVO showed 40.6% higher E-selectin (P < 0.001) solely at 3 months. By multivariable logistic regression, baseline Ks (odds ratio [OR] 0.010, 95% confidence interval [CI] 0.001-0.837, P = 0.042, per 10- 9 cm2), baseline D-dimer (OR 1.105, 95% CI 1.000-1.221, P = 0.049, per 100 ng/ml), and E-selectin levels after 3 months (OR 3.874, 95% CI 1.239-12.116, P = 0.020, per 1 ng/ml) were associated with RPVO. CONCLUSIONS: RPVO patients despite anticoagulation characterize with the formation of denser fibrin clots on admission and higher E-selectin at 3 months. Those parameters could be the potential novel RPVO risk factors that warrant further evaluation in an independent cohort.

Low bleeding acceptance is associated with increased death risk in patients with atrial fibrillation on oral anticoagulation.

Bleeding is the most feared complication of anticoagulation. We sought to investigate whether the bleeding risk acceptance has a prognostic value during long-term follow-up in the era of direct oral anticoagulants (DOACs) among patients with atrial fibrillation (AF). We studied 167 consecutive AF outpatients [aged 68.8 SD 10.6 years; 141 (84.4%) on DOACs]. The bleeding acceptance was assessed based on the Bleeding Ratio defined as the declared maximum number of major bleedings that a patient would be willing to accept to prevent one major stroke. We recorded cerebrovascular ischemic events, major or clinically relevant non-major bleeds (CRNMB), and mortality. A median Bleeding Ratio was 4 (IQR 2-5). During follow-up of 946 patient-years, cerebrovascular ischemic events and/or death were observed in 28 patients (3.3%/ year) and major bleeding or CRNMB in 33 (4.6%/ year). The Bleeding Ratio was lower in patients who experienced cerebrovascular events or death (p = 0.004), but not bleeding. Patients with the Bleeding Ratio 0-3 were more often non-persistent to the OAC therapy, and more likely to have cerebrovascular event or die than those with higher bleeding acceptance (odds ratio 2.55; 0.95% CI 1.08-6.02) which was driven by the impact on mortality. The multiple Cox proportional hazards model showed that lower Bleeding Ratio, higher CHA2DS2-VASc score, and older age predicted cerebrovascular events or death during follow-up. AF patients who are willing to accept fewer serious bleedings to avoid major stroke during anticoagulation are more likely to experience death and/or cerebrovascular ischemic events, but not bleeding, what might be related to non-persistence.

Elevated plasma protein carbonylation increases the risk of ischemic cerebrovascular events in patients with atrial fibrillation: association with a prothrombotic state.

INTRODUCTION: Plasma protein carbonylation that reflects oxidative stress has been demonstrated to be associated with the prothrombotic fibrin clot phenotype. However, the role of protein carbonyls (PC) in predicting ischemic stroke in atrial fibrillation (AF) is largely unknown. This study aimed to investigate whether PC increase the risk of stroke in anticoagulated AF patients during follow-up. METHODS: In 243 AF patients on anticoagulation (median age 69 years; median CHA2DS2-VASc of 4), we measured plasma PC using the assay by Becatti, along with plasma clot permeability (Ks), clot lysis time (CLT), thrombin generation, and fibrinolytic proteins, including plasminogen activator inhibitor type 1 (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI). Ischemic stroke, major bleeding, and mortality were recorded during a median follow-up of 53 months. RESULTS: Plasma PC levels (median, 3.16 [2.54-3.99] nM/mg protein) at baseline showed positive associations with age (P < 0.001), CHA2DS2-VASc (P = 0.003), and N-terminal B-type natriuretic peptide (P = 0.001), but not with type of AF or comorbidities except for heart failure (P = 0.007). PC levels were correlated with CLT (r = 0.342, P < 0.001), endogenous thrombin potential (r = 0.217, P = 0.001) and weakly with Ks (r = -0.145, P = 0.024), but not with fibrinogen, PAI-1, or TAFI levels. Stroke was recorded in 20 patients (1.9%/year), who had at baseline 36% higher PC levels (P < 0.001). Elevated PC (P = 0.003) at baseline were independently associated with stroke risk. CONCLUSION: Our findings suggest that in patients with AF enhanced protein carbonylation is associated with increased "residual" risk of stroke despite anticoagulation, which is at least in part due to unfavorably altered fibrin clot phenotype.

