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.

Positive antiphospholipid antibodies increase the risk of ischemic stroke in patients with atrial fibrillation.

BACKGROUND: Antiphospholipid antibodies (aPL), including lupus anticoagulant (LA), antibodies against ß2 glycoprotein I (anti-ß2GPI) and anticardiolipin antibodies (aCL) are associated with ischemic stroke (IS). Their prevalence and clinical relevance in atrial fibrillation (AF) remain unclear. AIM: To assess whether aPL are associated with increased risk of ischemic stroke (IS) in AF patients despite anticoagulation. MATERIAL AND METHODS: We conducted a post hoc analysis of aPL using blood samples from 243 consecutive AF patients enrolled in the cohort study. Markers of a prothrombotic state, including endogenous thrombin potential (ETP), fibrin clot permeability and lysis time were measured at baseline. During a median follow-up of 52 months, IS/transient ischemic attack (TIA) and major bleeding were recorded. RESULTS: We observed aPL at a moderate or high titer in 51 (21%) patients, including 17 (7%) with anti-ß2GPI, 19 (7.8%) with aCL antibodies, and 37 (15.2%) with LA. aPL-positive patients were more likely to have prior stroke (p=0.01) and be active smokers (p=0.03), along with increased ETP (p=0.02), without any changes in fibrin clot properties. Anti-ß2GPI (HR=4.38, 95% CI 1.58-12.19) and aCL (HR=4.70, 95% CI 1.80-12.30) at a moderate or high titer were associated with IS during follow-up (n=20; 1.9%/year). There were 23 major bleedings (2.1% per year) and 20 deaths (1.9% per year), which were not associated with aPL. CONCLUSION: Our study showed a relatively high prevalence of aPL-positivity in AF patients, which was linked to increased risk of IS/TIA. This suggests that screening for aPL might help optimizing anticoagulant therapy in such patients.

Complement activation is associated with right ventricular dysfunction and the severity of pulmonary embolism: links with prothrombotic state.

Background: Little is known about the role of complement activation in acute pulmonary embolism (PE). We investigated whether complement activation is associated with the severity of acute PE, along with the associated prothrombotic state, systemic inflammation and neutrophil extracellular traps (NETs) formation. Methods: We studied 109 normotensive, non-cancer PE patients (aged 58.1±15.0 years). On admission prior to initiation of anticoagulation, plasma soluble complement components, i.e., C3a and sC5b-9, were measured with enzyme-linked immunosorbent assay (ELISA), along with thrombin generation, fibrinolysis proteins (plasminogen, antiplasmin, plasminogen activator inhibitor-1), factor VIII (FVIII) activity, and fibrin clot properties, including clot permeability (Ks, a measure of clot density) and clot lysis time (CLT). Moreover, we determined inflammatory markers and citrullinated histone H3, a specific marker of NETs formation. Results: Patients in the lower tertile of C3a (≤1.45 ng/mL, n=37) had lower simplified Pulmonary Embolism Severity Index (sPESI) values and were less likely to have right ventricular (RV) dysfunction compared to the remaining subjects. The former subgroup also had 13% lower FVIII activity, but not fibrinogen, interleukin-6, fibrinolysis proteins, and thrombin generation. Plasma C3a levels correlated inversely with Ks and positively with CLT indicating formation of denser and poorly lysable clots in subjects with elevated C3a. Despite a positive association between C3a and sC5b-9, the latter parameter was solely associated with higher FVIII, but not with other variables. Conclusions: We showed that in acute PE enhanced complement activation characterizes patients with poorer short-term prognosis who display prothrombotic fibrin clot properties and elevated FVIII, which supports the involvement of complement proteins in acute thromboembolism.

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.

Thrombosis and Aging: Fibrin Clot Properties and Oxidative Stress.

