Endogenous Thrombin Potential Ratio Research Articles

P-275 Advanced hemostasis assessment in endometriosis: strengthening confidence in the safety of hormonal treatments

Abstract Study question Are women with endometriosis in a state of systemic imbalance between pro- and anti-coagulants? Summary answer Women with endometriosis do not seem to exhibit a coagulation imbalance, at least at a systemic level. What is known already A state of platelet hyperactivation potentially leading to a hypercoagulation status has been described in women with endometriosis. This phenomenon could induce an altered local environment, promoting platelet-mediated paracrine changes in endometrial tissue and also increasing cardiovascular risk, which could be worsened by the use of estrogen dependent hormonal treatments. However, despite a slight difference in some coagulation parameters has been observed between women affected and controls, their levels remain, generally, within the normal reference range. Study design, size, duration This prospective case-control study including 101 women seeking infertility consultation at the Infertility Unit of Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano between January and July 2023 aimed to assess hemostasis function beyond traditional coagulation parameters. Participants/materials, setting, methods Blood of women with endometriosis (n = 51) or tubal or male factor infertility (controls; n = 50) was obtained before any ART treatment, and a complete panel of coagulation tests were performed, including basic parameters (aPTT, PT, FVIII, FII, PC, AT, FIV-C, DD, vWF, ADMT) as well as more sophisticated ones, such as Thrombin Generation Assay and Thromboelastometry, providing data on endogenous thrombin potential (ETP), clot formation time (CFT) and maximal clot firmness (MCF). Main results and the role of chance There were no significant differences between cases and controls in terms of age (37 [34-39] vs 36 [33-39]; p = 0.53, respectively) and smoking habits (14% vs 28%; p = 0.14, respectively). Women affected had a lower BMI compared to controls (22.7 [20.9-24.9] vs 21.4 [19.6-23.8]; p = 0.03), as it is usually expected. Results from TGA were expressed as ETP ratio (with-/without- thrombomodulin), and high ones were indicative of thrombomodulin resistance, taken as indexes of procoagulant imbalance; however, it was not different between women with and without the disease (0.75 [0.65-0.82] vs 0.75 [0.66-0.82]; p = 0.82, respectively). Other markers of procoagulant imbalance evaluated by thromboelastometry were also similar between cases and controls, including CFT (66 sec [58-78] vs 65 sec [59-79]; p = 0.69, respectively) and MCF (65 mm [62-68] vs 66mm [63-68]; p = 0.95, respectively). Considering the vWF/ADAMTS13 ratio, recently proposed to be altered in endometriosis, no difference was seen between affected and no-affected women (0.95 [0.76-1.14] vs 0.91 [0.68-1.15]; p = 0.41, respectively). Finally, all the basic coagulation parameters were also similar between groups. Limitations, reasons for caution Our sample size does not allow us to perform sub-analyses regarding the type of endometriosis, although we cannot exclude differences according to the various manifestations of the disease. Also, not having a systemic haemostasis imbalance does not exclude the possibility of local coagulation imbalances. Wider implications of the findings An altered systemic haemostasis could be worsened by the use of hormonal treatments. Our result indicating that there is no coagulation imbalance at a systemic level reassures about the absence of further contraindications to hormonal drugs as a treatment for endometriosis. Trial registration number not applicable

Use of thrombomodulin-modified thrombin generation in uncomplicated pregnancy: the normal range and prothrombotic phenotype

Pregnancy is a hypercoagulable state associated with an increased risk of venous thrombosis. Thrombomodulin(TM)-modified thrombin generation is a promising laboratory method to detect the thrombotic tendency and prothrombotic phenotype. 141 women were enrolled: 30 healthy non-pregnant controls, 85 healthy pregnant women (26 in 1st trimester, 28 in 2nd trimester, 31 in 3rd trimester), and 26 patients with gestational diabetes mellitus (GDM). Thrombin generation was measured using platelet poor plasma (PPP) TM + and PPP TM- reagents. The parameters were endogenous thrombin potential (ETP), Lagtime, Peak Height, time to peak and ETP ratio(ETP(TM+)/ETP(TM−)). Protein S-depleted plasma samples with different activity were prepared and measured. Pregnancy was associated with a significant decrease of ETP in the presence of TM, compared with that found in the absence of TM. This was observed in all trimesters (1st trimester 1185.67 ± 284.95 nM*min vs.1510.39 ± 281.90 nM*min, p < .001; 2nd trimester 1458.96 ± 349.65 nM*min vs. 1929.10 ± 316.98 nM*min, p < .001; 3rd trimester 1391.60 ± 317.05 nM*min vs. 1854.88 ± 327.60 nM*min, p < .001). The ETP ratio was also markedly increased in all trimesters (0.78 ± 0.10, 0.76 ± 0.11 and 0.74 ± 0.12) compared with that of non-pregnant controls (0.51 ± 0.17, p < .001). The results of ETP ratio in protein S-depleted plasmas were 0.986, 0.943 and 0.880 with 0%, 16% and 40% of protein S activity, which indirect represented the thrombotic phenotype of PS deficiency in pregnancy. TM-modified thrombin generation serves as a useful test for hypercoagulation in pregnant women. The ETP ratio and the reference range of ETP in the presence of TM could provide the basis to predict the risk of thrombotic complications during pregnancy.

