Plasma Fibrin Clot Research Articles

Good metabolic control is associated with decreased circulating factor VIIa– antithrombin complexes in type 2 diabetes: a cross-sectional study

BackgroundDiabetes is associated with a prothrombotic state that contributes to cardiovascular (CV) events in type 2 diabetes (T2DM). Activated factor VII (FVIIa)– antithrombin (AT) complexes are indicative of tissue factor (TF) exposure and have been associated with thromboembolic risk in coronary artery disease. To our knowledge there have been no reports on FVIIa-AT complexes in T2DM, therefore we assessed factors that determine FVIIa-AT complexes in this disease and the impact of higher complexes on a prothrombotic state.MethodsIn 108 T2DM patients (mean age 63.8 years, 52.8% men, median HbA1c of 6.9 [interquartile range 6.1–8.2] %) and 83 age- and sex-matched non-diabetic subjects, we measured FVIIa-AT complexes. Metabolic control of T2DM involved fasting glucose, glycated hemoglobin (HbA1c), albumin/creatinine ratio (ACR), and lipid levels. To characterize a prothrombotic state, we determined thrombin generation parameters, fibrinolysis markers, and plasma fibrin clot properties.ResultsFVII-AT complexes in T2DM patients were similar to controls (73.6 [59.4–91.7] vs. 79.6 [59.2–97.1]pM, respectively, p = 0.30). The T2DM patients with FVIIa-AT in the top vs. the bottom quartile had a larger prevalence of active smoking and insulin use, along with higher fasting glucose (+ 36.4%), HbA1c (+ 27.4%), ACR (+ 72.8%), total cholesterol (+ 34.5%), and LDL-cholesterol (+ 80%). FVIIa-AT complexes showed no associations with in vitro thrombin generation potential, plasma fibrin clot properties, or fibrinolysis variables. On multivariable analysis HbA1c, ACR, and total cholesterol remained independently associated with FVIIa-AT complexes in T2DM.ConclusionsThis is the first study to show that in T2DM higher FVIIa-AT complexes are associated with markers of dyslipidemia and glycemia control, indicating that TF-induced coagulation activation could be suppressed by achieving treatment targets.Graphical abstract

Unfavorably Altered Fibrin Clot Phenotype in Women Following Postpartum Hemorrhage of Unknown Cause: Effect of Lower Coagulation Factors.

Increased clot permeability and susceptibility to lysis have been reported in women with heavy menstrual bleeding. We hypothesized that similar alterations in fibrin clot properties may also be present in women with postpartum hemorrhage (PPH) of unknown cause. To determine fibrin clot properties and their determinants in women after PPH of unknown cause. 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 (K s), and fibrinolysis efficiency were determined. Women with PPH showed reduced activity of FII (-10.3%), FV (-6.6%), FIX (-6.5%), FX (-7.2%), and FXI (-5.7%) compared with 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 (K s; +16.3%, p = 0.008) with lower maximum absorbance and shorter clot lysis time (CLT; -13.5%, p = 0.001) compared with the controls. On multivariable logistic regression, PPH was independently associated with higher C-reactive protein (per 1 mg/L, odds ratio [OR] = 1.70, 95% confidence interval [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 minute, OR = 0.94, 95% CI: 0.90-0.98). 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.

Reduced Plasma Selenoprotein P Is Associated With Type I Antithrombin Deficiency and a Prothrombotic State.

A positive association between antithrombin activity and selenium level was reported. Selenoprotein P, the most important selenium carrier, was identified within human plasma fibrin clots. To investigate the relationship between selenoprotein P and antithrombin and its role in modulation of fibrin clot properties in antithrombin-deficient patients. Proteomic analysis of plasma fibrin clots was performed with mass spectrometry. In 108 patients with genetically confirmed type I (57%) or type II (43%) antithrombin deficiency and in healthy controls (n = 50), we assessed plasma selenoprotein P levels and thiobarbituric acid-reactive substances by enzyme-linked immunosorbent assay, along with fibrin clot permeability, clot lysis time, and thrombin generation. Clot-bound antithrombin concentration was 0.46 ± 0.32 mg/g protein, while selenoprotein P level was 30-fold lower (0.015 ± 0.012 mg/g). Type I compared to type II antithrombin-deficient patients had higher clot-bound antithrombin and selenoprotein P levels (both P < .001), associated together (ρ = 0.93, P < .001). Individuals with type I compared to type II antithrombin deficiency or controls had about 40% lower plasma selenoprotein P levels (P < .001). In antithrombin-deficient patients, plasma selenoprotein P was associated with antithrombin antigen (ρ = 0.35, P < .001) and thiobarbituric acid-reactive substances (ρ = 0.42, P < .001). Plasma selenoprotein P correlated also with endogenous thrombin potential (r = -0.33, P < .001), fibrin clot permeability (r = 0.43, P < .001), and clot lysis time (r = -0.40, P < .001) in antithrombin-deficient patients but not in controls. Patients with type I antithrombin deficiency had higher clot-bound selenoprotein P and reduced plasma selenoprotein P levels. Plasma selenoprotein P was associated with prothrombotic fibrin clot phenotype and enhanced thrombin generation.