Reduced protein carbonylation on hormone therapy is associated with improved fibrinolysis in postmenopausal women: the impact of PAI-1 and TAFI activity.

Hormone therapy (HT) has been reported to reduce protein carbonylation (PC) in postmenopausal women, in whom fibrinolysis is impaired. We investigated whether PC affects fibrinolysis and if HT modulates this effect. We enrolled 150 women aged 55.5 ± 4.7 years in a randomized interventional open-label study, including 50 on standard oral HT, 50 on ultra-low-dose HT, and 50 controls. PC, along with global fibrinolysis (clot lysis time, CLT), fibrinolysis proteins, and prothrombotic markers were determined at baseline and at 24 weeks. Patients with the baseline top quartile PC (> 2.07 nM/mg protein) had 10.3% longer CLT, higher activity (but not antigen) of TAFI (+ 19.9%) and PAI-1 (+ 68.1%) compared to the remainder. No differences were observed in thrombin generation, factor VIII, plasminogen or α2-antiplasmin. On-treatment PC decreased by 16.4% (p < 0.0001), without differences related to the type of HT, compared to baseline and by 30% compared to controls, in whom PC and fibrinolysis markers remained unchanged. Patients with PC > 2.07 nM/mg had shortened CLT during HT compared to baseline, along with lower PAI-1 (-69%) and TAFI (-26%) activity. In this subgroup CLT was 5.8% shorter compared to controls with the highest PC. In postmenopausal women with increased PC, HT was accompanied by PC reduction and faster clot lysis together with decreased PAI-1 and TAFI activity.

SERPINE1 and MTHFR genetic variants in patients with embolic stroke of undetermined source: links with fibrin clot properties.

INTRODUCTION: The SERPINE1 c.-820G (4_5), MTHFR gene variants, and unfavourably altered fibrin clot features, have been suspected to be associated with embolic stroke of undetermined source (ESUS). We investigated the SERPINE1 c.-820G (4_5) gene variants alone and coexisting with MTHFR c.665C > T and c.1286A > C gene variants in relation to thrombophilic factors and plasma fibrin clot properties in Polish patients with ESUS. PATIENTS AND METHODS: Unrelated consecutive patients with ESUS (n = 206) were genotyped by TaqMan assay. Thrombophilia screening was performed four weeks or more after a thrombotic event while off oral anticoagulation. Factor VIII (FVIII) activity was determined by a coagulometric assay, while lipoprotein(a) was determined using immunoturbidimetry. We determined fibrin clot permeability (Ks) and clot lysis time (CLT). Apparently healthy individuals without a family history of stroke or venous thromboembolism (n = 30), and patients with a history of atrial fibrillation (n = 25) or carotid artery disease-related stroke (n = 21), served as controls. RESULTS: Among ESUS patients, the SERPINE1 c.-820G (4_5) minor allele frequency was 0.57. There were no differences in common factors associated with thrombophilia among ESUS patients regarding SERPINE1 variants. The overall prevalence of FVIII > 150IU/dL was 26% (n = 53) and elevated FVIII predominated in SERPINE1 variants carriers (n = 45; 84.9%), including 36 (68%) carriers of MTHFR variant. Moreover, 4.3-fold higher Lp(a) levels along with 50% reduced Ks and 46% prolonged CLT were found in patients with mutant SERPINE1 combined with mutant homozygotes in the MTHFR c.665C > T variant compared to the wild type SERPINE1 combined with mutant homozygotes in the MTHFR c.665C >T (P < 0.001). CONCLUSIONS: The SERPINE1 c.-820G (4_5) variants carriers have increased FVIII levels, while the SERPINE1 c.-820G (4_5) mutant homozygotes coexisting with MTHFR c.665C > T have more prothrombotic fibrin clot features and elevated Lp(a). Our study underlines the cumulative effect of genetic risk factors in patients with ESUS that might require specific antithrombotic therapy.

Protein Carbonylation Contributes to Prothrombotic Fibrin Clot Phenotype in Acute Ischemic Stroke: Clinical Associations.