Unfolded Von Willebrand Factor Interacts with Protein S and Limits Its Anticoagulant Activity

Background: The critical plasma anticoagulant protein S (PS) circulates in two pools: free (anticoagulant) or bound to complement component 4-binding protein (C4BP) (antiinflammatory). Acquired free PS deficiency occurs in patients with severe viral infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and is associated with increased risk of thrombosis. von Willebrand Factor (VWF) is a large multimeric protein , which unfolds under shear, exposing protein binding sites. Elevation of VWF antigen and activity are associated with infection-induced vascular endothelial activation, including during SARS-CoV-2 infection. Here, we identified a shear-dependent association between VWF and PS, and assessed the effect of VWF on PS anticoagulant activity. Methods: This study was approved by the University of Kentucky Institutional Review Board. Citrated plasma was collected from consenting adults, including 28 SARS-CoV-2+ inpatients, 49 SARS-CoV-2+ outpatients, and 31 healthy controls. Results: Initial studies in our SARS-CoV-2+ patient cohort revealed that free PS, a measure of the anticoagulant pool, was similarly reduced in both inpatients (p=0.037) and outpatients (p=0.008) compared to controls, while total PS (p=0.964) was unchanged. This indicated an increase in a plasma PS-binding protein. However, known abundant PS-binding proteins C4BPβ and protein C, an anticoagulant that functions with PS, were unchanged (p=0.647 and p=0.185, respectively), and Mer, a macrophage receptor that binds PS, was only mildly increased (p=0.042). Mass spectrometric and immunoblotting analyses revealed that PS interacts with VWF in pooled donor plasmas and that this interaction is enhanced >10,000,000-fold by shear-induced unfolding of VWF. Plasma VWF was elevated 3.8-fold in inpatients (p<0.0001) compared to outpatients and controls, suggesting endothelial activation, though no change in multimer distribution was apparent (n=4). We hypothesize that shear-induced unfolding of VWF exposes a binding site, which interacts either directly or indirectly with PS and contributes to acquired free PS deficiency. Consistent with this hypothesis, sheared-VWF dose-dependently inhibited free, but not total, PS antigen measurements, when measuring either purified proteins or plasma. We next assessed the functional consequences of the VWF/PS interaction using purified proteins. Sheared-VWF dose dependently reversed FXa inhibition by the PS/Tissue Factor Pathway Inhibitor-α complex, in a chromogenic FXa activity assay. Conversely, Activated Protein C (APC) cofactor function was unaffected, either on phospholipid vesicles or on the surface of activated platelets, as measured either by the cleavage of factor Va heavy chain by western blot and by the rate of thrombin activation. Finally, to assess PS function in patients, we measured plasma thrombin generation. Despite anticoagulation, plasma thrombin generation in inpatient samples was comparable to controls (Endogenous Thrombin Potential (ETP): p=0.257; Lag Time: p=0.095), suggesting a significant hypercoagulability, possibly exacerbated by PS deficiency. Consistent with this, free PS negatively correlated with plasma thrombin generation parameters measured with thrombomodulin (TM) supplementation (r=-0.443, p=0.0008), which is required for sensitivity to the APC/PS pathway; ETP ratio (ETP with TM/ ETP without TM) in inpatients was higher compared to controls indicating less APC/PS activity (p=0.009); and these patients had profoundly elevated D-dimer (p<0.0001). Conclusion: We propose a novel mechanism of acquired free PS deficiency, by which pathological shear forces unfold VWF, which interacts with PS, and at least partially blocks its anticoagulant function. It is possible that unfolded VWF further limits PS anticoagulant activity in vivo by sequestering it from APC, factor IXa, or other interacting partners. We anticipate that this mechanism also contributes to acquired free PS deficiency in other inflammatory conditions in which VWF is similarly unfolded.

Thrombin Generation to Predict the Risk of Venous Thromboembolism Recurrence, Major Bleeding and Death in the Elderly: A Prospective Multicenter Cohort Study

