Unfavorably altered fibrin clot phenotype in women following postpartum hemorrhage of unknown cause: effect of lower coagulation factors.

Background Increased clot permeability and susceptibility to lysis have been reported in women with heavy menstrual bleeding. We hypothesized that similarly altered fibrin clot properties in women with postpartum hemorrhage (PPH) of unknown cause. Objective To determine fibrin clot properties and their determinants in women after PPH of unknown cause. Methods We studied 52 consecutive women, aged 35 years (27-40), after at least 3 months since PPH of unknown cause and 52 matched controls for age, weight, and fibrinogen. Coagulation factors (F), antithrombin, thrombin generation, along with a comprehensive plasma fibrin clot analysis including fibrin polymerization, clot permeability (Ks), and fibrinolysis efficiency were determined. Results Women with PPH showed reduced activity of FII (-10.3%), FV (-6.6%), FIX (-6.5%), FX (-7.2%), and FXI (-5.7%) compared to the controls, though all values were within ranges (all p<0.05). There were no intergroup differences in fibrinogen, FVIII, FXIII, and thrombin generation. The PPH group formed with a delay looser plasma fibrin network (Ks; +16.3%, p=0.008) with lower maximum absorbance and shorter clot lysis time (CLT; -13.5%, p=0.001) compared to the controls. On multivariable logistic regression, PPH was independently associated with higher C-reactive protein (per 1 mg/L, OR=1.70, 95% CI 1.09-2.68), lower FII (per 1%, OR=0.93, 95% CI 0.89-0.98), lower FV (per 1%, OR=0.93, 95% CI 0.89-0.97), and shorter CLT (per 1 min, OR=0.94, 95% CI 0.90-0.98). Conclusion Prohemorrhagic fibrin clot properties, with lower, though normal coagulation factors characterize women with PPH of unknown cause, which suggests novel mechanisms contributing to this type of bleeding.

High-dose atorvastatin and rosuvastatin reduce neutrophil extracellular traps-related proteins in coronary artery disease: association with prothrombotic state.

INTRODUCTION: Neutrophil extracellular traps (NETs) formation is involved in atherothrombosis. OBJECTIVES: We sought to investigate whether statins affect NETosis in coronary artery disease (CAD) and if such changes show associations with statin-induced additional effects. PATIENTS AND METHODS: We studied 130 patients with advanced CAD before and at least 6 months after initiation of high-dose statin therapy with rosuvastatin 40 mg/d or atorvastatin 80 mg/d. Circulating citrullinated histone H3 (H3cit), myeloperoxidase (MPO), and neutrophil elastase (NE) were assessed as proteins associated with NETosis along with thrombin generation, plasma clot permeability (Ks), clot lysis time (CLT), and fibrinolysis inhibitors. RESULTS: Following statin therapy intensification, we observed reductions in H3cit (-30.4%), MPO (-28.1%), and NE (-25.5%, all P <0.001), all not associated with low-density lipoprotein cholesterol (LDL-C) lowering (-25%). However, H3cit was lower in 50 patients (38.5%) who achieved the target LDL-C <1.8 mmol/L (-16.5%, P = 0.004) and 19 (14.6%) with LDL-C <1.4 mmol/L (-25.5%, P = 0.001) compared with the remainder. Reductions in H3cit and MPO were associated with a 42.9% decrease in C-reactive protein (CRP) on high-dose statins (R = 0.855, P <0.001; R = 0.250, P = 0.004, respectively), along with increases in Ks and reduction in thrombin activatable fibrinolysis inhibitor (TAFI) activity, but not with CLT or thrombin generation (all |R| 0.24-0.4, P <0.01). On multivariable analysis, changes in CRP (ß = 0.771, P <0.001), TAFI activity (ß = 0.125, P = 0.013), and fibrinogen (ß = 0.106, P = 0.034) were independently associated with decrease in H3cit. CONCLUSIONS: We showed for the first time that high-dose statins can reduce NETs-related proteins in association with anti-inflammatory and antithrombotic actions in CAD patients.

Elevated lipopolysaccharide level is largely driven by time since symptom onset in acute ischemic stroke: the impact on clinical outcomes.