BACKGROUND: Acute ischemic stroke (AIS) is associated with enhanced oxidative stress and unfavorably altered fibrin clot properties. We investigated determinants of plasma protein carbonylation (PC) in AIS, its impact on the prothrombotic state, and prognostic value during follow-up. METHODS: We included 98 consecutive AIS patients aged 74±12 years (male:female ratio, 50:48 [51%:49%]) at the Neurology Center in Warsaw, Poland, between January and December 2014. As many as 74 (75.5%) patients underwent thrombolysis, and 24 were unsuitable for thrombolysis. We determined plasma PC, along with thrombin generation, fibrin clot permeability, and clot lysis time on admission, at 24 hours, and 3 months. Stroke severity was assessed using the National Institutes of Health Stroke Scale and stroke outcome with the modified Rankin Scale. Hemorrhagic transformation was assessed on the computed tomography scan within 48 hours from the symptom onset, while stroke-related mortality was evaluated at 3 months. RESULTS: On admission, PC levels (median, 4.61 [3.81-5.70] nM/mg protein) were associated with the time since symptom onset (r=0.41; P<0.0001) and with the National Institutes of Health Stroke Scale score (P=0.36; P=0.0003). Higher PC levels on admission correlated with denser fibrin clot formation and prolonged clot lysis time but not with thrombin generation. In thrombolysed patients, lower PC levels were observed after 24 hours (-34%) and at 3 months (-23%; both P<0.001). PC levels at baseline and after 24 hours predicted the modified Rankin Scale score >2 at 3 months (OR, 1.90 [95% CI, 1.21-3.00]; OR, 2.19 [95% CI, 1.39-3.44], respectively). Higher PC at baseline predicted hemorrhagic transformation of stroke (OR, 1.95 [95% CI, 1.02-3.74]) and stroke-related mortality (OR, 2.02 [95% CI, 1.08-3.79]), while higher PC at 24 hours predicted solely stroke-related mortality (OR, 2.11 [95% CI, 1.28-3.46]). CONCLUSIONS: Elevated plasma PC levels in patients with AIS, related to prothrombotic fibrin clot properties, are associated with stroke severity. Thrombolysis reduces the extent of PC. The current study suggests a prognostic value of PC in AIS.

Plasma thiol levels and methylenetetrahydrofolate reductase gene c.665C > T and c.1286A > C variants affect fibrin clot properties in Polish venous thromboembolic patients.

BACKGROUND AND AIMS: Aminothiols, including cysteine (Cys) and glutathione (GSH) in relation to fibrin clot phenotype were not investigated in patients with venous thromboembolism (VTE) and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene variants. We aimed to explore the associations between MTHFR variants and plasma oxidative stress indicators including aminothiols as well as fibrin clot properties with plasma oxidative status and fibrin clot properties in this group of patients. METHODS: In 387 VTE patients the MTHFR c.665C > T and c.1286A > C variants were genotyped, together with chromatographic separation of plasma thiols. We also determined nitrotyrosine levels and fibrin clot properties, including clot permeability (Ks), lysis time (CLT), and fibrin fibers thickness. RESULTS: There were 193 patients with MTHFR c.665C > T (49.9%) and 214 (55.3%) with c.1286A > C variants. Both allele carriers with total homocysteine (tHcy) levels >15 µM (n = 71, 18.3%), compared to patients with tHcy ≤15 µM had 11.5% and 12.5% higher Cys levels, 20.6% and 34.3% higher GSH levels as well as 28.1% and 57.4% increased nitrotyrosine levels, respectively (all P < 0.05). The MTHFR c.665C > T carriers with tHcy levels >15 µM compared to tHcy ≤15 µM had 39.4% reduced Ks and 9% reduced fibrin fibers thickness (both P < 0.05) with no differences in CLT. In the MTHFR c.1286A > C carriers with tHcy levels >15 µM, Ks was decreased by 44.5%, CLT prolonged by 46.1%, and fibrin fibers thickness was reduced by 14.5% compared to patients with tHcy ≤15 µM (all P < 0.05). Nitrotyrosine levels in MTHFR variants carriers correlated with Ks (r = -0.38, P < 0.05) and fibrin fibers diameter (r = -0.50, P < 0.05). CONCLUSIONS: Our study indicates that patients with MTHFR variants and tHcy >15 µM are characterized by elevated Cys and nitrotyrosine levels associated with prothrombotic fibrin clot properties.

