Thrombin Generation Assay Parameters Research Articles

Predelivery Haemostatic Biomarkers in Women with Non-Severe Postpartum Haemorrhage

Background: Postpartum haemorrhage (PPH) is a frequent complication of childbirth that is difficult to predict. Predelivery coagulation biomarkers may help to guide preventive strategies. Our objective was to evaluate the association of predelivery haemostatic biomarkers with non-severe PPH. Methods: A nested case-control study was conducted within the « Study of Biological Determinants of Bleeding Postpartum » in order to compare different haemostatic biomarkers in plasma from pregnant women with non-severe PPH (cases) and controls without PPH matched for age, body mass index, term, and mode of delivery. Blood was collected at entry in the delivery room. Global haemostatic assays (thrombin generation assay (TGA) and plasmin generation assay (PGA)) were then performed on freshly thawed aliquots of platelet-poor plasma. Results: A total of 370 pregnant women (185 cases and 185 controls) were included. Median [interquartile range] predelivery platelet count was lower in PPH cases than in controls (217 [181–259] versus 242 [196–280] G/L). TGA and PGA parameters were similar between cases and controls. In a subset analysis of vaginal deliveries (n = 144), median predelivery TGA thrombin peak was lower, and median predelivery PGA lag phase was longer in cases compared to controls. In multivariable analysis, only predelivery platelet count was independently associated with non-severe PPH. Conclusions: Predelivery platelet count is associated with non-severe PPH. Differences in other haemostatic parameters are tenuous, questioning their usefulness in predicting non-severe PPH.

Plasma levels of complement components C5 and C9 are associated with thrombin generation

BackgroundThe thrombin generation assay (TGA) evaluates the potential of plasma to generate thrombin over time, providing a global picture of an individual's hemostatic balance. ObjectivesThis study aimed to identify novel biological determinants of thrombin generation using a multiomics approach. MethodsAssociations between TGA parameters and plasma levels of 377 antibodies targeting 236 candidate proteins for cardiovascular risk were tested using multiple linear regression analysis in 770 individuals with venous thrombosis from the Marseille Thrombosis Association (MARTHA) study. Proteins associated with at least 3 TGA parameters were selected for validation in an independent population of 536 healthy individuals (Etablissement Français du Sang Alpes-Méditerranée [EFS-AM]). Proteins with strongest associations in both groups underwent additional genetic analyses and in vitro experiments. ResultsEighteen proteins were associated (P < 1.33 × 10⁻4) with at least 3 TGA parameters in MARTHA, among which 13 demonstrated a similar pattern of associations in EFS-AM. Complement proteins C5 and C9 had the strongest associations in both groups. Ex vivo supplementation of platelet-poor plasma with purified C9 protein had a significant dose-dependent effect on TGA parameters. No effect was observed with purified C5.Several single nucleotide polymorphisms associated with C5 and C9 plasma levels were identified, with the strongest association for the C5 missense variant rs17611, which was associated with a decrease in C5 levels, endogenous thrombin potential, and peak in MARTHA. No association of this variant with TGA parameters was observed in EFS-AM. ConclusionThis study identified complement proteins C5 and C9 as potential determinants of thrombin generation. Further studies are warranted to establish causality and elucidate the underlying mechanisms.

Testosterone therapy does not affect coagulation in male hypogonadism: a longitudinal study based on thrombin generation.

Testosterone therapy has been variably associated with increased thrombotic risk but investigations of global coagulation in this setting are lacking. To compare global coagulation of hypogonadal men before (T0) and 6 months after (T1) starting testosterone replacement therapy (TRT), and healthy controls. Observational prospective cohort study. Two tertiary endocrinological ambulatory care centers. Thirty-eight men with hypogonadism (mean age 55, SD 13) and 38 age-matched healthy controls. Thrombin generation assay (TGA) was performed at T0 and T1 in hypogonadal men and in controls. TGA is an in vitro procedure based on the continuous registration of thrombin generation and decay under conditions mimicking the process that occurs in vivo. The following TGA parameters were recorded: lag-time; thrombin-peak concentration; time-to-reach the peak, velocity index and endogenous thrombin potential (ETP), the latter representing the total amount of thrombin generated under the driving forces of procoagulants opposed by the anticoagulants. PC, antithrombin, factor (F)VIII, and fibrinogen were assessed. No changes of TGA parameters were observed between T0 and T1. Hypogonadal men displayed significantly higher ETP, fibrinogen, and significantly lower antithrombin levels both at T0 and T1 compared to controls. Thrombin-peak of hypogonadal men was significantly higher than controls at T0 but not at T1. ETP and antithrombin were correlated with testosterone levels. Hypogonadal men display a procoagulant imbalance detected by increased thrombin generation. Short-term TRT does not worsen global coagulation, suggesting that the treatment can be safely prescribed to men diagnosed with hypogonadism.