BACKGROUND: Gut dysbiosis leading to increased intestinal barrier permeability and translocation of lipopolysaccharide (LPS) in the circulation has been demonstrated in patients with acute myocardial infarction and pulmonary embolism. OBJECTIVES: We investigated changes in circulating LPS concentrations in acute ischemic stroke (AIS) and their consequences, including prognosis. METHODS: We studied 98 AIS patients, aged 74 ± 12 years, including 74 (75.5%) thrombolysed individuals. We determined serum LPS and zonulin, a marker of gut permeability, along with protein carbonyl (PC), fibrin clot properties, and thrombin generation on admission, at 24 hours and 3 months. Stroke severity was assessed using the National Institutes of Health Stroke Scale. Stroke functional outcome using modified Rankin scale and stroke-related mortality were evaluated at 3 months. RESULTS: Serum LPS and zonulin levels on admission were associated with time since symptom onset (r = 0.57; P < .0001; and r = 0.40; P < .0001). Baseline LPS levels correlated with PC (r = 0.51; P < .0001) but not with coagulation and fibrinolysis markers. LPS levels increased at 24 hours in thrombolysed patients (P < .001) and correlated with the National Institutes of Health Stroke Scale score (r = 0.31; P = .002) and PC (r = 0.32; P = .0057). Both LPS and zonulin levels measured at 24 hours increased the odds of having unfavorable modified Rankin scale scores (odds ratio [OR], 1.22; 95% CI, 1.04-1.42; and OR, 2.36; 95% CI, 1.24-4.49 per unit). Elevated LPS level, but not zonulin, was associated with stroke-related mortality (OR, 1.26; 95% CI, 1.02-1.55 per unit). CONCLUSION: In AIS patients intestinal permeability is mainly driven by increasing time since the symptom onset. Our findings suggest that LPS, with a trend toward its further rise following thrombolysis, adversely affects neurologic functional outcomes and 3-month mortality.

Elevated plasma factor XI is associated with postthrombotic syndrome.

INTRODUCTION: Postthrombotic syndrome (PTS), a common complication of deep vein thrombosis (DVT), is largely inflammatory by nature with contribution of prothrombotic mechanisms. The role of factor (F)XI in PTS has not been explored yet. We investigated whether elevated FXI is associated with PTS occurrence. MATERIALS AND METHODS: We enrolled 180 consecutive patients (aged 43 ± 13 years) with first-ever DVT. After 3 months FXI levels were measured, along with inflammatory markers, thrombin generation, plasma clot permeability (Ks), clot lysis time (CLT), and fibrinolysis proteins. We assessed PTS using the Villalta score and recorded symptomatic venous thromboembolism (VTE) at a 1-year and venous ulcers at a median 53 months follow-up. RESULTS: Baseline median FXI was 102 % [IQR 92-113 %] and showed positive association with Villalta score (R = 0.474, P < 0.001). Patients with PTS (n = 48, 26.7 %) had 16.1 % higher FXI (P < 0.001) and FXI ≥120 % occurred more often in PTS patients (odds ratio [OR] 5.55, 95 % confidence interval [CI] 2.28-13.47). There were associations of baseline FXI with Ks and CLT along with thrombin activatable fibrinolysis inhibitor (TAFI) activity, C-reactive protein, and interleukin-6, but not with fibrinogen, or thrombin generation. After age adjustment higher FXI was independently associated with PTS occurrence (OR per 1 % 1.06, 95 % CI 1.02-1.09) and VTE recurrence (OR 1.03, 95 % CI 1.01-1.06). At long-term follow-up, patients with venous ulcers had 13.6 % higher baseline FXI (P = 0.002). CONCLUSIONS: Elevated FXI in association with inflammation and prothrombotic fibrin clot properties may contribute to the development of PTS following DVT.

Poor patients' knowledge about venous thromboembolism and its therapy is associated with increased risk of major bleeding and discontinuation of anticoagulation: A cohort study.

It has been shown that patients' knowledge about venous thromboembolism (VTE) and its therapy is suboptimal, which might reduce compliance and worsen prognosis. We investigated whether low VTE patients' knowledge affects their clinical outcomes during long-term follow-up. We evaluated 151 consecutive patients (51.8 ±â€…15.7 years) after unprovoked VTE, who were recruited from the outpatient clinic (Krakow, Poland). All patients received anticoagulant treatment, mostly with direct oral anticoagulants (n = 113, 74.8%). The modified Jessa Atrial fibrillation Knowledge Questionnaire (JAKQ-VTE; 16 questions) was used to assess the knowledge of VTE and anticoagulant therapy. During a median follow-up of 58.0 months, VTE recurrence, major bleeding, and anticoagulation withdrawal were recorded. The median percentage of correct responses was 62.5% (12.5-100%) and was inversely correlated with age (P < .01). Diabetic patients and those with positive family history of VTE had lower overall scoring compared to the remainder (both P < .05). Major bleeding (n = 10, 6.6%) and anticoagulation withdrawal (n = 28, 18.5%), but not VTE recurrence (n = 12, 7.9%), were associated with lower overall scoring compared to the remainder (48.8% ±â€…12.5% vs 63.8% ±â€…16.3%, P = .003 and 55.3% ±â€…14.7% vs 64.4% ±â€…16.3%, P = .040, respectively). Major bleeding was independently associated with the female sex (hazard ratio [HR] 6.18; 95% confidence interval [CI] 1.15-33.19, P = .034), younger age (HR per 10 years 0.55; 95% CI 0.34-0.90, P = .016), OAC therapy discontinuation (HR 6.69; 95% CI 1.62-27.70), and lower overall scoring of JAKQ-VTE (HR 0.60 per 10 percentage points; 95% CI 0.40-0.92, P = .019). Insufficient knowledge about VTE and anticoagulant treatment predisposes to a higher risk of major bleeding and therapy discontinuation, but not VTE recurrence in unprovoked VTE patients during long-term follow-up.

