Fibrin clot properties in acute ischaemic stroke (AIS) are unfavourably altered, including faster formation of denser and poorly lysable fibre networks. We investigated clot properties in AIS patients treated with recombinant tissue plasminogen activator (rtPA) and their impact on clinical outcome. In 74 consecutive AIS patients eligible for rtPA treatment, we assessed ex vivo plasma fibrin clot formation, permeability (K_s), and rtPA-induced lysis, along with peak thrombin generation, fibrinolysis proteins and inhibitors at three time points – on admission, after 24 hours and 3 months since stroke. Clinical outcome was assessed using the NIHSS and mRS scores. Compared with the pretreatment values, fibrin networks assessed 24 hours since thrombolysis were formed more slowly (+20.5 % lag phase on turbidimetry), were less compact (+36.9 % K_s), composed of thinner fibres (-10.6 % lower maximum absorbancy [ΔAb]), which were lysed more rapidly (-20.8 % clot lysis time [CLT] and +7.1 % the rate of rtPA-induced D-dimer release from clots [D-D_rate]). Thrombin generation and fibrinolysis proteins remained elevated. Lower ΔAb (<0.86 at 405 nm), shorter CLT (0.072 mg/l/min) assessed at baseline predicted good outcome (mRS 0–2) at 3 months after adjustment for age and fibrinogen. Logistic regression adjusted for potential confounders showed that good outcome at 3 months was predicted by pretreatment D-D_rate, while pretreatment CLT predicted excellent outcome (mRS of 0–1). In conclusion, formation of denser fibrin clots displaying impaired lysability and pattern of their changes induced by thrombolysis may affect clinical outcome in AIS patients.

It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein—fibrinogen and fibrin that forms a plasma clot. The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC–MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC–MS/MS. The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.

We sought to investigate whether the G20210A prothrombin mutation modifies plasma fibrin clot properties in patients after venous thromboembolism (VTE) and how rivaroxaban treatment affects these alterations. We studied 34 prothrombin mutation heterozygous carriers and sex- and age-matched 34 non-carriers, all at least three months since the first VTE episode, before and during treatment with rivaroxaban. Clot permeability (K_s) and clot lysis time (CLT) with or without elimination of thrombin activatable fibrinolysis inhibitor (TAFI) were assessed at baseline, 2–6 hours (h) after and 20–25 h after intake of rivaroxaban (20 mg/day). At baseline, the prothrombin mutation group formed denser clots (K_s −12 %, p=0.0006) and had impaired fibrinolysis (CLT +14 %, p=0.004, and CLT-TAFI +13 %, p=0.03) compared with the no mutation group and were similar to those observed in 15 healthy unrelated prothrombin mutation carriers. The G20210A prothrombin mutation was the independent predictor for K_s and CLT before rivaroxaban intake. At 2–6 h after rivaroxaban intake, clot properties improved in both G20210A carriers and non-carriers (K_s +38 %, and +37 %, CLT −25 % and −25 %, CLT-TAFI −20 % and −24 %, respectively, all p<0.001), but those parameters were worse in the prothrombin mutation group (K_s −12.8 %, CLT +17 %, CLT-TAFI +13 %, all p<0.001). Rivaroxaban concentration correlated with fibrin clot properties. After 20–25 h since rivaroxaban intake most clot properties returned to baseline. Rivaroxaban-related differences in clot structure were confirmed by scanning electron microscopy images. In conclusion, rivaroxaban treatment, though improves fibrin clot properties, cannot abolish more prothrombotic fibrin clot phenotype observed in prothrombin mutation carriers following VTE.

Background and Purpose— Venous thromboembolism and ischemic stroke are associated with unfavorable fibrin clot structure and function. We hypothesized that denser fibrin networks displaying impaired lysability characterize patients with cerebral venous sinus thrombosis (CVST). Methods— We assessed plasma fibrin clot properties in 50 patients (aged 38.9±9.8 years, 36 women) after the first CVST unrelated to trauma or malignancy after anticoagulation withdrawal and 50 well-matched controls. Recurrences were recorded during follow-up (18–46; median, 36 months). Results— Clot permeability was lower in patients with CVST than in controls (K_s, 6.43±0.97 versus 7.3±1.2 10⁻⁹ cm²; P<0.001) and was associated with prolonged clot lysis time (103.0±16.8 versus 92.4±16.2 minutes; P<0.001), lower maximum rate of D-dimer release from clots (0.068 [0.064–0.071] versus 0.072 [0.067–0.078] mg/L per minute; P<0.001) and higher maximum D-dimer levels in the lysis assay (4.39±0.56 versus 4.19±0.46 mg/L, respectively; P=0.03). Patients with CVST had a slightly shorter lag phase (P=0.02) and higher maximum absorbance of fibrin gels on turbidimetry (P<0.001) compared with controls. Deficiencies in natural anticoagulants or antiphospholipid syndrome, and factor V Leiden occurred more often in the patients (P4.83 mg/L) predicted CVST recurrence (odds ratio, 5.1; 95% confidence interval, 1.63–16.19) after adjustment for fibrinogen. Conclusions— CVST is associated with the formation of more compact plasma fibrin clots and resistance to fibrinolysis, which may predispose to the recurrence.