A small-molecule hemostatic agent for the reversal of direct oral anticoagulant–induced bleeding

BackgroundThe bleeding risk associated with direct oral anticoagulants (DOACs) remains a major concern, and rapid reversal of anticoagulant activity may be required. Although specific and nonspecific hemostatic biotherapies are available, there is a need for small-molecule DOAC reversal agents that are simple and cost-effective to produce, store, and administer. ObjectivesTo identify and characterize a small molecule with procoagulant activity as a DOAC reversal agent. MethodsWe sought to identify a small procoagulant molecule by screening a chemical library with a plasma clotting assay. The selected molecule was assessed for its procoagulant properties and its ability to reverse the effects of the DOACs in a thrombin generation assay. Its activity as a DOAC reversal agent was also evaluated in a tail-clip bleeding assay in mice. ResultsThe hemostatic molecule (HeMo) dose-dependently promoted thrombin generation in plasma, with dose values effective in producing half-maximum response ranging between 3 and 5 μM, depending on the thrombin generation assay parameter considered. HeMo also restored impaired thrombin generation in DOAC-spiked plasma and reversed DOAC activity in the mouse bleeding model. HeMo significantly reduced apixaban-induced bleeding from 709 to 65 μL (vs 43 μL in controls; P < .01) and dabigatran-induced bleeding from 989 to 155 μL (vs 126 μL in controls; P < .01). ConclusionHeMo is a small-molecule procoagulant that can counterbalance hemostatic disruption by a thrombin inhibitor (dabigatran) or factor Xa inhibitors (apixaban and rivaroxaban). The compound’s effective clot formation and versatility make it a possible option for managing the inherent hemorrhagic risk during DOAC therapy.

Pharmacokinetic-Pharmacodynamic Modelling in Hemophilia A: Relating Thrombin and Plasmin Generation to Factor VIII Activity After Administration of a VWF/FVIII Concentrate.

Hemophilia A patients are treated with factor (F) VIII prophylactically to prevent bleeding. In general, dosage and frequency are based on pharmacokinetic measurements. Ideally, an alternative dose adjustment can be based on the hemostatic potential, measured with a thrombin generation assay (TGA), like the Nijmegen hemostasis assay. The objective of this study was toinvestigate the predicted performance of a previously developed pharmacokinetic-pharmacodynamic model for FVIII replacement therapy, relating FVIII dose and FVIII activity levels with thrombin and plasmin generation parameters. Pharmacokinetic and pharmacodynamic measurements were obtained from 29 severe hemophilia A patients treated with pdVWF/FVIII concentrate (Haemate P®). The predictive performance of the previously developed pharmacokinetic-pharmacodynamic model was evaluated using nonlinear mixed-effects modeling (NONMEM). When predictions of FVIII activity or TGA parameters were inadequate [median prediction error (MPE) > 20%], a new model was developed. The original pharmacokinetic model underestimated clearance and was refined based on a two-compartment model. The pharmacodynamic model displays no bias in the observed normalized thrombin peak height and normalized thrombin potential (MPE of 6.83% and 7.46%). After re-estimating pharmacodynamic parameters, EC50 and Emax values were relatively comparable between the original model and this group. Prediction of normalized plasmin peak height was inaccurate (MPE 58.9%). Our predictive performance displayed adequate thrombin pharmacodynamic predictions of the original model, but a new pharmacokinetic model was required. The pharmacodynamic model is not factor specific and applicable to multiple factor concentrates. A prospective study is needed to validate the impact of the FVIII dosing pharmacodynamic model on bleeding reduction in patients.