Introduction: Venous thromboembolism (VTE), comprising deep vein thrombosis (DVT) and pulmonary embolism (PE), constitutes a worldwide major health issue, and a leading cause of death. VTE incidence increases exponentially with age mainly due to the accumulation of risk factors and comorbidities predisposing to thrombosis. This leads to greater morbidity impact of VTE on elderly patients, who are also at higher risk of bleeding. Consequently, identification of older patients who might benefit from indefinite anticoagulation treatment is paramount. In order to facilitate the identification of these patients, the benefit/risk ratio should be carefully evaluated by considering clinical and laboratory information. Thrombin activity can be recorded by continuously measuring cleavage of a fluorescent substrate, resulting in a thrombin generation (TG) curve. Recent association studies show promising data of thrombin generation parameters predicting first VTE in elderly (Wang H et. al. RPTH 2021, 5:e12536). However, the predictive ability of thrombin generation for recurrent VTE, major bleeding and mortality in the elderly is unknown. The goal of this study was to prospectively investigate the performance of the TG assay one year after index VTE in predicting the risk of VTE, recurrence, major bleeding and mortality up to 2 years in elderly population.Methods: The study was conducted as part of the Swiss Cohort of Elderly Patients with VTE (SWITCO65+), a prospective multicenter cohort study to assess medical outcomes and quality of life in elderly patients with acute VTE in Switzerland. For the present study, the clinical outcomes were VTE recurrence, major bleeding and mortality, which were assessed parallel to clinical data of thrombosis and other general laboratory parameters including thrombophilia testing in over a 3-year period. Blood samples for assessment of TG parameters were drawn 12 month after the index VTE. Venous blood was drawn after minimal venostasis and processed by double centrifugation according to the recommendation of the subcommittee of the Scientific and Standardization Committee of ISTH. TG measurements were performed with the calibrated automated thrombogramm assay (Stago, Asnières-sur-Seine, France) in two experimental settings: 1pM tissue factor (TF) with/without thrombomodulin (TM) and 13.6 pM TF with/without activated protein C (APC). In addition, reference plasma (Cryocheck Reference Control Normal, PrecisionBiologic, Dartmouth, Canada) was tested in all experiments in order to correct day-to-day variations. Lag time, velocity index, time to peak, peak height, endogenous thrombin potential (ETP) were measured and peak and ETP ratio obtained in presence/absence of TM or APC were calculated. Results from the reference plasma were used to calculate the normalized ETP and peak ratio in 1 pM setting and peak ratio in 13.6 pM TF setting.Results: TG was assessed in 565 patients 12 months after the index VTE. At this time, 59% of patients were still anticoagulated. Eleven percent of them had cancer-related VTE, 20% provoked VTE and 68% unprovoked VTE. The prevalence of inherited risk factors for VTE was in line with previous reports on European patients with VTE.Patients still anticoagulated 12 months after the index VTE were less likely to develop recurrent VTE in the next 24 months than patients without anticoagulation. However, the incidence of major bleeding and mortality was comparable in anticoagulated and non-anticoagulated patients.TG was faster and lower in anticoagulated than in non-anticoagulated patients.Some thrombin generation parameters measured 12 months after the index VTE (Figure 1) were discriminatory for VTE recurrence, major bleeding and mortality (Table 1). In addition, several thrombin generation parameters measured in patients not under anticoagulation 12 months after the index VTE were associated with an increased risk of VTE recurrence, major bleeding and mortality up to 24 months. These associations remained after adjustment for potential confounding factors for the risk of VTE recurrence, major bleeding and mortality (Table 2).Conclusion: In elderly patients, several parameters of thrombin generation were associated with VTE recurrence, major bleeding and/or mortality. These findings may serve as the basis for validation in a prospective interventional outcome trial. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-TNF-α Treatment Reduces the Baseline Procoagulant Imbalance of Patients With Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) are characterized by an increased thrombosis risk of uncertain etiology. Coagulation derangement arising from inflammation may be a triggering factor. We hypothesized that strong inflammation inhibitors (eg, anti-tumor necrosis factor-α drugs) may affect coagulation. Forty patients with IBD were compared with 57 control patients for coagulation factors and endogenous thrombin potential (ETP), the latter being the most sensitive marker of in vivo pro- and anticoagulation balance. We measured ETP in the presence and absence of thrombomodulin (the physiologic protein C [PC] activator). Coagulation at different timepoints was also assessed for 28 of these patients during infliximab treatment. The median ETP (nM thrombin × minutes) and range (minimum-maximum) were each higher in patients at baseline than in control patients in both the absence (2120 [1611-3041] vs 1865 [1270-2337]) and the presence (1453 [464-2522] vs 831 [104-1741]) of thrombomodulin. The ETP ratio (with/without thrombomodulin) was high at baseline (0.73 [0.21-0.90] vs 0.45 [0.07-0.85]). The ETP and ETP ratio declined during treatment and were significantly lower at the end than at baseline. Factor (F) VIII and fibrinogen, which were high at baseline, decreased during treatment and at the end were significantly lower than at baseline. The FVIII/PC ratio, which was high in patients at baseline, declined during treatment and at the end was lower than at baseline. C-reactive protein recorded at the end of treatment was lower than at baseline. Patients with IBD have a procoagulant imbalance as shown by increased ETP at baseline. The ETP decreases during treatment with infliximab, which is related to decreased FVIII and FVIII/PC ratio. This effect is also related to the improvement of inflammation as shown by decreased fibrinogen and C-reactive protein.

Procoagulant Imbalance in Klinefelter Syndrome Assessed by Thrombin Generation Assay and Whole-Blood Thromboelastometry.

