Reviewing the Rich History of Fibrin Clot Research with a Focus on Clinical Relevance.

Fibrin, described on a single-lens microscopy for the first time by Malpighi in 1666 and named by de Fourcroy, has been extensively studied by biochemists, biophysicists, and more recently by clinicians who recognized that fibrin is the major component of most thrombi. Elucidation of key reactions leading to fibrin clot formation in the 1950s and 1960s grew interest in the clinical relevance of altered fibrin characteristics. Implementation of scanning electron microscopy to image fibrin clots in 1947 and clot permeation studies in the 1970s to evaluate an average pore size enabled plasma clot characterization in cohorts of patients. Unfavorably altered fibrin clot structure was demonstrated by Blombäck's group in coronary artery disease in 1992 and in diabetes in 1996. Fifteen years ago, similar plasma fibrin clot alterations were reported in patients following venous thromboembolism. Multiple myeloma was the first malignant disease to be found to lead to abnormal fibrin clot phenotype in the 1970s. Apart from anticoagulant agents, in 1998, aspirin was first shown to increase fibrin clot permeability in cardiovascular patients. The current review presents key data on the rich history of fibrin research, in particular, those that first documented abnormal fibrin clot properties in a variety of human disease states, as well as factors affecting fibrin phenotype.

Impaired fibrinolysis in patients with atrial fibrillation and elevated circulating lipopolysaccharide.

It is unknown whether elevated gut-derived serum lipopolysaccharide (LPS) can affect thrombin generation, fibrinolysis, and fibrin clot properties in atrial fibrillation (AF). We aimed to evaluate associations of circulating LPS with prothrombotic markers in AF patients. A total of 157 (women, 57.3%) ambulatory anticoagulant-naïve AF patients aged from 42 to 86 years were recruited. Clinical data together with serum LPS, inflammation, endothelial injury, coagulation and fibrinolysis markers, including fibrin clot permeability (Ks) and clot lysis time (CLT), were analyzed. A median LPS concentration was 73.0 (58.0-100.0) pg/mL and it showed association with CLT (r = 0.31, p < 0.001) and plasminogen activator inhibitor-1 (PAI-1, r = 0.57, p < 0.001), but not other fibrinolysis proteins, thrombin generation, inflammatory markers, or Ks. There were weak associations of LPS with von Willebrand factor (vWF, r = 0.2, p = 0.013), cardiac troponin I (r = 0.16, p = 0.045), and growth differentiation factor-15 (r = 0.27, p < 0.001). No associations of LPS and CHA2DS2-VASc or other clinical variables were observed. Multivariable regression adjusted for potential confounders showed that serum LPS ≥ 100 pg/mL was an independent predictor of prolonged CLT. This study is the first to demonstrate antifibrinolytic effects of elevated LPS in AF patients largely driven by enhanced PAI-1 release.

The thrombotic risk in Cushing's syndrome-questions, answers, and the algorithm to consider in its assessment: part I-thrombotic risk not related to surgery.

Introduction: Recently, it has been reported that there is a great diversity in strategies used for thromboprophylaxis in patients with Cushing's syndrome (CS). An aim of this review was to discuss these practices in light of the existing data on the thrombotic risk in patients with CS and guidelines for medically ill patients. Methods: The four relevant topics and questions on thrombotic risk in CS were identified. The current guidelines on prevention and diagnosis of venous thromboembolism (VTE) were reviewed for the answers. An algorithm to consider in the assessment of the thrombotic risk in patients with CS was proposed. Results: To address both generic and CS-specific risk factors for VTE, the algorithm includes the stepwise approach consisting of Padua Score, urine free cortisol, and CS-VTE score, with no indication for routine thrombophilia testing in the prediction of an index VTE episode. Having confirmed VTE, selected patients require thrombophilia testing to aid the duration of anticoagulant treatment. The separate part of the algorithm is devoted to patients with ectopic adrenocorticotropic hormone syndrome in whom exclusion of VTE precedes introducing routine thromboprophylaxis to prevent VTE. The cancer-related VTE also prompts thromboprophylaxis, with the possible vessel invasion. The algorithm presents a unifactorial and multifactorial approach to exclude high-bleeding risks and safely introduce thromboprophylaxis with low-molecular-weight heparin. Summary: Our article is the first to present an algorithm to consider in the thrombotic risk assessment among patients with Cushing's syndrome as a starting point for a broader discussion in the environment. A plethora of factors affect the VTE risk in patients with CS, but no studies have conclusively evaluated the best thromboprophylaxis strategy so far. Future studies are needed to set standards of care.