Low thrombin generation in postmenopausal women using estetrol

Objective Estetrol (E4) represents a novel estrogen of interest to relieve vasomotor symptoms. E4 activates the nuclear estrogen receptor α (ERα) but antagonizes the estradiol ERα-dependent membrane-initiated steroid signaling pathway. The distinct pharmacological properties of E4 could explain its low impact on hemostasis. This study aimed to assess the effect of E4 on coagulation in postmenopausal women, using the thrombin generation assay (TGA). Methods Data were collected from a multicenter, randomized, placebo-controlled, dose-finding study in postmenopausal women (NCT02834312). Oral E4 (2.5 mg, n = 42; 5 mg, n = 29; 10 mg, n = 34; or 15 mg, n = 32) or placebo (n = 31) was administered daily for 12 weeks. Thrombograms and TGA parameters were extracted for each subject at baseline and after 12 weeks of treatment. Results After 12 weeks of treatment, all treatment groups showed a mean thrombogram (±95% confidence interval [CI] of the mean) within the reference ranges, that is, the 2.5th–97.5th percentile of all baseline thrombograms (n = 168), as well as for TGA parameters. Conclusions The intake of E4 15 mg for 12 weeks led to significant but not clinically relevant changes compared to baseline as the mean values (±95% CI of the mean) remained within reference ranges, demonstrating a neutral profile of this estrogen on hemostasis.

Thrombin generation and all-cause mortality in The Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)

Introduction: Thrombin generation assay (TGA) is a laboratory method that provides the global evaluation of hemostasis. The association between thrombin generation and all-cause mortality is poorly investigated and results are contradictory. This study evaluated whether TGA parameters are associated with all-cause mortality in a prospective cohort. Methods: This study was conducted in 2,588 participants enrolled at baseline of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). TGA was performed using the Calibrated Automated Thrombogram (CAT) method, and its parameters lagtime, time-to-peak, peak, Endogenous Thrombin Potential (ETP) and normalized ETP (nETP) were evaluated according to the reference interval (RI). The association between TGA parameters and all-cause mortality was estimated by Cox regression and adjusted for confounders. Results: The mean follow-up time was 6.6 ± 2.7 years and 85 deaths occurred. After adjustment, time-to-peak values above the RI at low and high tissue factor (TF) concentrations were associated with higher risk of death [HR = 2.45 (95 % CI: 1.17–5.13) and HR = 2.24 (95 % CI: 1.02–4.93), respectively] and nETP and peak values below RI at high TF concentration were associated with higher risk of death [HR = 3.85 (95 % CI: 1.39–10.68) and HR = 2.56 (95 % CI: 1.17–5.61), respectively]. Conclusions: Delayed thrombin generation was associated with higher risk of all-cause mortality.

Thrombin generation assay in platelet-poor plasma in children with iron deficiency anemia.

Iron deficiency anemia (IDA) is the most common type of anemia in childhood and it leads to a hypercoagulable state. We investigated endogenous thrombin production in platelet-poor plasma before and after oral iron replacement in children with IDA using the thrombin generation assay (TGA). A total of 72 children diagnosed with IDA (IDA group) and 60 healthy children (control group) were included in the study. Blood samples were collected from the patients before and 1 month after oral iron replacement. TGA parameters [lag time, time to peak, peak height, endogenous thrombin potential (ETP)] were studied. In the IDA group, the lag time and time to peak decreased by 8.3% and 10.6%, respectively, and the endogenous thrombin potential (ETP) and peak height both increased by 30% compared to those of the control group. Compared to the values before iron replacement, 1 month after iron replacement, the lag time and time to peak increased by 8.7% and 5%, respectively, and the ETP and peak height decreased by 31% and 31.3%, respectively, and became similar to those of the control group. Children with IDA have increased endogenous thrombin production in platelet-poor plasma and a tendency for hypercoagulability. These changes are reversible, and the ETP values become similar to those of healthy children 1 month after iron replacement.

Effect of PCC on Thrombin Generation among Patients on Factor Xa Inhibitors with Major Bleeding or Needing Urgent Surgery (GAUGE): Design and Rationale.

