Plasma Fibrin Clot Properties Research Articles

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.

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.

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.

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.

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.

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.

Therapy with high-dose statins reduces soluble P-selectin: The impact on plasma fibrin clot properties

ObjectiveStudies on the effect of statins on platelets in patients with coronary artery disease (CAD) yielded inconsistent results. We sought to investigate whether high-dose statin therapy reduces plasma concentrations of soluble P-selectin (sP-selectin), a well-established platelet activation marker and if such changes can affect fibrin clot properties, which are unfavorably altered in CAD patients. MethodsWe studied 130 consecutive patients with advanced CAD who did not achieve the target LDL cholesterol on statins. At baseline and after 6–12 months of treatment with atorvastatin 80 mg/day or rosuvastatin 40 mg/day, soluble plasma sP-selectin, along with plasma fibrin clot permeability (Ks), clot lysis time (CLT), thrombin generation and fibrinolysis proteins were determined. ResultsBefore high-intensity statin treatment, lower Ks and longer CLT values were associated with increased sP-selectin (β −0.27 [95% CI -0.44 to −0.10] and β 0.21 [95% CI 0.01 to 0.41]; both p < 0.05, respectively) also after adjustment for potential confounders. sP-selectin, alongside fibrin features and other variables at baseline showed no association with lipid profile. On high-dose statin therapy, there was 32% reduction in sP-selectin levels (p < 0.001). On-treatment change (Δ) in sP-selectin correlated with ΔKs and ΔCLT (r = −0.32, p < 0.001 and r = 0.22, p = 0.011, respectively), but not with cholesterol and C-reactive protein lowering. We did not observe any associations between post-treatment sP-selectin levels and lipids, fibrin clot properties or thrombin generation. ConclusionsHigh-dose statin therapy reduces markedly sP-selectin levels in association with improved fibrin clot phenotype, which highlights the contribution of platelet-derived proteins to a prothrombotic state in hypercholesterolemia and statin-induced antithrombotic effects.

GnRH Antagonist Protocol Enhances Coagulation During Controlled Ovarian Stimulation for IVF.

Changes in coagulation and fibrinolysis have been reported in women undergoing controlled ovarian hyperstimulation (COH) supporting a potential hypercoagulable condition related to this treatment. This study aimed at evaluating the changes in fibrin clot properties and thrombin generation induced by two different COH protocols: long with gonadotropin-releasing hormone agonist (GnRH-a) and GnRH antagonist (GnRH-ant). Primary outcomes included determination of plasma fibrin clot properties, including clot permeability (Ks) and efficiency of fibrinolysis using clot lysis time (CLT), along with thrombin generation (prothrombin fragments 1 + 2) and endogenous thrombin potential (ETP) and fibrinolysis inhibitor levels. One hundred twenty-nine infertile women were included in the final analysis. The GnRH-ant protocol resulted in increased ETP (+ 9.8%) and reduced Ks (- 2.4%). Conversely, COH with the GnRH-a protocol reduced thrombin generation by decreasing both ETP (- 6.6%) and F1 + 2 (- 30.8%) together with favorably altered fibrin clot properties represented by increased Ks (+ 21.7%) and reduced CLT (- 13.8%) as well as decreased PAI-1 levels (by 2.5 times). The GnRH-ant compared to the GnRH-a protocol increased PAI-1 levels (+ 77.3%), thrombin generation (9.3% higher ETP), and Ks (+ 13.7%). In the GnRH-a group, post-COH Ks was 14.3% higher (Ks ≥ 7.92 × 10-9 cm2) in women with positive vs. negative pregnancy outcomes. Our results show that the GnRH-ant protocol enhanced thrombin generation and slightly decreased fibrin clot density. COH with the GnRH-a reduced thrombin generation and improved fibrin clot features. This trial was registered (NCT04166825). Clinical Trial Registration Number: NCT04166825.