Aortic stenosis (AS) has been associated with impaired fibrinolysis and increased oxidative stress. This study aimed to investigate whether oxidative stress could alter fibrin clot properties in AS. We studied 173 non-diabetic patients, aged 51–79 years, with isolated AS. We measured plasma protein carbonylation (PC) and thiobarbituric acid reactive substances (TBARS), along with plasma clot permeability (K_s), thrombin generation, and fibrinolytic efficiency, which were evaluated by two assays: clot lysis time (CLT) and lysis time (Lys50). Coagulation factors and fibrinolytic proteins were also determined. Plasma PC showed an association with AS severity, reflected by the aortic valve area and the mean and maximum aortic gradients. Plasma PC was positively correlated with CLT, Lys50, plasminogen activator inhibitor-1 (PAI-1), and tissue factor (TF) antigens. TBARS were positively correlated with maximum aortic gradient, Lys50, and TF antigen. Regression analysis showed that PC predicted prolonged CLT (>104 min; odds ratio (OR) 6.41, 95% confidence interval (CI) 2.58–17.83, p < 0.001) and Lys50 (>565 s; OR 5.83, 95% CI 2.23–15.21, p < 0.001). Multivariate regression analysis showed that mean aortic gradient, PC, α2-antiplasmin, PAI-1, and triglycerides were predictors of prolonged CLT, while PC, α2-antiplasmin, and fibrinogen were predictors of Lys50. Our findings suggest that elevated oxidative stress contributes to impaired fibrinolysis in AS and is associated with AS severity.

Little is known about factors that affect the composition of contracted blood clots in specific diseases. We investigated the content of polyhedral erythrocytes (polyhedrocytes) formed in blood clots and its determinants in type 2 diabetes (T2D) patients. In 97 patients with long-standing T2D [median HbA1c, 6.4% (interquartile range 5.9–7.8)], we measured in vitro the composition of blood clots, including a clot area covered by polyhedrocytes using scanning electron microscopy and the erythrocyte compression index (ECI), defined as a ratio of the mean polyhedrocyte area to the mean native erythrocyte area. Moreover, plasma fibrin clot permeability (Ks), clot lysis time (CLT), thrombin generation, oxidative stress [total protein carbonyl (total PC), total antioxidant capacity and thiobarbituric acid reactive substances (TBARS)], and platelet activation markers were determined. The impact of glucose concentration on polyhedrocytes formation was assessed in vitro. Polyhedrocytes content in contracted clots was positively correlated with glucose (r = 0.24, p = 0.028), glycated hemoglobin (r = 0.40, p = 0.024), total cholesterol (r = 0.22, p = 0.044), TBARS (r = 0.60, p = 0.0027), P-selectin (r = 0.54, p = 0.0078) and platelet factor-4, PF4 (r = 0.59, p = 0.0032), but not with thrombin generation, platelet count, Ks or CLT. Patients who formed more polyhedrocytes (≥ 10th percentile) (n = 83, 85.6%) had higher glucose (+ 15.7%, p = 0.018), fibrinogen (+ 16.6%, p = 0.004), lower red blood cell distribution width (RDW, − 8.8%, p = 0.034), reduced plasma clot density (− 21.8% Ks, p = 0.011) and impaired fibrinolysis (+ 6.5% CLT, p = 0.037) when compared to patients with lesser amount of polyhedrocytes (< 10th percentile). ECI and the content of polyhedrocytes were strongly associated with total PC (r = 0.79, p = 0.036 and r = 0.67, p = 0.0004, respectively). In vitro an increase of glucose concentration by 10 mmol/L was associated with 94% higher polyhedrocytes content (p = 0.033) when compared to the baseline (7.1 mM). After adjustment for age, sex and fibrinogen, multiple regression analysis showed that RDW was the only independent predictor of polyhedrocytes content in T2D (OR = 0.61, 95% CI 0.39–0.92). Poor glycemic control, together with enhanced platelet activation and oxidative stress, increase the content of polyhedrocytes in blood clots generated in T2D patients.