ContextKlinefelter syndrome (KS) is a condition at increased risk of thrombosis compared to 46,XY men.ObjectiveThis work aimed to investigate the coagulation balance of KS patients by thrombin generation assay (TGA) and thromboelastometry.MethodsAn observational, cross-sectional study was conducted at 3 tertiary endocrinological centers in Milan, Italy. Fifty-eight KS patients and 58 age-matched healthy controls were included. Anticoagulant or antiplatelet therapy and known coagulation disorders were exclusion criteria. TGA was performed in platelet-poor plasma (PPP) and platelet-rich plasma (PRP). Whole-blood thromboelastometry and activities of coagulation factors were assessed. Endogenous thrombin potential (ETP), the area under the thrombin generation curve, assessed with and without thrombomodulin (ETP-TM+ and ETP-TM–), and their ratio (ETP ratio), were considered as indexes of procoagulant imbalance.ResultsPatients with KS displayed higher PPP-ETP-TM+ (mean 1528 vs 0.1315 nM × min; P < .001), PPP-ETP ratio (0.78 vs 0.0.70; P < .001), factor (F)VIII (135% vs 0.107%; P = .001), fibrinogen (283 vs 0.241 mg/dL; P < .001), and FVIII/protein C ratio (1.21 vs 0.1.06; P < .05) compared to controls. Protein C was comparable in the 2 groups. Similar results were observed in PRP. The ETP ratio was positively associated with FVIII (ρ = 0.538, P < .001) in KS. Thromboelastometry parameters confirmed evidence of hypercoagulability in KS.ConclusionPatients with KS display a procoagulant imbalance expressed by increased thrombin generation both in PPP and PRP, which is at least in part explained by increased FVIII levels. The procoagulant imbalance, which was confirmed by thromboelastometry, may be responsible for the thrombotic events observed in these patients. Further investigation on the benefit/risk ratio of antithrombotic prophylaxis is warranted.

Proof of concept of a new scale for the harmonization and the standardization of the ETP‐based APC resistance

BackgroundThe evaluation of the activated protein C resistance (APCr) based on the endogenous thrombin potential (ETP) is recommended during the development of steroid contraceptives. Results are usually expressed as “normalized APC sensitivity ratio” (nAPCsr) using a reference plasma that should achieve an ETP ratio of 0.1 in presence of exogenous APC. Because of the interassay variability, achieving exactly an ETP ratio of 0.1 in each run is almost impossible, which significantly affects the theoretical 0‐10 scale of nAPCsr. ObjectivesTo compare the nAPCsr to the nAPCsr10, a newly proposed method to express the degree of APC resistance. MethodsIndividual plasma samples (n = 854) were analyzed to compare nAPCsr and nAPCsr10. These values were obtained using the validated ETP‐based APCr assay. ResultsThe Spearman correlation between nAPCsr and nAPCsr10 had a coefficient of 0.99. Linear regression showed the following equation y = 0.9315*x + 0.03942 (r2 = .97). When differences (nAPCsr10 – nAPCsr) were plotted against nAPCsr10, the mean difference equaled 0.16% or 4.95%. The correction obtained with the use of the nAPCsr10 showed that the results of the nAPCsr were statistically different (P < .0001). ConclusionsThis new scale provides a harmonization and normalization of the nAPCsr. Results show a better reproducibility with the nAPCsr10. It avoids the additional variability and the unharmonized scale introduced by the use of a reference plasma. This adapted method for the calculation of the APC resistance could provide the regulatory and scientific bodies with more reproducible and harmonized evaluations.

The relationship between coagulation disorders and the risk of bleeding in cirrhotic patients

Introduction and ObjectivesFor long, bleeding in cirrhotic patients has been associated with acquired coagulation disorders. The aim of our study was to investigate the impact of acquired coagulation disorders on bleeding risk in cirrhotic patients. Materials and methodsBlood samples were collected from 51 cirrhotic patients with (H+) or without (H−) bleeding events and 50 controls matched by age and sex. Thrombin generation was assessed as endogenous thrombin potential (ETP). Hemostatic balance was assessed by means of ratios of pro- to anticoagulant factors and by ETP ratio with/without protein C (PC) activator (ETP ratio). ResultsBleeding events occurred in 9 patients (17.6%). Compared with controls, VIII/anticoagulant factors, VII/PC and XII/PC were significantly higher in (H+) patients. No significant difference as regards all ratios across patient groups was detected. ETP ratio was significantly higher in (H+) patients than in controls (p=0.017). However, there was no significant difference between patient groups as regards ETP ratio. ConclusionHemostatic balance is shifted toward a hypercoagulability state even in cirrhotic patients who experienced bleeding. These findings provide evidence against traditional concept of hemostasis-related bleeding tendency in cirrhotic patients.

Reversal of hypercoagulability in patients with HCV-related cirrhosis after treatment with direct-acting antivirals.