Background Direct factor Xa inhibitors (FXaIs) account for most oral anticoagulant use and FXaI-associated bleeding events are common. Clinicians have variable national and regional access to specific FXaI reversal agents such as andexanet alfa. Many centers have adopted the use of prothrombin complex concentrates (PCCs) as hemostatic therapy for FXaI-associated major bleeding events. PCC does not impact circulating FXaI levels and its mechanism of action to achieve hemostasis in FXaI-associated bleeding is uncertain. While PCC increases quantitative thrombin generation assay (TGA) parameters, it does not correct FXaI-altered thrombin generation kinetics, nor does it normalize thrombin generation. Clinical data supporting the use of PCC are based on cohort studies reporting clinical hemostatic efficacy, which is difficult to measure. The benefits of PCC for FXaI-associated bleeding beyond supportive care are uncertain. Objective GAUGE is a prospective observational study designed to measure the effects of four-factor PCC administration (Octaplex) on TGA parameters among patients with FXaI-associated bleeding or needing urgent surgery. Methods Laboratory outcomes will include the mean paired change in TGA parameters from pre- to post-PCC administration and the proportion of participants whose post-PCC TGA values fall within a defined reference range. Clinical outcomes will include hemostatic efficacy, thromboembolic complications, and all-cause death at 30 days post-PCC. Conclusion Development of a viable and universally accessible FXaI bleed management strategy is crucial. GAUGE will provide in vivo data on the effects of PCC among patients with FXaI-associated bleeding.

Thrombin generation in real life bleeding patients on oral anticoagulants reversed (or not) with (activated) prothrombin complex concentrate

BackgroundBleeding during oral anticoagulant therapy is currently codified by expert guidelines. Monitoring of coagulation during bleeding events is challenging. Our study sought to assess thrombin generation assay (TGA) in direct oral anticoagulant-treated patients without bleeding (WB), bleeding without reversal therapy (BR-), and bleeding with reversal therapy (BR+). MethodsWe conducted a prospective, monocentric study from June 2015 to June 2018. For all bleeding groups, TGA was evaluated using platelet-poor plasma collected upon arrival at emergency (T0), and 30 min (T1), 6 h (T2) and 24 h (T3) after reversal therapy (if indicated) following activation by tissue factor 5 pM and phospholipids. ResultsOverall, 292 patients participated, including 91 BR+, 94 BR-, and 107 WB patients. At T0, vitamin K antagonist reversed (VKA-BR+) patients experienced a significant decrease in TGA parameters (ETP and peak) compared with VKA without bleeding (VKA-WB). Compared with healthy controls, VKA-BR+ patients reversed by four-factor prothrombin complex concentrate (4F-PCC) displayed comparable TGA ‘s ETP and peak at T1, T2, and T3, whereas direct anti-Xa BR+ patients reversed by 4F-PCC or activated prothrombin complex concentrate (aPCC) reached thrombin generation parameters that exceeded normal range at T2 and T3. ConclusionsIn VKA-treated patients reversed by 4F-PCC, TGA parameters were normalized, whereas in rivaroxaban or apixaban-treated patients reversed by 4F-PCC or aPCC, TGA parameters exceeded normal range. Further studies are needed to compare the efficacy and safety of a different dose of reversal therapy and the impact on coagulation parameters.

High levels of factor VIII activity in patients with acquired hemophilia A in remission are associated with unusually low coagulation potentials.

Elevated factor VIII activity (FVIII:C) is often observed in patients with acquired hemophilia A (PwAHA) in remission. However, comprehensive coagulation potentials in this patient group remain to be investigated. To evaluate comprehensive coagulation potentials in PwAHA. We investigated coagulation function in eleven PwAHA with high FVIII:C (> 150IU/dL) using thrombin generation assay (TGA) and/or rotational thromboelastometry (ROTEM), and compared findings with results obtained from contrived samples generated by spiking recombinant FVIII. The median FVIII:C and FVIII inhibitor titers during remission in enrolled PwAHA were 206IU/dL and 0.44 BU/mL, respectively. In all patients, lag time and time to peak were either prolonged or normal compared to contrived samples corresponding to their FVIII:C. However, higher values of peak thrombin and endogenous thrombin potentials compared to contrived samples were observed in two patients. ROTEM parameters were within normal ranges in all cases. One patient (FVIII:C 171IU/dL) developed venous thrombosis and pulmonary embolism, but TGA parameters showed low or normal coagulation potential compared to contrived samples corresponding to his FVIII:C. PwAHA with high FVIII:C could exhibit lower coagulation potentials than those corresponding to their FVIII:C.