Fibrinogen β chain and FXIII polymorphisms affect fibrin clot properties in acute pulmonary embolism.

Prothrombotic fibrin clot properties, including increased clot density, are in part genetically determined. We investigated whether fibrinogen alpha-chain gene (FGA) c.991A>G (rs6050), fibrinogen beta chain gene (FGB) -455G>A (rs1800790) and factor XIII gene (F13) c.103G>T (rs5985) polymorphisms affect plasma fibrin clot properties in patients with acute pulmonary embolism (PE). As many as 126 normotensive patients with PE, free of cancer, were genotyped by TaqMan assay. Fibrin clot permeability (Ks ), clot lysis time (CLT) and endogenous thrombin potential (ETP) were assessed on admission. The minor allele frequencies were as follows: FGA rs6050 (n=62, 0.31), FGB rs1800790 (n=40, 0.17) and F13 rs5985 (n=49, 0.23). There were no differences related to any of the polymorphisms with regard to demographic, clinical and laboratory data, except for fibrinogen concentration, which was higher in carriers of F13 rs5985 polymorphism (p=.024), and PE combined with deep-vein thrombosis, which was less prevalent in FGB rs1800790 polymorphism carriers (p=.004). Carriers of FGB rs1800790 A allele and F13 rs5985T allele had lower Ks , prolonged CLT and higher ETP compared with major homozygotes (all p<.05). After adjustment for fibrinogen, all differences remained significant (all p<.01). There were no associations between the FGA rs6050 polymorphism and Ks , CLT or ETP. Our study showed that FGB rs1800790 and F13 rs5985 polymorphisms contribute to the prothrombotic fibrin clot phenotype and these effects are strong enough to be observed in the acute phase of PE.

Certain Associations Between Iron Biomarkers and Total and γ' Fibrinogen and Plasma Clot Properties Are Mediated by Fibrinogen Genotypes.

Introduction: Evidence for the relationship between body iron and cardiovascular disease (CVD) is inconsistent and mechanisms involved remain poorly understood. Therefore, we first investigated whether there are linear or non-linear relationships between iron status and total and γ' fibrinogen as well as plasma fibrin clot properties and, second, determined whether there are interactions with iron biomarkers and fibrinogen and FXIII single nucleotide polymorphisms (SNPs) in relation to fibrinogen concentration and functionality.Methods: In this cross-sectional analysis of 2,010 apparently healthy Black South Africans we quantified total and γ' fibrinogen, serum iron, ferritin and transferrin using standardized methods and calculated transferrin saturation (TS). Clot architecture and lysis were explored with a global analytical turbidity assay. The SNPs were determined through an Illumina BeadXpress® platform.Results: Total, but not %γ', fibrinogen negatively correlated with serum iron concentrations, although both decreased over iron tertiles. %γ' fibrinogen correlated negatively with transferrin and decreased over the transferrin tertiles. A weak negative association between total fibrinogen and TS was detected with fibrinogen decreasing over the TS tertiles and categories based on TS. Lag time correlated positively with transferrin and increased over transferrin tertiles, when adjusting for fibrinogen. Before adjusting for fibrinogen, lag time was shorter in those with adequate iron status based on TS than other iron subcategories. Clot lysis time (CLT) negatively correlated with ferritin and was longer in the first than in the third ferritin tertile. Among iron status categories based on ferritin, only CLT differed and was longer in those with adequate iron than with iron-overload. CLT negatively correlated with TS, albeit weakly, shortened over the TS tertiles and was shorter in those with adequate iron based on TS categories. Interactions were observed between FGB SNPs and some of the markers of iron status investigated, in relation to the clot properties with the most prominent associations detected in homozygous carriers of the variant alleles for whom increased iron status was more beneficial than for those harboring the wild-type alleles. Iron modulated the influence of the SNPs so that for the majority iron was beneficial in respect of clot properties, but even more so for a minority group harboring specific variant alleles.Conclusion: This is the first large-scale epidemiological study to relate fibrinogen concentration and functionality to markers of iron status and to take genetic factors into consideration. We have detected a relationship between iron biomarkers and fibrinogen as well as clot characteristics that are influenced by the genetic make-up of an individual.