The long-term impact of sustained virological response (SVR) after direct-acting antivirals (DAAs) on the hypercoagulability associated with HCV cirrhosis is unknown. We longitudinally evaluated the effect of DAAs treatment on cirrhotic coagulopathy. Pro- and anticoagulant factor levels and thrombin generation were assessed in patients with HCV-related cirrhosis at baseline, end of therapy (EOT), at 12, 24 and 48weeks (W) after EOT. Fifty-eight patients were enrolled (86% Child's A). SVR was 100%. Median factor VIII activity significantly decreased at EOT, 12weeks and 24weeks compared with baseline, whereas protein C significantly increased at 24weeks and 48weeks. Cirrhotic patients showed a slight but sustained increase in endogenous thrombin potential (ETP) with a statistically significant difference at EOT, 12weeks, 24weeks and 48weeks compared with baseline. Conversely, thrombomodulin-modified ETP was elevated before treatment and decreased over time to normal levels at 24weeks and 48weeks. The ETP ratio decreased slowly at EOT and 12weeks, and was significantly decreased at 24weeks and 48weeks compared with baseline (P<.001 for both comparisons), being not statistically different from ETP ratio measured in healthy controls. Child's B patients showed a significantly higher ETP ratio compared to Child's A at baseline and did not show any significant improvement in ETP ratio through 12weeks. Two Child's B patients developed PVT with an incidence rate of 1.1% p-yrs (95%CI, 0.18 to 3.58). DAAs therapy in HCV-related cirrhotic patients is associated with significant changes in thrombin generation suggesting a reversal of hypercoagulability particularly in Child's A patients.

Detection of procoagulant imbalance. Modified endogenous thrombin potential with results expressed as ratio of values with-to-without thrombomodulin.

Each individual possesses his/her own endogenous-thrombin-potential (ETP) (i. e. the ability to generate thrombin) which depends on the relative strength of the pro- and anticoagulant drivers operating in plasma. This ability depends in turn on the clinical conditions in which the balance between the two drivers is variably affected. One of the major determinants of this balance is the factor (F)VIII-protein C(PC) axis and its effect can be conveniently explored by the thrombin generation procedures with results expressed as ETP ratio with/without thrombomodulin (TM) (ETP-TM ratio). Furthermore, owing to the many feedback mechanisms mediated by thrombin (e. g. activation of PC, FXI, FV, FVIII, platelets etc.) it is also possible that any perturbation of the balance between pro- and anticoagulants that may occur in plasma even outside the FVIII-PC axis could result in an increased ETP-TM ratio and therefore may suggest a procoagulant imbalance. Indeed, other non-coagulation moieties (e. g. microparticles, neutrophil extracellular traps, pro-inflammatory cytokines and others) circulating in blood of patients with various clinical conditions may also contribute to the procoagulant imbalance even when FVIII and/or PC are apparently normal. It can be postulated that dual ETP measurements performed in the presence and absence of TM with results expressed as their ratio may be the candidate procedure to detect subtle procoagulant imbalance in many clinical conditions characterised by an increased risk of thromboembolism. This article aimed at reviewing the clinical conditions in which evidence for the value of the ETP-TM ratio has been provided.

Fresh Frozen Plasma Transfusion Ameliorates INR and aPTT but Does Not Increase Thrombomodulin-Corrected Thrombin Generation in Patients with Cirrhosis

▪Introduction: In vitro studies have shown that transfusion of fresh frozen plasma (FFP) has limited efficacy in correcting coagulopathy in cirrhosis. This study aims to assess the effect of FFP transfusion on endogenous thrombin potential (ETP) in patients with cirrhosis with abnormal coagulation test results. Methods: Using a stringent protocol for control of pre-analytic variables, blood samples were collected before and up to 8 hours after FFP transfusion. All samples were tested for INR, aPTT and ETP. The ETP assay was performed in platelet-poor plasma, using an automated fluorometer (CAT, Netherlands), using the technique developed by Hemker and modified by Tripodi. ETP was defined as the area under ROC curve and results were expressed as the ratio of ETP with thrombomodulin addition to ETP without (used to mimic in vivo conditions when patients are transfused). Results: 42 patients were included (male n=28; Child-B n= 11; Child-C n=31). Patients underwent FFP transfusion prior to high-risk invasive procedures (n=25), for treatment of bleeding (n=10) or for both reasons (n=6). The mean dose of FFP was 11.3 ml/kg (95% CI 9.16 - 20.46). After transfusion, INR decreased from 2.6±1,4 to 2.09±0.5 (p<0.0001) and aPTT from 1.47±0.5 to 1.27±0.26 (p<0.0001). At baseline, normal or above normal values of ETP ratio (³0.66) were found in 37 (88%) patients, and mean values remained largely unchanged after FFP transfusion (0.75±0.22; median 0.82 before to 0.76±0.16; median 0.81 after transfusion; p=0.56). The results for INR, aPTT and ETP are shown in figure 1. During the one-week follow-up period, 4 patients (9,5%) had acute reactions to transfusion, including fluid overload and allergic reactions. Of the 31 patients that underwent invasive procedures, four had severe bleeding. Discussion: FFP transfusion at standard doses ameliorated INR and aPTT, but did not increase thrombin generation corrected by thrombomodulin. That might be explained by the fact the former tests detect only 5% of the amount of thrombin generated. Besides, they are performed without the addition of thrombomodulin, an endothelial protein with anticoagulant effects, to which patients with cirrhosis have shown to be resistant. Patients were exposed to the risks of transfusions, without a clear benefit in the improvement in thrombin generation and decreasing the risk of bleeding. Our results call into question the common use of INR and aPTT for guiding the transfusion policy of FFP when treating coagulopathy in cirrhosis.Figure 1: Results showing comparison before and after FFP transfusionfor (A) INR, (B) aPTT and (C) ETP ratio with and without thrombomodulin. DisclosuresNo relevant conflicts of interest to declare.