Elevated factor XI is associated with recurrent left ventricular thrombus of unknown origin.

BACKGROUND: Elevated factor XI (FXI) has been shown to predispose to thromboembolism. We investigated whether it is associated with left ventricular thrombus (LVT) formation, its recurrence and subsequent thromboembolic events. METHODS: In 54 patients with prior LVT of unknown origin, who stopped anticoagulation and 54 controls, we determined FXI, along with plasma clot permeability (Ks ), fibrinolysis time (CLT), endogenous thrombin potential (ETP), von Willebrand factor (vWF) and fibrinolysis proteins. During follow-up, the primary endpoint involving the recurrence of LVT a symptomatic ischemic stroke or systemic embolism was recorded. RESULTS: Elevated (>120%) FXI levels were more often observed in LVT patients when compared to the control group (14 [25.9%] vs. 6 [11.1%], p = .048) in association with the presence of active FXI. FXI correlated with age (r = .406, p = .002), Ks (r = -.542, p < .001) and CLT (r = .406, p = .002), also after adjustment for age, but not with ETP, vWF or fibrinolysis proteins. During follow-up of 77.6 ± 18.5 months the primary endpoint occurred in 17 (31.5%) LVT patients, including 11 (20.4%) recurrent LVT, and in 4 (7.4%) controls (annual incidence rate 4.9% vs. 1.1%, respectively; p = .002). On multivariate logistic regression analysis, elevated FXI was independently associated with the primary endpoint (OR 1.18; 95% CI 1.09-1.28). CONCLUSIONS: Elevated FXI in association with a prothrombotic state characterizes patients with prior LVT of unknown origin and predisposes to its recurrence and/or ischemic stroke during follow-up. It might be speculated that the measurement of FXI helps identify patients who could benefit from prolonged anticoagulation and FXI inhibitors in the future.

Altered fibrin clot properties are associated with the progression of chronic kidney disease in atrial fibrillation.

INTRODUCTION: Formation of denser and resistant to lysis fibrin clot networks has been shown in chronic kidney disease (CKD) and atrial fibrillation (AF). We investigated whether such prothrombotic fibrin clot properties are associated with faster progression of CKD in AF patients. MATERIAL AND METHODS: We recruited 265 AF patients (men 49.1 %, median age of 64.0 years, median estimated glomerular filtration rate [eGFR] of 77.0 ml/min/1.73 m2), including 137 patients on non-vitamin K antagonist oral anticoagulants (NOACs) (51.7 %) and 109 patients (41.1 %) on vitamin K antagonists (VKAs). At baseline while off anticoagulation, we determined fibrin clot permeability (Ks), and clot lysis time (CLT), along with plasminogen activator inhibitor-1 (PAI-1), endogenous thrombin potential (ETP), and von Willebrand factor (vWF). The kidney function was assessed at baseline and after a median follow-up of 50.0 months. RESULTS: During follow-up, a median eGFR decreased by 8.0 (5.0-11.0) ml/min/1.73 m2, 1.8 ml/min/1.73 m2/year and this change correlated with age (R = 0.19, P = 0.002), Ks (R = 0.46, P < 0.0001), and CLT (R = -0.17, P = 0.005), but not ETP, fibrinogen, PAI-1 or vWF. A decrease in eGFR was lower in patients who used NOACs at baseline but not in those who started NOACs during follow-up (n = 101) as compared to the remaining patients. On multiple linear regression analysis, adjusted for age and fibrinogen, baseline Ks, eGFR, hypertension, and NOACs use independently predicted a decrease in eGFR. CONCLUSIONS: This study is the first to show that more compact fibrin clot networks may contribute to faster progression of CKD in AF, indicating novel kidney-related harmful effects of prothrombotic clot properties in humans.