Plasma fibrin clot properties and cardiovascular mortality in patients with type 2 diabetes: a long\u2010term follow\u2010up study

BackgroundPatients with type 2 diabetes mellitus (T2DM) are at high risk of cardiovascular mortality, but the mechanisms behind this remain unclear. Prothrombotic fibrin clot properties have been shown in T2DM and cardiovascular disease. We hypothesized that formation of denser clots, which are resistant to fibrinolysis, has a negative impact on cardiovascular mortality in T2DM.MethodsWe studied 133 T2DM patients aged 43–83 years. Plasma fibrin clot turbidity, permeation, compaction, and efficiency of clot lysis using 3 assays including the determination of maximum concentration (D-Dmax) and rate of increase in D-dimer concentration (D-Drate) released during tissue plasminogen activator-induced degradation, were evaluated at the time of enrollment, along with thrombin generation and fibrinolytic proteins. During a median follow-up period of 72 months, cardiovascular mortality was recorded.ResultsCardiovascular deaths (n = 16, 12%) occurred more frequently in patients with increased D-Dmax (> 4.26 mg/l, hazard ratio [HR] 5.43, 95% confidence interval [CI] 1.99–14.79), or decreased D-Drate (< 0.07 mg/l/min, HR 2.97, 95% CI 1.07–8.23), or increased peak thrombin (> 283.5 nM, HR 5.65, 95% CI 2.07–15.51). These predictors had an even more potent impact on cardiovascular mortality in patients with prior cardiovascular disease (64.7%) and with corresponding risks as follows: HR 6.18, 95% CI 2.02–18.96; HR 8.98, 95% CI 2.99–26.96; and HR 5.35, 95% CI 1.62–17.72, respectively. Other investigated fibrin variables and fibrinolytic proteins did not associate with cardiovascular mortality. In multivariable analysis, cardiovascular mortality was predicted by D-Dmax > 4.26 mg/l, age > 65 years, prior cardiovascular disease, and C-reactive protein > 3 mg/l.ConclusionsThis study is the first to show that formation of denser fibrin clots resistant to fibrinolysis could be a risk factor for long-term cardiovascular mortality in T2DM.

Effect of enoxaparin on plasma fibrin clot properties and fibrin structure in patients with acute pulmonary embolism

BackgroundLow-molecular-weight heparins (LMWHs) influence the fibrin network structure in in vitro models. There have been no reports on LMWH-induced modifications of fibrin clot characteristics and their determinants in acute pulmonary embolism (PE). AimWe investigated how enoxaparin alters fibrin clot properties in acute PE patients. MethodsClots were generated from plasma of 46 acute PE patients, aged 47-77 years treated with enoxaparin 1 mg/kg bid. Fibrin clot permeability (Ks) and clot lysis time (CLT), along with coagulation and fibrinolysis proteins were determined. Plasma fibrin clot nanostructure was assessed using scanning electron microscopy (SEM). ResultsBoth Ks and CLT were associated with anti-factor (F)Xa activity (r = 0.75, p < 0.0001 and r = −0.37, p = 0.011). Anti-FXa was positively associated with fibrin fiber diameter and the pore area, and inversely with fibrin fiber density on SEM images. Multiple regression analysis adjusted for age, body-mass index, and fibrinogen levels showed that anti-FXa activity, antithrombin activity, and FVIII activity determined Ks, while anti-FXa activity, plasminogen activator inhibitor-1 level, and presence of right ventricular dysfunction determined CLT. ConclusionsWe identified new laboratory and clinical factors contributing to prothrombotic plasma fibrin clot characteristics during enoxaparin treatment, which might help elucidate mechanisms underlying therapy failure in patients with acute PE.