Profile of Perioperative Coagulation in Major Liver Resection for Cancer: A Prospective Observational Study

Background: The liver plays a key role in the complex and dynamic process of blood coagulation. Consequently, after liver resection the equilibrium between procoagulant and anticoagulant activity is often deranged. Major hepatectomies have been associated with excessive intraoperative blood loss, hemodynamic instability, massive fluid shifts, and coagulopathy. Alterations of the coagulation profile are always present post-operatively even in case of normal pre-operative coagulation function. Cumulative evidence supports the superiority of global coagulation analysis compared with conventional coagulation for clinical decision-making. Aim: The purpose of this study was to assess the perioperative coagulation status of patients undergoing major hepatectomies for primary or metastatic hepatic malignancy. Conventional coagulation tests, plasma levels of coagulation factors, and TGA measurements were used for longitudinal documentation of the coagulation activity. Methods: Patients included in the study were referred for major liver resection which was defined as a resection of three or more liver segments, and were followed up to the 10th post-operative day by serial measurements of conventional coagulation tests, plasma levels of coagulation factors, and thrombin generation assay parameters. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were determined by STA Neoplastin R, and STA-Cephascreen, Fibrinogen by STA-Liquid Fib, D-dimer STA Liatest D-DI Plus, all from Diagnostica Stago, France. Coagulation factor activity for FII, FV, FVII, FVIII, FIX, FX, was measured by clotting assay using coagulation factor deficient plasma, vWFAg , and vWFAc by vWFAg and Innovance vWFAc, from Siemens Healthcare Diagnostics, Germany. Thrombin generation (TG) was assessed in samples of PPP with the TF 5 pM PPP-Reagent on Calibrated Automated Thrombogram (Stago, France). Results: Changes over time for FII, FV, FVII and FX were statistically significant (p <0.05). Factor VIII level was above normal values at baseline. A steady rise was observed for fibrinogen, FVIII, and vWF antigen throughout the study period, but only vWF antigen exhibited statistically significant changes over time. Levels of coagulation inhibitors, AT, PC and PS, decreased significantly during the first postoperative day and then increased reaching baseline values by POD5 for protein C and S, but AT, remained low by POD10. INR increased after surgery, reaching a peak on the first post-operative day and decreasing toward baseline levels from then on (p <0.001). The aPTT remained within normal range throughout the study period (Table 1). TG conducted at baseline, on POD1, POD5, and POD10 showed elevated of Lag time and time to peak at baseline, whereas the rest thrombin generation parameters were within reference range. ETP decreased significantly over time, reaching lower values by POD5 and sustaining those low values by POD10. Peak height fluctuated in a similar manner, but changes over time did not bear any statistical significance. Lag-time, which was prolonged at baseline, exhibited a steady rise throughout the study period, bearing borderline statistical significance (Table 2) . A statistically significant positive correlation was detected between AT and time to Peak (ttPeak) at baseline, however any other significant difference between coagulation factors, natural coagulation inhibitors, and TGA parameters was observed. Conclusion: This study shows that patients with hepatic malignancy have systematic endothelial activation and hyper-coagulability as indicated by increased FVIII levels and lag time prolongation in thrombin generation. These patients are in a hyper-coagulable state in the early post-operative period, as shown by increased vWF, FVIII, D-dimer, fibrinogen and decreased levels of natural coagulation inhibitors AT, PC, and PS. Measurements of INR, PT, aPTT are not useful in this setting and could be misleading. Global coagulation analysis may be more appropriate for clinical guidance. To the best of our knowledge this is the first report in literature documenting the pre-existing coagulation disturbances in patients with hepatic carcinoma and their impact on the coagulation status after major hepatectomy. However, the clinical implications of our findings deserve further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Global Hemostatic Assays for Risk Stratification of Thrombosis in Myeloproliferative Neoplasms (MPN): A Systematic Review and Meta-Analysis