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.

Altered fibrin clot phenotype in young adults with intracerebral hemorrhage of unknown cause: A case-control study.

BACKGROUND: Intracerebral hemorrhage (ICH) of undetermined etiology occurs infrequently in young and middle-aged adults. We hypothesized that slight decreases in coagulation factors and formation of less compact fibrin clots prone to faster lysis predispose to this type of ICH. METHODS: We recruited 44 consecutive patients aged <50 years following ICH of unknown cause at least 3 months since the event. Subjects free of ICH (n = 47) matched for age, sex, BMI, and hypertension served as the control group. We assessed plasma fibrin clot permeability, turbidity and fibrinolytic capacity, along with thrombin generation, coagulation factors (F) II, FV, FVII, FVIII, FIX, FX, FXI, antithrombin, and fibrinolysis proteins. RESULTS: ICH patients (median age 41 years, 45.5 % women) had 8.4 % lower FII (p = 0.0001) and 10.1 % lower FVII activity (p = 0.0003), 9.4 % higher antithrombin activity (p = 0.0004) and 13.5 % lower platelet count (p = 0.02). Other factors and thrombin generation did not differ between the two groups. The ICH survivors were characterized by impaired fibrin polymerization reflected by 10.1 % longer lag phase of the turbidimetry curve (p = 0.0002), decreased fiber density indicated by 11.8 % lower maximum absorbance (p = 0.004), as well as 11.1 % shorter clot lysis time (p = 0.014) and 10.0 % faster increase of maximal D-Dimer levels (p = 0.000001). CONCLUSIONS: We demonstrated a prohemorrhagic fibrin clot phenotype, along with lower FII, FVII and higher antithrombin activity in adults below 50 years of age who suffered from ICH of unknown cause, which might indicate novel mechanisms contributing to ICH in younger individuals.

Prothrombotic plasma fibrin clot phenotype is associated with spontaneous echo contrast in atrial fibrillation: The role of protein carbonylation.

INTRODUCTION: Spontaneous echo contrast (SEC) and left atrial appendage thrombus (LAAT) increase the risk of stroke and its severity in patients with atrial fibrillation (AF). Formation of denser fibrin networks and impaired fibrinolysis are associated with stroke risk in AF. This study investigated whether the prothrombotic fibrin clot phenotype characterizes patients with SEC/LAAT. METHODS: We studied 139 anticoagulated patients with AF (median age, 70 years), who underwent transesophageal echocardiography (TEE). SEC and LAAT were recorded. We assessed plasma fibrin clot properties, i.e. permeability (Ks) and clot lysis time (CLT), von Willebrand Factor (vWF) antigen, endogenous thrombin potential (ETP), proteins involved in thrombosis and fibrinolysis, as well as plasma carbonylated protein content (PC). RESULTS: SEC/LAAT was identified in 36 subjects (25.9 %) and was associated with heart failure (HF), AF duration, higher CHA2DS2VASc score, N-terminal prohormone of brain natriuretic peptide, and growth differentiation factor 15. Patients with SEC/LAAT had lower Ks (-15 %) and prolonged CLT (+19 %), along with higher fibrinogen (+24 %), ETP (+3 %), and plasminogen activator inhibitor-1 antigen (+16 %) compared with the remainder. Thrombin-activatable fibrinolysis inhibitor antigen, plasminogen, α2 - antiplasmin, and tissue plasminogen activator antigen were similar between the two groups. PC content was 50 % higher in SEC/LAAT and correlated with Ks (r = -0.47, p < 0.001) and CLT (r = 0.40, p < 0.001). On multivariate analysis, Ks, CLT, and PC levels, along with HF, remained independently associated with SEC/LAAT. CONCLUSIONS: We demonstrated a formation of denser and poorly lysable fibrin networks in AF patients with SEC/LAAT despite anticoagulation. We suggest that this phenomenon is in part related to enhanced oxidative stress.

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.

Laboratory Testing for Fibrinogen Disorders: From Routine Investigations to Research Studies.