Effects of direct oral anticoagulants on thromboelastographic parameters and fibrin clot properties in patients with venous thromboembolism.

Data concerning the impact of direct oral anticoagulants (DOACs) on the thromboelastography (TEG) indices in venousthromboembolism (VTE) patients are limited. The goal of this study was to compare the impact of DOACs on clot properties measured in whole blood using TEG kaolin test and in plasma obtained from real-life VTE patients. We assessed 53 patients, including 20 on rivaroxaban, 20 on apixaban, and 13 on dabigatran. Using the TEG® 5000, coagulation status was evaluated in whole blood samples, while plasma fibrin clot permeability (Ks) and its susceptibility to lysis (CLT) were assessed in citrated plasma. Plasma concentrations of rivaroxaban (99 [48 - 311] ng/ml) and apixaban (85 [40 - 105] ng/ml) were positively associated with Ks and inversely with CLT, while dabigatran concentrations (71 [39 - 98] ng/ml) correlated positively with Ks. Moreover, Ks was associated with clot formation times (R and K), time to maximum clot strength (TMA), coagulation index (CI) reflecting the overall coagulation status, and whole blood clot lysis time (TEG-CLT). Blood clot lysis index (CL30) was associated with plasma CLT in all patients on DOACs, while TMA correlated with CLT only in patients on apixaban. Higher DOAC concentrations were associated with longer retardation of whole blood clot formation and longer time needed to reach maximum level of its strength as well as with more permeable clots. We concluded that plasma fibrin clot properties correlate with corresponding TEG indices suggesting that TEG might be helpful in providing prognostic information about DOAC influence in VTE patients.

Plasma Fibrin Clot Properties as Determinants of Bleeding Time in Human Subjects: Association with Histidine-Rich Glycoprotein.

Aims Fibrin formation and histidine-rich glycoprotein (HRG) are involved in primary hemostasis and wound healing. Little is known regarding the relationship of clot characteristics, bleeding time, and wound healing. Methods and Results We studied 154 patients with coronary artery disease (CAD) and 154 subjects free of CAD matched for age, obesity, and current smoking. We evaluated bleeding time (BT) using standardized skin incisions on a forearm, along with plasma clot permeability (Ks), clot lysis time (CLT), and histidine-rich glycoprotein (HRG). Compared with controls, BT was 45% shorter in CAD cases. CAD patients had 32% lower Ks), clot lysis time (CLT), and histidine-rich glycoprotein (HRG). Compared with controls, BT was 45% shorter in CAD cases. CAD patients had 32% lower p < 0.001). After adjusting for potential confounders, Ks), clot lysis time (CLT), and histidine-rich glycoprotein (HRG). Compared with controls, BT was 45% shorter in CAD cases. CAD patients had 32% lower n = 79, 25.6%) was independently predicted by both short and prolonged BT in CAD cases (OR 21.87, 95% CI 7.41-64.55 and OR 10.17, 95% CI 2.88-35.97) and controls (OR 5.94, 95% CI 2.29-15.41 and OR 14.76, 95% CI 4.29-50.77, respectively). Conclusions The study shows that plasma fibrin clot density and HRG may influence BT and that appropriate skin wound healing is associated with medium BT. Translational Perspective. Elucidation of the complex relationships between plasma fibrin clot phenotype and wound healing might have important practical implications.