Background: Arterial and venous thromboembolism are major complications of myeloproliferative neoplasms (MPN), which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The pathogenesis of thrombosis in MPN is not fully understood. Global hemostatic assays, including thrombin generation assay (TGA), rotational thromboelastometry (ROTEM), and thromboelastography (TEG), have been evaluated as predictive markers of hypercoagulability in MPNs. However, validation of these and other laboratory surrogates is lacking. The objective of this systematic review is to summarize the literature and to examine the utility of global hemostatic assays as predictive markers for hypercoagulability and occurrence of thrombosis in MPNs. Methods: A systematic literature search was performed using MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews from inception to April 14, 2022, restricted to the English language. Criteria for eligible studies were studies that reported the results of TGA, ROTEM, and TEG in ET, PV, or PMF adult patients. Studies were required to have ≥10 subjects in each group. Two reviewers (A.T. and T.C.) independently searched the literature and extracted data from eligible studies. Disagreements were resolved by consensus or a third reviewer (C.K) when necessary. The methodological quality of included studies was appraised using the Newcastle - Ottawa Quality Assessment Scale (NOS) assessment tool. A meta-analysis was planned given sufficient data from included studies. The study protocol is available on PROSPERO (CRD42022325893). Results: A total of 815 records were retrieved from the literature search. After screening by title and abstract, 745 records were excluded. The remaining 70 references underwent full text review, 30 of which met eligibility criteria and were included in the analysis. These 30 studies (15 full-texts and 15 abstracts) collectively enrolled 926 controls, 700 ET, 556 PV, and 192 PMF patients. The number of studies that reported TGA, ROTEM, and TEG were 26, 3, and 3, respectively. The number of participants in each study ranged from 25 to 212 including the controls. Two studies originated from Oceania, 1 from North America, and 27 from Europe. JAK2 mutation was reported only in 15 studies and only two studies reported quantitative variant allele frequencies. There were 5 studies that did not have healthy controls. All of these studies were cross sectional analyses; no longitudinal prospective age/sex matched controlled studies have been published. Of the 26 TGA studies, comparison of TGA parameters were made between MPN vs. controls (15 studies), MPN with thrombosis vs. without thrombosis (3 studies), JAK2 positive vs. negative MPNs (11 studies), and MPN with vs. without treatment (6 studies). There is heterogeneity in the types of samples (platelet poor plasma or platelet rich plasma), trigger reagents (amounts of tissue factor, phospholipid, and thrombomodulin), and the automated platforms that were used. Significant differences in the endogenous thrombin potential (9 studies), peak height (10 studies), lag time (6 studies), and velocity index (3 studies) were found between MPN and controls. Thirteen studies reported rates of arterial and venous thrombotic outcomes in MPN patients, which ranged from 19 to 45%. Only 1 study found an association of TGA parameters and thrombosis. Two ROTEM studies showed ET having higher maximum clot firmness and shorter clot formation time compared to controls. Two TEG studies showed increased alpha angle between MPN and controls and between PV and controls. Risk of bias assessment with NOS showed summary scores ranging 1-3 for abstracts and 4-9 for full text studies. We were not able to perform meta-analysis due to insufficient data and the high heterogeneity in study objectives and laboratory methods. Conclusions: Studies were limited by small sample sizes and inconsistent results and do not comprehensively reflect the effects of treatment, duration of disease, or clonal status. The role of these global hemostatic assays in predicting thrombotic events remains inconclusive. Future studies are necessary to establish the usefulness and validity of these surrogate markers as predictors of thrombotic complications in the MPNs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigation of thrombin generation assay to predict vaso-occlusive crisis in adulthood with sickle cell disease.

IntroductionSickle cell disease (SCD) is an inherited hemoglobinopathy disorder. The main consequence is synthesis of hemoglobin S leading to chronic hemolysis associated with morbidity. The aim of this study was to investigate Thrombin Generation Assay (TGA) to assess hypercoagulability in SCD and TGA parameters as biomarkers of vaso-occlusive crisis (VOC) risk and hospitalization within 1 year.Materials and methodsWe performed TGA in platelet poor plasma (PPP) with 1 pM of tissue factor and 4 μM of phospholipid-standardized concentration, in duplicate for patients and controls. We measured thrombomodulin (TM), soluble endothelial Protein C Receptor and Tissue Factor Pathway Inhibitor (TFPI).ResultsA total of 113 adult patients with SCD, 83 at steady state and 30 during VOC, and 25 healthy controls matched on age and gender were included. Among the 83 patients at steady state, (36 S/S-1 S/β0, 20 S/Sα3.7, and 19 S/C-7 S/β+) 28 developed a VOC within 1 year (median: 4 months [2.25–6]). We observed an increase of peak and velocity associated with a shortening of lagtime and time to peak (TTP) and no difference of endogenous thrombin potential (ETP) in patients compared to controls. TFPI (p < 0.001) and TM (p = 0.006) were significantly decreased. TGA confirmed hypercoagulability in all SCD genotypes and clinical status. The association of ETP > 1,207 nM.min and peak >228.5 nM presented a sensitivity of 73.5% and a specificity of 93.9% to predict VOC development within 1 year.ConclusionWe have demonstrated a hypercoagulable state in SCD associated with chronic hemolysis. These preliminary findings suggest that TGA parameters, as ETP and peak, could be used to predict VOC development within 1 year.