Enhanced neutrophil extracellular traps formation in AF patients with dilated left atrium.

BACKGROUND: Atrial fibrillation (AF) is associated with cardiac remodelling and prothrombotic state. Enhanced neutrophil extracellular traps (NETs) formation has been reported in AF, contributing to thromboembolism. PURPOSE: We investigated whether increased left atrium (LA) diameter and reduced left ventricular ejection fraction (LVEF) affect NETs formation and prothrombotic state in AF patients. METHODS: In 243 AF patients (median CHA2 DS2 -VASc = 4) we measured LA diameter and LVEF, 123 of them with LVEF<50%. Moreover, we determined 3 markers of NETosis: circulating citrullinated histone H3 (H3cit), myeloperoxidase (MPO) and peptidylarginine deiminase 4 (PAD4), along with prothrombotic markers, including endogenous thrombin potential, plasma fibrin clot permeability (Ks ) and clot lysis time (CLT). Ischaemic cerebrovascular events, major bleeding and death were recorded during a median follow-up of 53 months, on anticoagulation. RESULTS: LA diameter correlated positively with H3cit, MPO and PAD4, while LVEF was inversely associated with the same NETosis markers. After adjustment for age and body mass index, concentrations of MPO (per 10 units; ß = -1.9, 95%CI -3.40;-0.42) and H3cit (per 10 units; ß = 2.02, 95%CI 0.61-3.42) were independently associated with LVEF and LA diameter. LA diameter, but not LVEF, correlated inversely with Ks and positively with CLT. The Cox regression analysis revealed that H3cit >6.16 ng/mL (HR = 21.76, 95%CI 2.85-166.28, p = .003) and LA diameter > 46 mm (HR = 2.89, 95%CI 1.04-8.03, p = .043) independently predicted cerebrovascular ischaemic events (1.9%/year). CONCLUSIONS: This hypothesis-generating study suggests that in AF enlarged LA diameter and reduced LVEF are associated with enhanced NETs formation, which might have clinical importance and contribute to thromboembolic events despite anticoagulation.

Increased factor XI but not factor XII is associated with enhanced inflammation and impaired fibrin clot properties in patients with eosinophilic granulomatosis with polyangiitis.

OBJECTIVES: In eosinophilic granulomatosis with polyangiitis (EGPA) a prothrombotic state, including formation of denser fibrin networks with reduced lysability has been observed. Little is known about the intrinsic pathway in EGPA. We investigated whether coagulation factors (F)XI and FXII are associated with eosinophil-driven prothrombotic state. METHODS: In 34 consecutive EGPA patients with remission we assessed FXI and FXII levels along with plasma fibrin clot permeability (Ks), fibrin clot morphology using scanning electron microscopy, and efficiency of fibrinolysis, expressed as lysis time (t50%) and maximum rate of increase in D-dimer levels (D-Drate). RESULTS: Increased FXI level (>130%, the upper reference limit) was found in 8 (23.5%) patients. Compared to patients with FXI levels ≤130%, those with increased FXI had higher eosinophil count (+365%) and reduced percentage of neutrophils (-20.4%), along with reduced Ks (-20.5%). In patients with FXI>130% clots were composed of thinner fibrin fibers (-17.5%). FXI was not associated with C-reactive protein and fibrinogen levels or anti-neutrophil cytoplasmic antibodies titers. There were no correlations between FXI and FXII levels as well as between FXII and eosinophil count (all p>0.05). CONCLUSIONS: To our knowledge, this study is the first to show association between FXI and a prothrombotic state in EGPA. Given clinical trials on FXI inhibition as an antithrombotic option, our findings suggest that this therapeutic approach could be useful in diseases with hypereosinophilia.

Metabolic effects of the left atrial appendage exclusion (the heart hormone study).

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The epicardial exclusion ligation in AF patients was associated with significant changes in insulin, leptin, and adiponectin over long follow-up.

Impaired fibrin clot lysis is associated with increased mortality in patients after coronary artery bypass grafting: A long-term follow-up study.