Neutrophil Extracelluar Traps Contributes to the Hypercoagulable State and Liver Damage in Liver Cirrhosis

Background: We recently reported that phosphatidylserine on blood and endothelial cells played an important role in the hypercoagulable state of liver cirrhosis (Wu et al, Liver Int 2016). Recent studies showed that enoxaparin could reduce the incidence of portal vein thrombosis and alleviate liver decompensation. Thus, we speculate that there may be other mechanisms involved in the hypercoagulability of cirrhosis. Neutrophil extracelluar traps (NETs) play an important role in thrombosis and organ dysfunction. Whether the benefit of enoxaparin is due to inhibiting NETs in cirrhotic patients remains unknown. Our objectives were to study the formation of NETs and their role in the hypercoagulable state and liver damage in cirrhotic patients.Methods:Cirrhotic patients (n = 36), healthy controls (n = 10), and mice treated with CCl4 (n = 50) were studied. Immunofluorescence confocal microscope was used to analyze and quantify NETs formation in neutrophil cells from human and mice. TAT and fibrin formation assays in normal and cirrhotic plasma were performed to analyze the procoagulant activity of NETs. Calibrated automated thrombography (CAT) was performed with and without 6 nM thrombomodulin I to analyze TM resistance changes induced by NETs. Liver injury was assessed by plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, determined spectrophotometrically, and by morphometric evaluation of left lateral liver lobes. DNase I and activated protein C (APC) were used to abolish NETs.Results: We observed that cirrhotic platelets induced 1.9 fold higher NETs formation in neutrophil cells (p = 0.021). Increased NETs promoted higher TAT (Fig 1A, B) and fibrin formation in normal and cirrhotic plasma (p < 0.05). DNase I abolished the procoagulant activity of NETs in corn trypsin inhibitor (CTI ) or anti-TF pre-treated plasma (p < 0.001) (Fig 1C). TM pre-induced with NETs for 24 h showed a higher endogenous thrombin potential (ETP) than no treatment in normal plasma using CAT (p = 0.032). APC and de-N-sulfated heparin (de-N-Hep), but not DNase I, can decrease the ETP ratio (with/without TM) in cirrhotic plasma (p < 0.05) (Fig 1D). Mice treated with CCl4 showed a higher ALT, AST, necrotic area (Fig 2), and NETs formation than control (p < 0.05). DNase I and enoxaparin decreased ALT, AST, necrotic area (Fig 2) and NETs formation (p < 0.05).Conclusions:Cirrhotic patients have enhanced NETs formation compared to healthy controls. NETs initiated intrinsic and extrinsic coagulation pathway and impaired anticoagulant function of TM. Additionally, NETs partly contributed to tissue injury in the liver of cirrhotic mice. DNase I and enoxaparin can disconnect NETs and decreased the toxin to liver. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hypercoagulability in patients with Cushing disease detected by thrombin generation assay is associated with increased levels of neutrophil extracellular trap-related factors.

Patients with Cushing disease (CD) are at increased risk of venous thromboembolism (VTE). It was surmised, but not conclusively shown that the risk is related to plasma hypercoagulability secondary to the glucocorticoids effect. This study is aimed at detecting hypercoagulability in patients with CD. Case-control study of 48 CD patients and controls enrolled at two Italian clinics for whom we assessed the thrombin-forming-potential in the presence of optimal activation of protein C obtained by adding into the assay system its main endothelial activator, thrombomodulin. These experimental conditions mimic more closely than any other test the in vivo situation. We observed enhanced thrombin-generation in CD patients, as shown by the modification of thrombin-generation parameters [i.e., shortened lag-time and time-to-peak, increased thrombin peak and endogenous thrombin potential (ETP)]. Moreover, the ETP ratio (with/without thrombomodulin), recognized as an index of hypercoagulability, was increased in patients as compared to controls. We attempted to explain such hypercoagulability by measuring both procoagulant and anticoagulant factors, and some other non-coagulation parameters (i.e., neutrophil extracellular traps (NET), recently associated with the VTE risk and/or increased hypercoagulability. We showed that the hypercoagulability in patients with CD is associated with increased levels of factor VIII and NET-related variables. We detected plasma hypercoagulability in patients with CD and found experimental explanation for its occurrence. Whether this hypercoagulability can entirely explain the occurrence of VTE in patients with CD should be investigated by ad-hoc clinical trials. However, until these studies will be available the evidence supports the concept that patients with CD are candidates for antithrombotic prophylaxis.