A prothrombotic state and denser clot formation in patients following acute limb ischemia of unknown cause

IntroductionFibrin clot structure differs between healthy individuals and those following thromboembolic events. Dense and poorly lysable fibrin clots have also been reported in peripheral artery disease. We studied fibrin clot properties and its determinants in individuals with a history of acute lower limb ischemia (ALI) of unknown cause. Materials and methodsIn this case-control study, we enrolled 43 patients who experienced ALI of unknown cause, and two age-and sex-matched reference groups: (1) patients with cryptogenic non-lacunar stroke (n = 43) and (2) individuals without any history of thromboembolism (n = 43, control group). Plasma fibrin clot properties, along with thrombin generation and fibrinolysis markers were assessed following ≥3 months of anticoagulation. ResultsCompared with the control group, the ALI group exhibited more compact plasma fibrin clots (13.4% lower permeability [Ks], p = .001), decreased formed clot lysis (12.5% lower D-Drate, p = .001) and unaltered clot lysis potential, along with enhanced thrombin generation potential (49% higher peak thrombin concentration, p < .0001). There were no differences in these variables between ALI and stroke patients. Patients with ALI had slightly higher α2-antiplasmin and lower plasminogen activator inhibitor 1 levels compared with the stroke and control groups (all p < .01). ConclusionsPatients who experienced ALI of unknown cause display a prothrombotic fibrin clot phenotype, including increased clot density and hypofibrinolysis associated with higher thrombin generation, which might suggest potential benefits from prolonged anticoagulation in this disease.

Plasma Fibrin Clot Properties Are Unfavorably Altered in Women following Venous Thromboembolism Associated with Combined Hormonal Contraception.

The use of hormonal contraception is associated with an increased risk of venous thromboembolism (VTE). Unfavorably altered fibrin clot phenotype has been reported in patients following unprovoked VTE who are at risk of recurrences. It remains unknown whether fibrin clot characteristics in women with contraception-related VTE differ from those in unprovoked VTE. We studied three age-matched groups of women: (1) after contraception-related VTE, (n = 48) (2) after unprovoked VTE (n = 48), and (3) controls (n = 48). Plasma fibrin clot permeability (Ks), turbidity of clot formation, efficiency of fibrinolysis using clot lysis time (CLT), and rate of increase in D-dimer during lytic clot degradation (D-Drate), along with thrombin generation and fibrinolysis proteins were determined. Compared with the controls, patients following contraception-related and unprovoked VTE formed faster (lag phase, -8.8% and -20.4%, respectively) fibrin clots of increased density (Ks, -8.6% and -13.4%, respectively) displaying impaired fibrinolysis as evidenced by prolonged CLT (+11.5% and +14.5%, respectively) and lower D-Drate (-7.1% and -5.6%, respectively), accompanied with higher plasminogen activator inhibitor-1 (PAI-1, +14.9% and +17.8%, respectively) and elevated peak thrombin generation (+63.8% and +36.7%, respectively). The only differences between women with unprovoked and contraception-related VTE were lower fibrin mass in plasma clots (D-Dmax, -8.6%), along with higher peak thrombin generation (+19.8%) and shorter lag phase (-6.8%) in the latter group. This study suggests that women after contraception-related VTE, similar to those following unprovoked VTE, have denser fibrin clot formation and impaired clot lysis. These findings might imply higher risk of VTE recurrence in women with the prothrombotic clot phenotype.

Antiphosphatidylserine/prothrombin complex antibodies as a determinant of prothrombotic plasma fibrin clot properties in patients with antiphospholipid syndrome