BACKGROUND: Mortality after coronary artery bypass grafting (CABG) is primarily thromboembolic by nature. We investigated whether impaired fibrinolysis observed in cardiovascular diseases is associated with long-term mortality following CABG. METHODS: The study population comprised 292 consecutive patients (aged 64.6 ± 8.1 years) who underwent scheduled CABG. We measured plasma clot lysis time (CLT) preoperatively as a measure of fibrinolysis capacity. Cardiovascular and all-cause deaths were recorded during a median follow-up of 13.8 years. RESULT: CLT positively correlated with age (r = .56, p < .001), fibrinogen (r = .25, p = .002) and EuroSCORE I (r = .32, p < .001). The cardiovascular and overall mortality rates were 3.0 and 4.9 per 100 patient-years (32.4% vs 52.8%) respectively. In patients who died from cardiovascular and all causes, CLT was prolonged compared with survivors (both p < .050). Multivariable Cox regression analysis adjusted for potential confounders showed that long-term cardiovascular and all-cause deaths were associated with CLT (HR per 10 min 1.206; 95% CI 1.037-1.402, p = .015 and HR 1.164; 96% CI 1.032-1.309, p = .012), low-density lipoprotein cholesterol (HR per 1 mmol/L 1.556; 95% CI 1.205-2.010, p < .001 and HR 1.388; 96% CI 1.125-1.703, p = .002), C-reactive protein (HR per 10 mg/L 1.171; 95% CI 1.046-1.312, p = .006 and HR 1.127; 95% CI 1.005-1.237, p = .022) and EuroSCORE I (HR 1.173; 95% CI 1.016-1.355, p = .030 and HR 1.183; 95% CI 1.059-1.317, p = .003 respectively). Type 2 diabetes was solely associated with overall mortality (HR 1.594; 96% CI 1.088-2.334, p = .017). CONCLUSIONS: In this study, we showed that reduced fibrin clot susceptibility to fibrinolysis is weekly associated with long-term mortality in advanced CAD.

Fibrinogen ß chain and FXIII polymorphisms affect fibrin clot properties in acute pulmonary embolism.

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 < .05). After adjustment for fibrinogen, all differences remained significant (all p < .01). There were no associations between the FGA rs6050 polymorphism and Ks , CLT or ETP. CONCLUSION: Our study showed that FGB rs1800790 and F13 rs5985 polymorphisms contribute to the prothrombotic fibrin clot phenotype and these effects are strong enough to be observed in the acute phase of PE.

Isoprostane-8 and GDF-15 as novel markers of post-PE syndrome: Relation with prothrombotic factors.

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 < .05). After 5-7 days, only hypofibrinolysis was noted in post-PE syndrome patients. When measured at 3 months, prolonged CLT and reduced Ks were observed in post-PE syndrome patients, accompanied by 23.8% higher growth differentiation factor 15 and 35.8% higher plasminogen activator inhibitor-1 (all P < .05). 8-isoprostane levels ≥108 pg/ml (odds ratio=4.36; 95% confidence interval 1.63-12.27) and growth differentiation factor 15 ≥ 1529 pg/ml (odds ratio=3.89; 95% confidence interval 1.29-12.16) measured at 3 months were associated with higher risk of developing post-PE syndrome. CONCLUSIONS: Enhanced oxidative stress and prothrombotic fibrin clot properties could be involved in the pathogenesis of the post-PE syndrome. Elevated growth differentiation factor 15 assessed at 3 months might be a new biomarker of this syndrome.

Diabetes concomitant to aortic stenosis is associated with increased expression of NF-κB and more pronounced valve calcification.

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 < 0.001). In VIC cultures treated with glucose in combination with reactive oxygen species (ROS) inhibitor (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.

Aortic valvular stenosis: Novel therapeutic strategies.

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 pro-inflammatory cytokines, clotting factors and proteins involved in calcification, strategies targeting these cell activations 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.

Fibrin clot properties among women with endometriosis and the impact of ovarian stimulation.