SERPINE1 and MTHFR genetic variants in patients with embolic stroke of undetermined source: links with fibrin clot properties.

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. 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. 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). 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.

Blood Lines: Intraspecific and Interspecific Variations in Anticoagulant Actions of Agkistrodon Viperid Venoms.

This study investigated the intraspecific and interspecific variability in the venom effects of Agkistrodon viperid snake species and subspecies (eleven venoms total) on plasma clotting times, fibrinogen levels, and fibrin clot strength. Significant delays in plasma clotting time were observed for A. conanti, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorus. Notably, the phylogenetically disjunct lineages A. conanti, A. contortrix mokasen, and A. howardgloydi exhibited the most potent anticoagulant effects, indicating the independent amplification of a basal trait. Inhibition assays with the activated clotting enzymes Factors XIa, IXa, Xa, and IIa (thrombin) revealed that FXa inhibition is another basal trait amplified independently on multiple occasions within the genus, but with A. howardgloydi, notably more potent than all others. Phospholipid degradation and zymogen destruction were identified as mechanisms underlying the variability in venom effects observed experimentally and in previous clinical reports. Thromboelastography demonstrated that the venoms did not clot fibrinogen directly but affected fibrin clot strength by damaging fibrinogen and that thrombin was subsequently only able to cleave into weak, unstable clots. The ability to activate Protein C, an endogenous anticoagulant enzyme, varied across species, with some venoms exceeding that of A. contortrix contortrix, which previously yielded the protein diagnostic agent Protac®. Phylogenetic analysis suggested that both fibrinogen degradation and Protein C activation were each amplified multiple times within the genus, albeit with negative correlation between these two modes of action. This study highlights the evolutionary, clinical, and biodiscovery implications of venom variability in the Agkistrodon species, underscoring their dynamic evolution, emphasising the need for tailored clinical approaches, and highlighting the potential for novel diagnostic and therapeutic developments inspired by the unique properties of snake venoms.

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

IntroductionSpontaneous 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. MethodsWe 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). ResultsSEC/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. ConclusionsWe 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.

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

BackgroundIntracerebral 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. MethodsWe 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. ResultsICH 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). ConclusionsWe 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.

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.

Impact of tissue factor expression and administration routes on thrombosis development induced by mesenchymal stem/stromal cell infusions: re-evaluating the dogma.

Hyperactive coagulation might cause dangerous complications such as portal vein thrombosis and pulmonary embolism after mesenchymal stem/stromal cell (MSC) therapy. Tissue factor (TF), an initiator of the extrinsic coagulation pathway, has been suggested as a predictor of this process. The expression of TF and other pro- and anticoagulant genes was analyzed in xeno- and serum-free manufactured MSCs. Furthermore, culture factors affecting its expression in MSCs were investigated. Finally, coagulation tests of fibrinogen, D-dimer, aPPTs, PTs, and TTs were measured in patient serum after umbilical cord (UC)-MSC infusions to challenge a potential connection between TF expression and MSC-induced coagulant activity. RESULTS: Xeno- and serum-free cultured adipose tissue and UC-derived MSCs expressed the highest level of TF, followed by those from dental pulp, and the lowest expression was observed in MSCs of bone marrow origin. Environmental factors such as cell density, hypoxia, and inflammation impact TF expression, so in vitro analysis might fail to reflect their in vivo behaviors. MSCs also expressed heterogeneous levels of the coagulant factor COL1A1 and surface phosphatidylserine and anticoagulant factors TFPI and PTGIR. MSCs of diverse origins induced fibrin clots in healthy plasma that were partially suppressed by an anti-TF inhibitory monoclonal antibody. Furthermore, human umbilical vein endothelial cells exhibited coagulant activity in vitro despite their negative expression of TF and COL1A1. Patients receiving intravenous UC-MSC infusion exhibited a transient increase in D-dimer serum concentration, while this remained stable in the group with intrathecal infusion. There was no correlation between TF expression and D-dimer or other coagulation indicators. The study suggests that TF cannot be used as a solid biomarker to predict MSC-induced hypercoagulation. Local administration, prophylactic intervention with anticoagulation drugs, and monitoring of coagulation indicators are useful to prevent thrombogenic events in patients receiving MSCs. Trial registration NCT05292625. Registered March 23, 2022, retrospectively registered, https://www. gov/ct2/show/NCT05292625?term=NCT05292625&draw=2&rank=1 . NCT04919135. Registered June 9, 2021, https://www. gov/ct2/show/NCT04919135?term=NCT04919135&draw=2&rank=1 .

