Fibrinogen conversion into insoluble fibrin and the formation of a stable clot is the final step of the coagulation cascade. Fibrin clot porosity and its susceptibility to plasmin-mediated lysis are the key fibrin measures, describing the properties of clots prepared ex vivo from citrated plasma. Cardiovascular disease (CVD), referring to coronary heart disease, heart failure, stroke, and hypertension, has been shown to be associated with the formation of dense fibrin networks that are relatively resistant to lysis. Denser fibrin mesh characterized acute patients at the onset of myocardial infarction or ischaemic stroke, while hypofibrinolysis has been identified as a persistent fibrin feature in patients following thrombotic events or in those with stable coronary artery disease. Traditional cardiovascular risk factors, such as smoking, diabetes mellitus, hyperlipidaemia, obesity, and hypertension, have also been linked with unfavourably altered fibrin clot properties, while some lifestyle modifications and pharmacological treatment, in particular statins and anticoagulants, may improve fibrin structure and function. Prospective studies have suggested that prothrombotic fibrin clot phenotype can predict cardiovascular events in short- and long-term follow-ups. Mutations and splice variants of the fibrinogen molecule that have been proved to be associated with thrombophilia or increased cardiovascular risk, along with fibrinogen post-translational modifications, prothrombotic state, inflammation, platelet activation, and neutrophil extracellular traps formation, contribute also to prothrombotic fibrin clot phenotype. Moreover, about 500 clot-bound proteins have been identified within plasma fibrin clots, including fibronectin, α2-antiplasmin, factor XIII, complement component C3, and histidine-rich glycoprotein. This review summarizes the current knowledge on the mechanisms underlying unfavourable fibrin clot properties and their implications in CVD and its thrombo-embolic manifestations.

Fibrin formed as a result of fibrinogen polymerization is the main protein component of a clot in a test tube and intravascular thrombi in vivo. Fibrin clot structure characterized by fiber diameter and pore size differs between healthy persons and those with thromboembolic diseases, in part due to the quality and quantity of fibrinogen and the magnitude of thrombin generation. A key measure of plasma clot structure is its permeability, reflected by the Darcy constant (Ks). Reduced Ks is a typical feature of the prothrombotic fibrin clot phenotype, which is associated with faster formation of denser fibrin mesh, relatively resistant to lysis. Low Ks has been reported in patients with prior or acute myocardial infarction (MI), stroke, or venous thromboembolism (encompassing deep vein thrombosis [DVT] and pulmonary embolism [PE]), as well as in those with prothrombotic conditions (eg, in several thrombophilic states) and in the presence of cardiovascular risk factors (eg, obesity). Antithrombotic and anticoagulant agents, along with statins, have been shown to increase Ks. Growing evidence indicates associations between the properties of plasma fibrin clots and morphology of intravascular thrombi in patients with MI. Recently, reduced Ks has been shown to predict recurrent thromboembolic episodes in patients with a history of stroke, PE, DVT, and their serious complications, including postthrombotic syndrome and thromboembolic pulmonary hypertension. We discuss the current evidence for the significance of clot density measured in vitro as a prognostic marker in a number of clinical conditions associated with elevated thromboembolic risk.

Objective—: Pulmonary embolism (PE) is a life-threatening manifestation of venous thromboembolism with a high recurrence rate after anticoagulation cessation. Recently, we have reported that prothrombotic clot phenotype in venous thromboembolism patients is associated with an increased risk of recurrent deep-vein thrombosis. Approach and Results—: We tested whether abnormal clot properties are predictive of recurrent PE. We investigated 156 consecutive white patients aged 18 to 65 years after the first-ever provoked or unprovoked PE (n=89), with or without deep-vein thrombosis. Plasma fibrin clot permeability (K _s ), turbidity measurements, calibrated automated thrombography, and efficiency of fibrinolysis using clot lysis time, maximum D-dimer levels, and rate of increase in D-dimer levels were evaluated at ≥3 months of anticoagulant therapy, at least 4 weeks since the anticoagulation withdrawal. The primary end point was recurrent PE during a median follow-up of 50 months. Recurrent PE was diagnosed in 23 (14.7%; 5%/yr) patients. Recurrent PE was associated with formation of denser fibrin networks reflected by lower K _s ( P =0.007) and impaired fibrinolysis, as evidenced by prolonged clot lysis time ( P =0.012) and reduced maximum rate of increase in D-dimer levels in the lysis assay ( P =0.004). Patients with recurrent PE had higher plasma D-dimer ( P <0.001) and thrombin peak ( P =0.007) compared with the remainder, whereas turbidity measurements and maximum D-dimer levels did not differ in the recurrence. Multivariate model showed that independent predictors of recurrent PE were female sex, unprovoked venous thromboembolism, higher plasma D-dimer, reduced K _s , and reduced maximum rate of increase in D-dimer levels in the lysis assay (all P <0.05). Conclusions—: Altered fibrin clot properties including formation of more compact clots displaying impaired susceptibility to lysis may predispose to recurrent PE.

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 HbA_1c ≥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 HbA_1c, 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 HbA_1c and fructosamine levels but also with AVA and transvalvular gradient, indicating that strict long-term glycaemic control is needed in AS patients with concomitant type 2 diabetes. This study suggests that maintaining these variables within the normal range may slow the rate of AS progression. Graphical