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 < 0.001) and prolonged CLT (+13.5%, P = 0.02), compared with the non-endometriosis group. Moreover, women with endometriosis had higher plasminogen activator inhibitor-1 (PAI-1; +272%, P = 0.004) concentrations and alpha-2-antiplasmin activity (+39.9%, P < 0.001) in contrast to the other group. Ovarian stimulation led to reduction in F1+2 (-48.1%, P < 0.001), improvement of fibrin clot phenotype reflected by higher Ks (+25.9%, P < 0.001) and shortened CLT (-11.9%, P < 0.001), along with lower PAI-1 (-54%, P = 0.016) compared with the baseline in women with endometriosis. 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.

Factor XIII and Fibrin Clot Properties in Acute Venous Thromboembolism.

Coagulation factor XIII (FXIII) is converted by thrombin into its active form, FXIIIa, which crosslinks fibrin fibers, rendering clots more stable and resistant to degradation. FXIII affects fibrin clot structure and function leading to a more prothrombotic phenotype with denser networks, characterizing patients at risk of venous thromboembolism (VTE). Mechanisms regulating FXIII activation and its impact on fibrin structure in patients with acute VTE encompassing pulmonary embolism (PE) or deep vein thrombosis (DVT) are poorly elucidated. Reduced circulating FXIII levels in acute PE were reported over 20 years ago. Similar observations indicating decreased FXIII plasma activity and antigen levels have been made in acute PE and DVT with their subsequent increase after several weeks since the index event. Plasma fibrin clot proteome analysis confirms that clot-bound FXIII amounts associated with plasma FXIII activity are decreased in acute VTE. Reduced FXIII activity has been associated with impaired clot permeability and hypofibrinolysis in acute PE. The current review presents available studies on the role of FXIII in the modulation of fibrin clot properties during acute PE or DVT and following these events. Better understanding of FXIII's involvement in the pathophysiology of acute VTE might help to improve current therapeutic strategies in patients with acute VTE.

Accumulation of advanced glycation end products (AGEs) is associated with the severity of aortic stenosis in patients with concomitant type 2 diabetes.

BACKGROUND: Accumulation of advanced glycation end products (AGEs) leads to chronic glycation of proteins and tissue damage, particularly in patients with diabetes mellitus (DM). We aimed to evaluate whether increased accumulation of AGEs in patients with aortic stenosis (AS) and concomitant type 2 diabetes (DM) is associated with AS severity. METHODS: We prospectively enrolled 76 patients with severe AS (47.1% males; nonDM), aged 68 [66-72] years, and 50 age-matched DM patients with a median blood glucose level of 7.5 [5.9-9.1] mM and glycated hemoglobin (HbA1c) of 6.8 [6.3-7.8]%, scheduled for aortic valve replacement. Valvular expression of AGEs, AGEs receptor (RAGE), interleukin-6 (IL-6), and reactive oxygen species (ROS) induction were evaluated ex vivo by immunostaining and calculated as the extent of positive immunoreactive areas/total sample area. Plasma levels of AGEs and soluble RAGE (sRAGE) were assessed by ELISAs. RESULTS: Subjects with DM had increased valvular expression of both AGEs (6.6-fold higher, 15.53 [9.96-23.28]%) and RAGE (1.8-fold higher, 6.8 [4.9-8.45]%) compared to nonDM patients (2.05 [1.21-2.58]% and 2.4 [1.56-3.02]%, respectively; both p < 0.001). Plasma levels of AGEs (12-fold higher) and sRAGE (1.3-fold higher) were elevated in DM patients, compared to nonDM (both p < 0.0001). The percentage of valvular ROS-positive (2.28 [1.6-3.09] vs. 1.15 [0.94-1.4]%, p < 0.0001) but not IL-6-positive areas was higher within DM, compared to nonDM valves. In DM patients, the percentage of valvular AGEs- and RAGE-positive areas correlated with HbA1c (r = 0.77, p < 0.0001 and r = 0.30, p = 0.034). Similarly, plasma AGEs and sRAGE levels were associated with HbA1c in the DM group (r = 0.32, p = 0.024 and r = 0.33, p = 0.014, respectively). In all DM patients, we found an association between the amount of valvular AGEs and the disease severity measured as aortic valve area (AVA; r = 0.68, p < 0.0001). Additionally, in DM patients with HbA1c > 7% (n = 24, 48%) we found that valvular expression of AGEs correlated with mean transvalvular pressure gradient (PGmean; r = 0.45, p = 0.027). Plasma AGEs levels in the whole DM group correlated with AVA (r = - 0.32, p = 0.02), PGmean (r = 0.31, p = 0.023), and PGmax (r = 0.30, p = 0.03). CONCLUSIONS: Our study suggests that poorly-controlled diabetes leads to increased AGEs and RAGE valvular accumulation, which at least partially, might result in AS progression in DM patients.