Evidence that low protein C contributes to the procoagulant imbalance in cirrhosis

Cirrhosis is associated with a plasmatic procoagulant imbalance, detected in vitro by thrombin generation tests performed in the presence vs. absence of such activators of protein C as thrombomodulin or Protac. This imbalance is thought to be due to decreased protein C and increased factor VIII, but this has never been directly demonstrated. To test this hypothesis we analyzed plasma from 50 patients with cirrhosis before and after in vitro addition of purified protein C meant to restore normal levels. Results for two thrombin generation assays were expressed as ratios of endogenous thrombin potential (ETP) with-to-without thrombomodulin or as Protac-induced coagulation inhibition (PICI%). By definition, high ETP ratios or low PICI% reflect a resistance to the anticoagulant action of thrombomodulin or Protac, respectively, and can be taken as indexes of in vitro procoagulant imbalance. The median (range) protein C level before addition was 40% (4-101%) and increased to 156% (110-305) after addition (p<0.001). The procoagulant imbalance, which was high before protein C addition [ETP ratio=0.83 (0.44-1.00)], was reduced after addition [ETP ratio=0.60 (0.14-0.84)], p<0.001. ETP-ratios were inversely correlated with protein C activity (rho=-0.46, p<0.001). Similar results were obtained with the Protac assay. The results provide evidence that low protein C contributes to the procoagulant imbalance in plasma from patients with cirrhosis. The findings may have clinical implications for the treatment or prophylaxis of thrombosis in these patients.

Coagulation Proteins Influencing Global Coagulation Assays in Cirrhosis: Hypercoagulability in Cirrhosis Assessed by Thrombomodulin-Induced Thrombin Generation Assay

Background. Liver disease is accompanied by profound hemostatic disturbances. We investigated the influences of pro- and anticoagulation factors on global coagulation tests including prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin generation assay (TGA) in cirrhosis. We also investigated whether cirrhotic patients exhibit hypo- or hypercoagulability using the TGA. Methods. The TGA was performed on a calibrated automated thrombogram, given lag time, endogenous thrombin potential (ETP), and peak thrombin in 156 cirrhotic patients and 73 controls. Results. PT was determined according to the factor (F) II, FV, FVII, FIX, and protein C levels. We observed that aPTT was dependent on FII, FIX, and FX levels. The ETP was dependent on FII, antithrombin, and protein C with 5 pM tissue factor (TF) stimulation, and FIX and protein C at 1 pM TF. The ETP ratio with 1 pM TF increased significantly in cirrhosis, indicating hypercoagulability, whereas that with 5 pM TF did not increase in cirrhosis. Conclusion. PT and the TGA are sensitive to protein C levels. Even with prolonged PT, the TGA can detect hypercoagulability in cirrhosis. Further studies should evaluate global coagulation status in cirrhosis patients using the newly devised TGA system.

African–Caribbean ethnicity is associated with a hypercoagulable state as measured by thrombin generation

African-Caribbean ethnicity is associated with an increased risk of both first and recurrent venous thromboembolism (VTE). The aim of this study was to evaluate thrombin generation in African-Caribbeans compared with whites in patients with deep vein thrombosis (DVT) and healthy volunteers. Thrombin generation was measured in a case-control study of 80 patients who had completed anticoagulation therapy for a first DVT (50 white and 30 African-Caribbean) and 66 controls. Peak thrombin with and without thrombomodulin was significantly higher in African-Caribbeans with DVT compared with whites with DVT (P < 0.001 for both comparisons) and African-Caribbean controls (P < 0.001, 0.003, respectively). Endogenous thrombin potential (ETP) with and without thrombomodulin was significantly higher in African-Caribbeans with DVT than whites with DVT (P ≤ 0.001 for both comparisons). Maximum velocity and ETP ratio were increased in African-Caribbeans with DVT compared with whites with DVT (P < 0.001 and 0.030, respectively) and African-Caribbean controls (P < 0.001 and 0.019, respectively). Within the control group, peak thrombin was significantly increased in African-Caribbeans compared with whites (P = 0.017). ETP, peak thrombin with thrombomodulin and maximum velocity were also increased in African-Caribbeans compared with white controls (P = 0.045 for all comparisons). African-Caribbeans with DVT had significantly higher factor VIII levels compared with whites with DVT and controls. African-Caribbean ethnicity confers a hypercoagulable state as measured by thrombin generation. This supports epidemiological findings of increased risk of first and recurrent VTE. Thrombin generation requires adjustment for ethnicity in studies undertaken in ethnically diverse populations.