BackgroundAntiphosphatidylserine/prothrombin complex (aPS/PT) antibodies are recognized as a marker for antiphospholipid syndrome (APS). Dense and poorly lysable fibrin clots occur in thrombotic APS. Compact clots predict thromboembolism, but determinants of the unfavorable clot phenotype remain unknown in APS. We hypothesized that elevated aPS/PT antibodies determine unfavorable clot features. MethodsIn a cohort study involving 124 consecutive patients with thrombotic APS, we measured at baseline plasma fibrin clot permeability (Ks), efficiency of fibrinolysis (clot lysis time, CLT), and turbidity (off anticoagulation) along with immunoglobulin (Ig)G/IgM aPS/PT. During follow‐up, symptomatic thromboembolic events were recorded. ResultsElevated IgG and IgM aPS/PT antibodies >30 international enzyme units (UI) were detected in 54.8% and 42.7% of APS patients, including 76.2% and 54% of lupus anticoagulant‐ (LA, n = 63) positive patients, respectively. Elevated IgG and IgM aPS/PT antibodies predicted low Ks (lower quartile, <6 × 10−9 cm2; odds ratio [OR] = 5.93, 95% confidence interval [CI] 2.09‐16.82 and OR = 11.79, 95% CI 4.10‐33.92) and prolonged CLT (top quartile, ≥116 min; OR = 4.85, 95% CI 2.42‐25.07 and OR = 6.04, 95% CI 2.42‐15.07). No such associations were observed for anticardiolipin or β2‐glycoprotein I antibodies or LA presence. During follow‐up (median 72.5, range 66‐83 months), thromboembolic events observed in 32 (26.7%, 4.6%/year) patients were independently predicted by IgG aPS/PT antibodies >30 UI (hazard ratio [HR] = 3.04, 95% CI 1.20‐8.88) and low Ks (HR = 3.00, 95% CI 1.41‐6.50). ConclusionsWe identified aPS/PT antibodies as a determinant of denser and poorly lysable plasma fibrin clot formation in APS patients. The association of elevated aPS/PT antibodies with thromboembolism in APS could be at least in part mediated by prothrombotic clot properties.

Plasma phospholipid fatty acids are associated with altered fibrin clot properties in a population-based setting

Evidence regarding the relationship of plasma clot properties with fatty acids is contradictory, owing to different experimental protocols employed. The relationship of fibrinogen and plasma fibrin clot properties with plasma phospholipid fatty acids were cross-sectionally investigated in a population-based setting in 900 individuals. Composite saturated fatty acids (driven by stearic acid) and composite n-3 and n-6 polyunsaturated fatty acids (driven by docosahexaenoic and arachidonic acid respectively) were associated with prothrombotic clot properties. Composite monounsaturated fatty acids (driven by oleic acid) were associated with a profibrinolytic clot phenotype. Fibrinogen and BMI partly mediated these relationships. Individual plasma fatty acids from the same composite group had opposing associations with clot properties indicating that associations with composite fatty acid groups is dependent on the relative composition of the comprising fatty acids. The relationship of the plasma phospholipid fatty acid profile with disease should not be interpreted without considering the role of regulatory mechanisms.

Altered fibrin clot properties in advanced lung cancer: impact of chemotherapy.

Faster formation of dense and poorly lyzable fibrin networks have been reported in patients at risk of thromboembolism, including cancer patients. We sought to investigate whether chemotherapy affects plasma fibrin clot properties and their determinants in lung cancer patients. In this observational study we enrolled 83 consecutive patients with advanced inoperable lung cancer. Plasma fibrin clot permeability (Ks), turbidimetric analysis of clot formation, clot lysis time (CLT), microparticle-associated tissue factor (MP-TF) activity, and thrombin generation parameters were investigated at enrolment and 3-4 months after standard chemotherapy. Lung cancer patients after 4 (range, 4-5) cycles of chemotherapy had 35.6% higher D-dimer, 22.1% lower MP-TF activity, and unaltered fibrinogen compared with baseline. Chemotherapy resulted also in 7.5% increased Ks, 8.6% prolonged lag phase, and 5.4% shortened CLT, while thrombin generation was unchanged. Chemotherapy-related differences in clot structure were confirmed by scanning electron microscopy images. Fibrin clot properties after chemotherapy did not differ among histological types of lung cancer, cancer stages or chemotherapy regimens. Interestingly, never smoking (n=13, 16%) was associated with looser post-treatment fibrin structure as reflected by 12.3% higher Ks. Multiple linear regression showed that more advanced cancer stage, higher peak thrombin generation, and higher white blood cell count determined post-treatment change in Ks, while active smoking was associated with change in CLT. Three-month chemotherapy in lung cancer patients improves clot properties despite unaffected thrombin generation, suggesting that anticancer treatment might quickly produce antithrombotic actions.