Vasoinhibin's Apoptotic, Inflammatory, and Fibrinolytic Actions Are in a Motif Different From Its Antiangiogenic HGR Motif.

Vasoinhibin, a proteolytic fragment of the hormone prolactin, inhibits blood vessel growth (angiogenesis) and permeability, stimulates the apoptosis and inflammation of endothelial cells, and promotes fibrinolysis. The antiangiogenic and antivasopermeability properties of vasoinhibin were recently traced to the HGR motif located in residues 46 to 48 (H46-G47-R48), allowing the development of potent, orally active, HGR-containing vasoinhibin analogues for therapeutic use against angiogenesis-dependent diseases. However, whether the HGR motif is also responsible for the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin has not been addressed. Here, we report that HGR-containing analogues are devoid of these properties. Instead, the incubation of human umbilical vein endothelial cells with oligopeptides containing the sequence HNLSSEM, corresponding to residues 30 to 36 of vasoinhibin, induced apoptosis, nuclear translocation of NF-κB, expression of genes encoding leukocyte adhesion molecules (VCAM1 and ICAM1) and proinflammatory cytokines (IL1B, IL6, and TNF), and adhesion of peripheral blood leukocytes. Also, intravenous or intra-articular injection of HNLSSEM-containing oligopeptides induced the expression of Vcam1, Icam1, Il1b, Il6, and Tnf in the lung, liver, kidney, eye, and joints of mice and, like vasoinhibin, these oligopeptides promoted the lysis of plasma fibrin clots by binding to plasminogen activator inhibitor-1 (PAI-1). Moreover, the inhibition of PAI-1, urokinase plasminogen activator receptor, or NF-κB prevented the apoptotic and inflammatory actions. In conclusion, the functional properties of vasoinhibin are segregated into 2 different structural determinants. Because apoptotic, inflammatory, and fibrinolytic actions may be undesirable for antiangiogenic therapy, HGR-containing vasoinhibin analogues stand as selective and safe agents for targeting pathological angiogenesis.

Formation of denser plasma fibrin clots is associated with coronary artery ectasias and aneurysms - preliminary results from the CARE-ANEURYSM study