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.

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.

Elevated factor XI is associated with increased risk of recurrent cerebral venous sinus thrombosis: a cohort study.

Cerebral venous sinus thrombosis (CVST) has no identified cause in 15% of cases. Elevated factors (F) VIII and FXI have been associated with thromboembolism, but data on CVST are limited. We hypothesized that elevated plasma FVIII and FXI predispose to first and recurrent CVST. In 50 CVST survivors aged < 60 years, following anticoagulant cessation and in 50 controls, we determined plasma FVIII and FXI, along with fibrin clot properties: lysis time, permeability, maximum D-dimer (D-Dmax), and maximum rate of D-dimer increase (D-Drate). We recorded CVST recurrence during a follow-up of 58.5 (55.0-60.0) months. Plasma FVIII was 22.7% higher in CVST than in controls, with elevated FVIII > 150% in 13 (26%) vs. 4 (8%) patients, respectively (p = 0.02). Median FXI tended to be higher in CVST vs. controls (110.5 [99.0-117-0]% vs. 104.5 [97.0-116.0]%, p = 0.07), while FXI > 120% was observed more commonly in the former group (12 [24%] vs. 4 [8%], respectively, p = 0.03). Patients with FVIII > 150% were less likely to achieve complete recanalization compared with the remainder (2 [15.4%] vs. 28 [75.7%], respectively; p < 0.001). Eight patients (16%) experienced CVST recurrence. They had higher baseline FXI, but not FVIII, as compared with the remainder (125.5 [114.5-140.0]% vs. 107.5 [102.0-117.0]%, respectively, p = 0.01). Patients with FXI > 120% were four times more likely to have recurrent CVST (5 [62.5%] vs. 7 [16.7%], respectively; p = 0.01). Plasma FXI > 120% could represent a novel risk factor for first and recurrent CVST. Given advances in anti-FXI agents, CVST might be another indication for this emerging treatment.

Effect of Three-Day Atorvastatin Administration on Coagulation Factors in Patients With Prior Venous Thromboembolism and Healthy Subjects: A Preliminary Study.

ABSTRACT: Statins exert antithrombotic effects, which might contribute to reduced risk of venous thromboembolism (VTE). Rosuvastatin 20 mg/d administered for 4 weeks has been reported to decrease coagulation factors (F) VII, FVIII, and FXI in VTE patients. Moreover, in accordance with recent registry data in non-VTE subjects, statins usage was associated with lower FXI. We investigated whether 3 doses of a statin decrease coagulation factors activity and if such changes can alter fibrin clot properties in VTE patients and healthy subjects. We enrolled 28 consecutive first-ever prior VTE patients after 6 months of anticoagulation and 25 healthy controls well-matched for demographics and lipid profiles (aged 44 [interquartile range 34-51] years) in an interventional nonrandomized study. Before and after 3 doses of atorvastatin 40 mg/d, activity of FVII, FVIII, FIX, and FXI was measured, along with fibrin clot properties, including permeability (Ks) and clot lysis using 3 various assays. After a 3-day statin administration, we observed the decrease of FVII (by 6.2%, P = 0.046) and FXI (by 8.6%, P = 0.044), irrespective of low-density lipoprotein cholesterol reduction (by 24%, P < 0.001), whereas other coagulation factors remained unaltered. Reduction of FVII and FXI activity was inversely correlated with Ks alterations (R = -0.292, P = 0.034 and R = -0.335, P = 0.014, respectively). After adjustment for age, studied group, and fibrinogen level, the reduction of FXI was independently associated with an increase of fibrin clot permeability (B = -0.084, P = 0.027). In conclusion, a 3-day 40 mg atorvastatin administration is sufficient to reduce FVII and FXI activity in our pilot study, which is associated with favorable fibrin clot properties modification.

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.

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.