DOAC-Remove abolishes the effect of direct oral anticoagulants on activated protein C resistance testing in real-life venous thromboembolism patients.

Background Direct oral anticoagulants (DOACs) may cause false results of activated protein C resistance (APC-R) ratio. DOAC-Remove, a new reagent based on activated carbon, has been designed to eliminate the interference of DOACs on coagulation assays. The aim of the study was to investigate whether the use of DOAC-Remove enables to determine APC-R in patients treated with DOACs. Methods We assessed 74 venous thromboembolism (VTE) patients, including 25 on rivaroxaban, 25 on apixaban and 24 taking dabigatran. APC-R was determined using the Russell Viper Venom Time (RVVT)-based clotting test. APC-R and DOAC concentrations were tested at baseline and following DOAC-Remove. Thrombophilia, including factor V Leiden (FVL) mutation was tested. Results FVL mutation was found in 20 (27%) patients. The APC-R ratio at baseline was measurable in 43 patients (58.1%), including 20 (80%) on rivaroxaban, 19 (76%) on apixaban and four (16.7%) on dabigatran. In patients with measurable APC-R at baseline, the ratio >2.9 was found in 23 patients (53.5%). In 16 (37.2%) subjects APC-R ratio <1.8 suggested FVL mutation which was genetically confirmed. Four (9.3%) FVL carriers on dabigatran showed negative/equivocal APC-R results. In 11 (14.9%) patients taking rivaroxaban or apixaban, in whom blood was collected 2-5 h since the last dose, we observed unmeasurable APC-R. DOAC-Remove almost completely eliminated all plasma DOACs. After addition of DOAC-Remove all APC-R ratios were measurable. In four FVL carriers on dabigatran with false negative APC-R, DOAC-Remove resulted in APC-R ratios <1.8. Conclusions DOAC-Remove effectively reduces DOACs concentration in plasma, which enables FVL testing using APC-R.

The effect of DOAC-Stop on lupus anticoagulant testing in plasma samples of venous thromboembolism patients receiving direct oral anticoagulants.

Background Direct oral anticoagulants (DOACs) cause false positive lupus anticoagulant (LA) results. We assessed the impact of DOAC-Stop, reversing in vitro effects of DOACs, on LA testing in anticoagulated patients. Methods We assessed 75 venous thromboembolism patients aged 44.5±14.6 years. Blood samples were collected 2-28 h since intake of DOACs, including 50 patients on rivaroxaban, 20 on dabigatran and five on apixaban. LA testing was performed at baseline and after DOAC-Stop treatment. Positive LA was defined as the normalized (patient/standard plasma clotting time) LA screening and screening (LA1)/confirmation (LA2) ratios exceeding 1.2. Results LA diluted Russell's viper venom time (dRVVT) normalized screening test revealed abnormal results in 73 (97.3%) and activated partial thromboplastin time (APTT)-LA in 49 (65.3%) patients. In six (8%) patients, antiphospholipid syndrome (APS) was diagnosed. dRVVT LA1/LA2 was abnormal in 35 (50.7%) patients taking DOACs. The APTT ratio was normal in all studied subjects. DOAC-Stop completely removed dabigatran and reduced by 98% rivaroxaban and by 92.3% apixaban concentrations (all p<0.05). After DOAC-Stop screening dRVVT remained prolonged in 34 (49.3%) patients (p<0.001), while dRVVT LA1/LA2 was abnormal in six (8.7%) subjects, with no association with DOAC concentrations at baseline and after DOAC-Stop. The APTT-LA screening test remained prolonged in five (7.2%) patients, while the APTT LA1/LA2 ratio was normal in those subjects. DOAC-Stop did not influence LA testing in APS patients. Conclusions Application of DOAC-Stop effectively reduced plasma DOAC concentrations leading to appropriate dRVVT results in up to 97% of VTE patients.