Abstract Background Coronary artery ectasias and aneurysms (CAEAs) are defined as diffused or localized coronary artery dilations, that exceed diameter of the adjacent segment by at least 50%. In the Caucasian population, CAEAs are present in 2-5% of all-comer patients undergoing coronary angiography and are associated with increased risk of myocardial infarction and death. Purpose To determine whether CAEAs are associated with increased thrombin generation potential and unfavorably modified plasma fibrin clot properties. Methods In this prospective study 19 patients with CAEAs, 19 sex-, age-, fibrinogen- and diabetes mellitus-matched patients with coronary artery disease (CAD) but without CAEAs, and 20 healthy control subjects were assessed. Plasma fibrin clot permeability (Ks) reflecting an average pore size within fibrin network, plasma fibrinolytic potential expressed as clot lysis time (CLT), and thrombin generation capacity defined as endogenous thrombin potential (ETP) were assessed. The CARE-ANEURYSM study, founded by the Polish National Science Centre (no. UMO-2020/39/O/NZ5/02863), was approved by the Ethical Committee and conducted in accordance with the Declaration of Helsinki. Patients provided written informed consent. Results As many as 84.2% of CAEA patients were men, mean age was 69.6 years, and 73.7% individuals had CAD. On average, CAEA affected 3 coronary segments and exceeded vessels referral diameter by 2.15-fold. Patients with CAEA had 46.5% reduced Ks (median, 3.98 [3.30-4.63] vs 7.43 [6.15-8.88] x10-9cm2, p&amp;lt;0.001), a tendency to prolonged CLT (112 [103-116.5] vs 99 [89-114] min, p=0.07), and 51% higher ETP (1685 [1502-1786] vs 1119 [1025-1189] nM×min, p&amp;lt;0.001) compared to healthy control (Fig. 1A-C). Moreover, individuals with CAEA compared to CAD patients had 19.5% lower Ks (median 3.98 [3.30-4.63] vs 5.33 [4.86-6.78] x10-9cm2, p=0.016) and 21% longer CLT (112 [103-116.50] vs 92.52 [81.50-97.50] min, p=0.015) and (1685 [1502-1786] vs 1119 [1693-1966] nM×min, p&amp;lt;0.001) (Fig. 1A-C). In the CAEA group, Ks was associated with the CAEA maximal diameter (R=-0.595, p=0.007) and the ratio of CAEA maximal diameter to referral vessel diameter (R=0.743, p&amp;lt;0.001; Figu.1D). There were no associations between the number of coronary artery segments affected by the lesion and fibrin clot properties or ETP. However, CAEA patients in whom the ratio of the CAEA maximal diameter to the referral vessel diameter exceed 2 (n=9) had 24.5% lower Ks (3.45 [1.88-3.68] vs 4.57 [4.08-5.35] x10-9cm2, p=0.004) and 14.7% higher ETP (1779 [1685-1812] vs 1515 [1431-1674] nM×min, p=0.028) with no difference in CLT (p&amp;gt;0.05). Conclusion CAEAs are associated with denser fibrin clot structure and enhanced thrombin generation, which indicate that CAEA patients are characterized by prothrombotic state. Further studies are needed to elucidate whether prothrombotic clot phenotype may be associated with increased risk of thromboembolic events in CAEA patients.Figure 1.

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

BackgroundLipopolysaccharide (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. MethodsWe 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. ResultsMedian 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. ConclusionsLow-grade endotoxemia is detectable in patients with acute PE and may contribute to increased thrombin generation and PAI-1-mediated hypofibrinolysis.

PB1076 Congenital Protein C Deficiency is Associated with Prothrombotic Plasma Fibrin Clot Phenotype

PB1076 Congenital Protein C Deficiency is Associated with Prothrombotic Plasma Fibrin Clot Phenotype

Increased factor XI but not factor XII is associated with enhanced inflammation and impaired fibrin clot properties in patients with eosinophilic granulomatosis with polyangiitis.

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. 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). 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). 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.

Plasma thiol levels and methylenetetrahydrofolate reductase gene c.665C>T and c.1286A>C variants affect fibrin clot properties in Polish venous thromboembolic patients.

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. 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. 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 >15μM (n=71, 18.3%), compared to patients with tHcy ≤15μM 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 >15μM compared to tHcy ≤15μM 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 >15μM, 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 ≤15μM (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). Our study indicates that patients with MTHFR variants and tHcy >15μM are characterized by elevated Cys and nitrotyrosine levels associated with prothrombotic fibrin clot properties.

Pharmacodynamics Monitoring of Emicizumab in Patients with Hemophilia A.

Emicizumab is a bispecific antibody mimicking coagulation factor VIII (FVIII) employed to treat patients with hemophilia A (PwHA) regardless of FVIII inhibitor status. The identification of biological markers reflecting the hemostatic competence of patients under emicizumab therapy would have a great clinical value. Unfortunately, emicizumab over-corrects standard coagulation assays, precluding their use for evaluating the hemostatic correction achieved in vivo. Here, we investigated whether global coagulation assays (GCA) would allow monitoring the biological response to non-factor replacement therapy with emicizumab. Six adults PwHA received a weekly dose of emicizumab of 3 mg/kg during weeks (W) 1 4 and 1.5 mg/kg from W5 onwards. Response to treatment was monitored weekly by emicizumab plasma concentration, thrombin generation (TG), and fibrin clot formation (FCF) and structure. TG and FCF results were compared to patient baseline, FVIII replacement, and healthy donors. TG and FCF significantly increased in PwHA after the loading period, reaching a plateau that lasted until the end of monitoring. Similarly, fibrin clot network became denser with thinner fibrin fibers. However, TG contrary to FCF remained at the lower limits of reference values. Remarkably, despite having similar plateau concentrations of emicizumab some patients showed markedly different degrees of TG and FCF improvement. Our study enriches the knowledge on the use of GCA to monitor non-factor replacement therapy, indicating that TG and FCF could act as direct markers of emicizumab biological activity. GCA allow to capture and visualize the individually variable response to emicizumab, leading a step forward to the personalization of patient treatment.