Significance: Aging is a complex process associated with an increased risk of many diseases, including thrombosis. This review summarizes age-related prothrombotic mechanisms in clinical settings of thromboembolism, focusing on the role of fibrin structure and function modified by oxidative stress. Recent Advances: Aging affects blood coagulation and fibrinolysis via multiple mechanisms, including enhanced oxidative stress, with an imbalance in the oxidant/antioxidant mechanisms, leading to loss of function and accumulation of oxidized proteins, including fibrinogen. Age-related prothrombotic alterations are multifactorial involving enhanced platelet activation, endothelial dysfunction, and changes in coagulation factors and inhibitors. Formation of more compact fibrin clot networks displaying impaired susceptibility to fibrinolysis represents a novel mechanism, which might contribute to atherothrombosis and venous thrombosis. Alterations to fibrin clot structure/function are at least in part modulated by post-translational modifications of fibrinogen and other proteins involved in thrombus formation, with a major impact of carbonylation. Fibrin clot properties are also involved in the efficacy and safety of therapy with oral anticoagulants, statins, and/or aspirin. Critical Issues: Since a prothrombotic state is observed in very elderly individuals free of diseases associated with thromboembolism, the actual role of activated blood coagulation in health remains elusive. It is unclear to what extent oxidative modifications of coagulation and fibrinolytic proteins, in particular fibrinogen, contribute to a prothrombotic state in healthy aging. Future Directions: Ongoing studies will show whether novel therapies that may alter oxidative stress and fibrin characteristics are beneficial to prevent atherosclerosis and thromboembolic events associated with aging.

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.

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.

Low-grade endotoxemia in acute pulmonary embolism: Links with prothrombotic plasma fibrin clot phenotype.

BACKGROUND: Lipopolysaccharide (LPS) can traverse the intestinal barrier and enter bloodstream, causing endotoxemia and triggering inflammation. Increased circulating LPS was reported in arterial thromboembolism. We investigated whether increased LPS levels occur in acute pulmonary embolism (PE) and if it is associated with a prothrombotic state. METHODS: We studied 120 normotensive PE patients (aged 59 [48-68] years) on admission, after 5-7 days, and after a 3-month anticoagulation. Serum LPS levels, along with zonulin, a marker of gut permeability, endogenous thrombin potential (ETP), fibrin clot permeability (Ks), clot lysis time (CLT), fibrinolysis proteins, and platelet markers were assessed. RESULTS: Median LPS concentration on admission was 70.5 (61.5-82) pg/mL (min-max, 34-134 pg/mL), in association with C-reactive protein (r = 0.22, p = 0.018), but not with fibrinogen, D-dimer or platelet markers. Patients with more severe PE had higher LPS levels compared with the remainder. Median zonulin level was 3.26 (2.74-4.08) ng/mL and correlated with LPS (r = 0.66, p < 0.0001). Patients with baseline LPS levels in the top quartile (≥82 pg/mL; n = 29) compared to lower quartiles had 18.6 % increased ETP, 14.5 % reduced Ks, and 25.3 % prolonged CLT, related to higher plasminogen activator inhibitor type 1 (PAI-1) levels. LPS decreased by 23.4 % after 5-7 days and by 40.4 % after 3-month anticoagulation together with reduced zonulin by 18.4 % and 22.3 %, respectively, compared to baseline (all p < 0.001). LPS levels were not related with fibrin characteristics and other variables assessed at 3 months. CONCLUSIONS: Low-grade endotoxemia is detectable in patients with acute PE and may contribute to increased thrombin generation and PAI-1-mediated hypofibrinolysis.

Increased Serum Levels of Phoenixin-14, Nesfatin-1 and Dopamine Are Associated with Positive Pregnancy Rate after Ovarian Stimulation.

BACKGROUND: We study the relationship between phoenixin (PNX-14), nesfatin-1 (NES-1), dopamine (DA) and oxytocin (OT) levels together with pregnancy rates in women after ovarian stimulation (OS). METHODS: In a prospective case-control study, 56 infertile women were enrolled from the Department of Gynecological Endocrinology University Hospital. Infertile women age < 40 years old, with polycystic ovary syndrome (PCOS), confirmed tubal patency and suitable sperm quality were included. Blood samples were drawn twice-before the initiation of OS and before the human chorionic gonadotropin (hCG) administration. Assessments of PNX-14, NES-1, DA and OT serum levels were performed. Pregnancy rates after OS were observed. RESULTS: Pregnant women showed higher baseline NES-1 and OT levels (+29.2% and +44%) but not PNX-14 and DA levels when compared to non-pregnant ones. In pregnant women, positive correlations between OT and prolactin, PRL (r = 0.47, p = 0.04), as well as between OT and NES-1 (r = 0.55, p = 0.02), were observed at baseline. At baseline, an OT level increase was associated with a positive pregnancy rate (per 100 pg/mL, OR = 1.39, 95% CI 1.04-1.74), while after OS, higher PNX-14 was a predictor of pregnancy (by 10 pg/mL, OR = 1.23, 95%CI 1.07-1.39). Post-stimulation PNX-14, NES-1 and DA concentrations were higher in pregnant women compared to non-pregnant ones (+17.4%, +26.1%, and +45.5%, respectively; all p < 0.05). In the pregnant group, OT levels were 2.7-times lower than in the remainder (p = 0.03). Moreover, in pregnant participants, a negative association between NES-1 and PNX (r = -0.53, p = 0.024) was observed. CONCLUSION: Elevated PNX-14, NES-1 and DA along with decreased OT levels were observed in women who achieved pregnancy.