Increased anticoagulant response to low‐molecular‐weight heparin in plasma from patients with advanced cirrhosis

Cirrhotic patients may present thrombotic complications that warrant anticoagulant therapy. However, the efficacy of low-molecular-weight heparin (LMWH) in this clinical setting is still unclear. To evaluate the in vitro effect of LMWH on thrombin generation (TG) in cirrhotic patients at different stages of liver disease. Thirty cirrhotics (10 Child Pugh A, 10 Child Pugh B and 10 Child Pugh C), 10 subjects with inherited type 1 antithrombin (AT) defect and 10 healthy controls were studied. TG was determined at baseline and with anti-Xa levels after the addition of enoxaparin at 0.35 and 0.7 U anti-Xa mL. The endogenous thrombin potential (ETP) ratio at 0.35 and 0.7 U anti-Xa mL was obtained by dividing ETP with LMWH by ETP at baseline. Mean AT levels in all cirrhotic subgroups and in patients with AT deficiency were significantly lower than in controls. The 0.35 ETP ratio was significantly lower in cirrhotic patients than in controls (0.26 ± 0.1 vs. 0.48 ± 0.1, P < 0.001) and the reduction paralleled the severity of liver disease, in spite of the concomitant decrease in AT and anti-Xa activity. AT-deficient subjects showed a significantly increased 0.35 ETP ratio compared with both cirrhotic patients and controls (0.69 ± 1 vs. 0.26 ± 0.1, P < 0.001, and vs. 0.48 ± 0.1, P = 0.04 respectively). LMWH at 0.7 U anti-Xa mL completely inhibited TG in 9/30 cirrhosis patients with more advanced liver disease (Child Pugh B and C), whereas complete TG abolition was seen in only 1/10 controls. Cirrhotic patients show an increased response to LMWH, which correlates with the severity of liver disease, in spite of reduced AT and anti-Xa activity levels. Thrombin generation may be a useful tool to monitor the response to LMWH in cirrhotic patients.

PWE-295 Thrombin generation is normal in cirrhotics with acute variceal haemorrhage: results from a prospective study

IntroductionCirrhotics have multifactorial derangements in haemostasis commonly leading to transfusion of blood components for both prophylaxis and treatment of bleeding. Thrombin generation assays have recently demonstrated that the haemostatic balance...

Enhanced thrombin generation in patients with cirrhosis-induced coagulopathy.

Prothrombin time (PT) and the international normalized ratio (INR) are still routinely measured in patients with liver cirrhosis to 'assess' their bleeding risk despite the lack of correlation with the two. Thrombin generation (TG) assays are global assays of coagulation that are showing promise in assessing bleeding and thrombosis risks. To study the relationship between the INR and TG profiles in cirrhosis-induced coagulopathy. Seventy-three patients with cirrhosis were studied. All TG parameters were compared with those from a normal control group. Contact activation was prevented using corn trypsin inhibitor. TG was also assayed in the presence of Protac(®). The endogenous thrombin potential (ETP) ratio was derived by dividing the ETP with Protac® by the ETP without Protac®. The INR (mean 1.7) did not correlate with the ETP and the velocity of TG (P > 0.05). There was no difference between the lag time and ETP of the two groups (P > 0.05). The velocity of TG was increased in cirrhosis (67.95 ± 34.8 vs. 45.05 ± 25.9 nM min⁻¹ ; P = 0.016) especially in patients with INRs between 1.21 and 2.0. Both the ETP with Protac(®) and the ETP ratio were increased in cirrhosis (mean 1074 ± 461.4 vs. 818 ± 357.9 nM min, P = 0.004 and 0.80 ± 0.21 vs. 0.44 ± 0.15, P ≤ 0.0001, respectively). Despite a raised INR, TG parameters are consistent with a hypercoagulable profile in cirrhosis-related coagulopathy. This confirms that the PT or INR should not be used to assess bleeding risk in these patients, and other parameters, such as TG, need to be explored as clinical markers of coagulopathy.

Endogenous Thrombin Potential for Predicting Risk of Venous Thromboembolism in Carriers of Factor V Leiden

Measurement of endogenous thrombin potential (ETP) detects hypercoagulability and can be used to identify activated protein C resistance due to factor V Leiden (FVL). However, not all carriers of FVL suffer thrombosis and therefore we sought to determine if the test for ETP could be modified in such a way as to enable detection of FVL patients who were at increased risk of venous thromboembolism. Protac, an activator of both protein C and factor V, was incorporated into the traditional thrombin generation reaction and ratios (reaction with Protac:reaction without Protac) were calculated. Plasma samples from 42 FVL heterozygotes (12 with a history of thrombosis and 30 with no prior thrombosis) and 38 controls (non-FVL with no history of thrombosis) were analysed. The mean ETP ratio was significantly higher in FVL heterozygotes (0.90 ± 0.06) compared to normal controls (0.41 ± 0.10; p = 0.00004). Multivariate analysis indicated that the average ETP ratio was significantly and inversely correlated with factor V levels in FVL heterozygotes (p = 0.002) but not controls. Within the FVL group, patients with a history of thrombosis had higher ETP ratios (0.92 ± 0.06) compared to those without (0.89 ± 0.05), however, this did not reach statistical significance (p = 0.09). Further investigation into the use of ETP for detecting risk of thrombosis in people who are genetically predisposed is warranted. The recent introduction of diagnostic ETP measurements in the form of the calibrated automated thrombin generation from Thrombinoscope and the TechnoThrombin from Baxter should facilitate such studies.