Intravascular Cells and Circulating Microparticles Induce Procoagulant Activity Via Phosphatidylserine Exposure in Venous Thromboembolism and Contribute to Altered Plasma Fibrin Clot Properties

Introduction:Venous thromboembolism(VTE), encompassing pulmonary embolism (PE) and deep vein thrombosis (DVT), is a major contributor to global morbidity and mortality, especially among cancer patients. However, the factors that contribute to PE, a prevalent and fatal complication of DVT, remain poorly understood.Despite a common pathology relating PE to DVT, this leaves a grey area in the differences among DVT, confirmed PE, or DVT with PE, and the definitive relationship between VTE and this hypercoagulable state.However, relatively little is known about the definitive role of phosphatidylserine (PS), one of the blood constituents in the coagulant pathway, in the hypercoagulability of VTE and their altered plasma fibrin clot properties. Our objectives were to assess the levels of PS exposure on microparticles (MPs), blood cells, endothelial cells(ECs) and cell-derived MPs in each group of VTE and to evaluate their procoagulant activity (PCA), as well as the correlation between initial PS exposure and their altered plasma fibrin clot properties.Methods:DVT alone (n=27), definitely PE alone (n=22), DVT with PE (n=19) and healthy controls (n=30) were enrolled in the present study. PS exposure on MPs, blood cells and cultured ECs treated with VTE serum in vitro was analyzed with flow cytometry and confocal microscopy. MPs were classified based on their cellular origin: platelets (CD41a+, PMPs), neutrophils (CD66b+, NMPs), endothelial cells(CD31+CD41a-, EMPs), erythrocytes (CD235a+, RMPs), monocytes (CD14+, MMPs), T lymphocytes (CD3+, T LyMPs), and B lymphocytes (CD19+, B LyMPs). PCA was evaluated by clotting time, extrinsic/intrinsic FXa and prothrombinase production assays, as well as fibrin formation assays. Inhibition assays of PCA of PS+MPs, blood cells and ECs were performed by lactadherin. Abnormal fibrin clot properties measured ex vivo in plasma was examined by fibrin clot analysis and scanning electron microscopy (SEM).Results: There was no significant difference in MP cellular origin between healthy and VTE subjects. However, the total number of PS+MPs was significantly increased (P < 0.001 for total MP and all MP subtypes) in VTE patients compared with healthy controls. In addition, circulating PS+ MPs cooperated with PS+blood cells and cultured ECs to markedly shorten coagulation time (Figure 1A) and dramatically increase FXa/thrombin generation and fibrin formation in each VTE group (all P < 0.05). Confocal microscopy images showed that the FVa/FXa complex and fibrin strands co-stained with PS on ECs. Moreover, blockade of exposed PS on MPs and intravascular cells with lactadherin inhibited PCA by approximately 80% (Figure 1B). Fibrin clot structure was evaluated in 15 VTE patients (DVT=5, PE=5 and DVT with PE=5) and 5 healthy subjects.Compared with those from DVT and PE, clots from plasma of DVT with PE subjects showed lower fiber density and faster clot lysis time, whereas there were no differences in lag time, rate of clot formation and maximum absorbance of turbidity.Conclusions: Our results suggest that compared with healthy subjects, each group of VTE patients had markedly higher levels of circulating PS+MPs, PS+blood cells. Additional, ECs treated with VTE patient serum had elevated percentages of PS+cells versus those treated with serum from healthy controls. Blockade of PS with lactadherin significantly inhibits PCA of blood cells, MPs and VTE serum-treated ECs. In addition, differences in fibrin clot properties and clot structure among DVT, PE and DVT with PE subjects may provide important insights into the pivotal mechanisms that regulate embolization. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.