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.

PAI-1 Overexpression in Valvular Interstitial Cells Contributes to Hypofibrinolysis in Aortic Stenosis.

Aortic stenosis (AS) is associated with hypofibrinolysis, but its mechanism is poorly understood. We investigated whether LDL cholesterol affects plasminogen activator inhibitor 1 (PAI-1) expression, which may contribute to hypofibrinolysis in AS. Stenotic valves were obtained from 75 severe AS patients during valve replacement to assess lipids accumulation, together with PAI-1 and nuclear factor-κB (NF-κB) expression. Five control valves from autopsy healthy individuals served as controls. The expression of PAI-1 in valve interstitial cells (VICs) after LDL stimulation was assessed at protein and mRNA levels. PAI-1 activity inhibitor (TM5275) and NF-κB inhibitor (BAY 11-7082) were used to suppress PAI-1 activity or NF-κB pathway. Clot lysis time (CLT) was performed to assess fibrinolytic capacity in VICs cultures. Solely AS valves showed PAI-1 expression, the amount of which was correlated with lipid accumulation and AS severity and co-expressed with NF-κB. In vitro VICs showed abundant PAI-1 expression. LDL stimulation increased PAI-1 levels in VICs supernatants and prolonged CLT. PAI-1 activity inhibition shortened CLT, while NF-κB inhibition decreased PAI-1 and SERPINE1 expression in VICs, its level in supernatants and shortened CLT. In severe AS, valvular PAI-1 overexpression driven by lipids accumulation contributes to hypofibrinolysis and AS severity.

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.

Neutrophil-activating Peptide 2 as a Novel Modulator of Fibrin Clot Properties in Patients with Atrial Fibrillation.

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). 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. NAP-2 levels were 89% higher in AF patients than in controls (626 [448-796] vs. 331 [226-430] ng/ml; p < 0.0001). NAP-2 levels were not associated with demographics, CHA2DS2-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 p < 0.05). NAP-2 levels were positively associated with fibrinogen in AF patients (r = 0.41, p = 0.0006) and controls (r = 0.65, p < 0.01), along with citH3 (r = 0.36, p < 0.0001) and 3-nitrotyrosine (r = 0.51, p < 0.0001) in the former group. After adjustment for fibrinogen, higher citH3 (per 1 ng/ml ß = -0.046, 95% CI -0.029; -0.064) and NAP-2 (per 100 ng/ml ß = -0.21, 95% CI -0.14; -0.28) levels were independently associated with reduced Ks. 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.

Antithrombin Deficiency Is Associated with Prothrombotic Plasma Fibrin Clot Phenotype.

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 < 0.01). Prothrombin fragment 1 + 2 levels were 26.5% higher in patients with antithrombin deficiency than in controls along with 94% increased endogenous thrombin potential (ETP) and 108% higher peak thrombin (all p < 0.01). Antithrombin deficiency was associated with 18% reduced Ks and 35% prolonged CLT (both p < 0.001). Patients with type I (n = 65; 43.9%) compared with type II antithrombin deficiency (n = 83; 56.1%) had 22.5% lower antithrombin activity (p < 0.001) and despite similar fibrinogen levels, 8.4% reduced Ks, 18% prolonged CLT, and 30% higher ETP (all p < 0.01). Reduced Ks was associated with lower antithrombin antigen level (ß = - 6.1, 95% confidence interval [CI]: -1.7 to -10.5), while prolonged CLT was associated with lower antithrombin antigen (ß = - 69.6, 95% CI: -9.6 to -129.7), activity (ß = - 2.4, 95% CI: -0.3 to -4.5), higher PAI-1 (ß = 12.1, 95% CI: 7.7-16.5), and thrombin-activatable fibrinolysis inhibitor levels (ß = 3.8, 95% CI: 1.9-5.7). Addition of exogenous antithrombin reduced ETP (-42%) and peak thrombin (-21%), and improved Ks (+8%) and CLT (-12%; all p < 0.01). CONCLUSION: 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.