Congenital and acquired fibrinogen disorders often have heterogeneous clinical phenotypes and are challenging from a laboratory perspective. Fibrinogen determination using the Clauss method remains the gold standard, while the reproducibility and significance of the thrombin time and the reptilase time are limited. Molecular testing for causative mutations in fibrinogen genes is now recommended to confirm the diagnosis of congenital fibrinogen disorders. Research assays are used to evaluate alterations to fibrin formation and properties of plasma and purified fibrinogen-derived clots, characterized by fiber thickness, the number of branches, and pore sizes. Fibrin clot permeability (permeation, porosity) using a hydrostatic pressure system represents the most commonly used method for evaluating fibrin network density. Reduced clot permeability, which denotes the reduced size of an average pore in the network, results in tighter fibrin networks, typically associated with impaired susceptibility to lysis, leading to a thrombotic tendency. Biophysical properties of fibrin clots are largely assessed using rheometry, with atomic force microscopy and nanorheology being increasingly used in disease states. Thromboelastography and thromboelastometry, a simple modification of rheometry, have been used, mainly in intensive care units, for more than 50 years. Given growing evidence for altered fibrin clot properties in diseases with elevated risk of venous and arterial thromboembolism and in some bleeding disorders, further work on standardization and validation of the assessment of fibrin clot characteristics is needed. This review summarizes the current methods used to evaluate fibrinogen abnormalities in both diagnostic and research laboratories.

Apixaban Versus Vitamin K Antagonists in Patients With Antiphospholipid Syndrome: A Cohort Study.

ABSTRACT: Current guidelines recommend that direct anticoagulants should not be used in prevention of recurrent thrombosis in patients with antiphospholipid syndrome (APS). However, except for triple-positive APS and rivaroxaban use, little evidence supports such recommendation. In a real-life cohort study, we evaluated the risk of thromboembolism and bleeding in patients with APS on apixaban versus vitamin K antagonists (VKA). We enrolled 152 patients with APS (aged 44 years [interquartile range 36-56], 83% women), including 66 patients treated with apixaban 5 mg bid and 86 with warfarin (target international normalized ratio [INR] 2-3). During a median follow-up of 53 months, we recorded venous thromboembolism, ischemic stroke, or myocardial infarction, along with major bleeding. We observed 4 thrombotic events (6.1%, 3 venous thromboembolism and 1 ischemic stroke) in patients on apixaban and 12 events (14%, 9 venous thromboembolism, 2 ischemic strokes and 1 myocardial infarction) in VKA patients. Patients with APS on apixaban had similar risk of recurrent thromboembolism compared with those on warfarin (hazard ratio [HR] = 0.327, 95% confidence interval [CI]: 0.104-1.035). Thromboembolic events occurred less commonly in statin users (8% vs. 50%, P = 0.01) and more frequently in triple-positive APS (50% vs. 22.1%, P = 0.028) and in patients with higher D-dimer at baseline ( P = 0.023); the latter difference was present in the apixaban group ( P = 0.02). Patients on apixaban had similar risk of major bleeding compared with warfarin (HR = 0.54, 95% CI: 0.201-1.448). In real-life patients with APS, apixaban appears to be similar to VKA for the prevention of thromboembolism and risk of bleeding, which might suggest that some patients with APS could be treated with apixaban.

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

BACKGROUND: Antiphospholipid antibodies (aPL), including lupus anticoagulant, antibodies against ß2 glycoprotein I (anti-ß2GPI), and anticardiolipin (aCL) antibodies are associated with ischemic stroke (IS). Their prevalence and clinical relevance in atrial fibrillation (AF) remain unclear. OBJECTIVES: To assess whether aPL are associated with increased risk of IS in AF patients despite anticoagulation. METHODS: We conducted a post hoc analysis of aPL using blood samples from 243 consecutive AF patients enrolled in a cohort study. Markers of a prothrombotic state, including endogenous thrombin potential, fibrin clot permeability, and lysis time, were measured at baseline. During a median follow-up of 52 months, IS/transient ischemic attack 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 lupus anticoagulant. aPL-positive patients were more likely to have prior stroke (P = .01) and be active smokers (P = .03), along with increased endogenous thrombin potential (P = .02), without any changes in fibrin clot properties. Anti-ß2GPI (hazard ratio, 4.38; 95% CI, 1.58-12.19) and aCL (hazard ratio, 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% per year). There were 23 major bleedings (2.1% per year) and 20 deaths (1.9% per year), which were not associated with aPLs. CONCLUSION: Our study showed a relatively high prevalence of aPL positivity in AF patients, which was linked to an increased risk of IS/transient ischemic attack. This suggests that screening for aPL might help optimize anticoagulant therapy in such patients.

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.