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

Neutrophil-activating peptide2 (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 β = -0.046, 95% CI -0.029; -0.064) and NAP-2 (per 100 ng/ml β = -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.

Deficiency of antithrombin increases risk of venous thromboembolism. We hypothesized that antithrombin deficiency affects fibrin clot structure and function. 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. 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 (β = - 6.1, 95% confidence interval [CI]: -1.7 to -10.5), while prolonged CLT was associated with lower antithrombin antigen (β = - 69.6, 95% CI: -9.6 to -129.7), activity (β = - 2.4, 95% CI: -0.3 to -4.5), higher PAI-1 (β = 12.1, 95% CI: 7.7-16.5), and thrombin-activatable fibrinolysis inhibitor levels (β = 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). 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.

Elevated factor XIa as a modulator of plasma fibrin clot properties in coronary artery disease.

Patients with coronary artery disease (CAD) display a prothrombotic fibrin clot phenotype, involving low permeability and resistance to lysis. The determinants of this phenotype remain elusive. Circulating tissue factor (TF) and activated factor XI (FXIa) are linked to arterial thromboembolism. We investigated whether detectable active TF and FXIa influence fibrin clot properties in CAD. In 118 CAD patients (median age 65 years, 78% men), we assessed Ks, an indicator of clot permeability, and clot lysis time (CLT) in plasma-based assays, along with the presence of active TF and FXIa. We also analysed proteins involved in clotting and thrombolysis, including fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and thrombin activatable thrombolysis inhibitor (TAFI). During a median 106 month (interquartile range 95-119) follow-up, myocardial infarction (MI), stroke, systemic thromboembolism (SE) and cardiovascular (CV) death were recorded. Circulating TF and FXIa, detected in 20.3% and 39.8% of patients, respectively, were associated with low Ks and prolonged CLT. Solely FXIa remained an independent predictor of low Ks and high CLT on multivariable analysis. Additionally, fibrinogen and PAI-1 were associated with low Ks, while PAI-1 and TAFI-with prolonged CLT. During follow-up low Ks and prolonged CLT increased the risk of MI and the latter also a composite endpoint of MI, stroke/SE or CV death. To our knowledge, this study is the first to show that circulating FXIa is associated with prothrombotic fibrin clot properties in CAD, suggesting additional mechanisms through which FXIa inhibitors could act as novel antithrombotic agents in CAD.

Effect of high-dose statin therapy on coagulation factors: Lowering of factor XI as a modifier of fibrin clot properties in coronary artery disease

BackgroundMultiple pleiotropic effects of statins include antithrombotic properties with formation of looser fibrin networks more susceptible to lysis. Recently, rosuvastatin 20 mg/d has been reported to decrease coagulation factors (F) VII, FVIII and FXI in venous thrombosis patients. ObjectivesWe investigated how high-dose statin therapy recommended in coronary artery disease (CAD) alters plasma levels of coagulation factors and if such changes might affect fibrin clot properties. MethodsWe studied 130 advanced CAD patients, who initially did not achieve the target low-density lipoprotein cholesterol (LDL-C). Before high-dose statin therapy (rosuvastatin 40 mg/d or atorvastatin 80 mg/d) and 6–12 months after its initiation, FII, FV, FVII, FVIII, FIX, FX, FXI and fibrinogen were assessed. We evaluated the impact of statin-induced alterations to the factors on plasma fibrin clot permeability (Ks) reflecting a fibrin pore size, and clot lysis time (CLT) reflecting fibrinolytic potential. ResultsAt baseline LDL-C (median 3.2, interquartile range 2.7–3.7 mmol/L) was independently associated solely with FXI (β = 0.58, P < 0.001). Median LDL-C reduction by 25% (P < 0.001) on high-dose statin treatment was accompanied by lowering of FVII, FVIII, and FXI (for all P < 0.001). On high-dose statin treatment, Ks (R = 0.65, P < 0.001) inversely associated with CRP (β = −0.41, P < 0.001), LDL-C (β = −0.26, P = 0.001), and FXI (β = −0.18, P = 0.016). In turn, CLT (R = 0.45, P < 0.001) was positively associated with LDL-C (β = 0.19, P = 0.043) and FXI (β = 0.17, P = 0.049). ConclusionsHigh-dose statin therapy in CAD patients decreases FVII, FVIII, and FXI. The statin-induced reduction in FXI may contribute to less prothrombotic fibrin clot phenotype, indicating additional antithrombotic effect of high-dose statins.