Perioperative Coagulation Profile in Major Liver Resection for Cancer: A Prospective Observational Study

Hepatectomy-induced coagulation disturbances have been well studied over the past decade. Cumulative evidence supports the superiority of global coagulation analysis compared with conventional coagulation tests (i.e., prothrombin time or activated partial thromboplastin time) for clinical decision making. Cancer, however, represents an acquired prothrombotic state and liver resection for cancer deserves a more thorough investigation. This prospective observational study was conducted to assess the perioperative coagulation status of patients undergoing major hepatectomies for primary or metastatic hepatic malignancy. Patients were followed up to the 10th post-operative day by serial measurements of conventional coagulation tests, plasma levels of coagulation factors, and thrombin generation assay parameters. An abnormal coagulation profile was detected at presentation and included elevated FVIII levels, decreased levels of antithrombin, and lag time prolongation in thrombin generation. Serial hematological data demonstrated increased Von Willebrand factor, FVIII, D-dimer, fibrinogen and decreased levels of natural anticoagulant proteins in the early post-operative period predisposing to a hyper-coagulable state. The ratio of the anticoagulant protein C to the procoagulant FVIII was low at baseline and further declined post-operatively, indicating a prothrombotic state. Though no bleeding complications were reported, one patient experienced pulmonary embolism while under thromboprophylaxis. Overall, patients with hepatic carcinoma presenting for elective major hepatectomy may have baseline malignancy-associated coagulation disturbances, aggravating the hyper-coagulable state documented in the early post-operative period.

Comparison among three different bleeding scores and the thrombin generation assay to assess the different hemorrhagic phenotypes in patients with FVII deficiency

Defining the bleeding risk in patients with FVII-deficiency is not easy. Aim of this study is to define correlation and differences between three different scores and the thrombin generation assay (TGA) in correctly evaluating the hemorrhagic phenotype in a group of FVII-deficient patients. Fifty-seven patients patients with FVII-deficiency whose hemorrhagic phenotype was assessed by Mariani, ISTH/SSC- Bleeding Assessment Tool (BAT) and Di Minno scores, and by the TGA, were enrolled in this study. TGA parameters (LagTime, Peak, ttPeak, ETP - endogenous thrombin potential) highlighted how both LagTime and ttPeak can discriminate major bleeders from the others, while the same conclusion could not be reached by the ETP and Peak. However, no TGA parameter was found to be useful in separating the mild hemorrhagic phenotype from the moderate one. Scores and TGA were found to be able to only define the severe hemorrhagic phenotypes. None of the methods was able to exactly discriminate the other phenotypes. Given these results, there is therefore a risk of either underestimating or overestimating the potential bleeding risk in patients with non-severe FVII-deficiency.

Report of surgeries, their outcome and the thrombin generation assay in patients with Factor XI deficiency: A retrospective single-centre study.

In patients with FXI deficiency, the risk of surgery-related bleeding is poorly correlated with plasma FXI activity (FXI:C); the latter can therefore not be used as a reliable predictor of bleeding in surgeries. The aim of this retrospective study was to determine whether thrombin generation assay (TGA) could be used to evaluate the risk of surgery-related bleeding in FXI-deficient patients. TGA parameters were compared to FXI:C values, haemostatic treatments and surgical outcomes. All patients followed at the haemophilia treatment care centre (Lyon, France) with a FXI:C<50IU/dL, and for whom a baseline TGA was performed between January 2014 and December 2019, were included. Among the 175 surgeries reported herein in 49 patients, FXI concentrates were used for 11 (6%) surgeries and fresh frozen plasma was used for five (3%) surgeries; these surgeries were performed in patients with two or three impaired TGA parameters. No haemostatic treatment was prescribed for 119 (68%) surgeries. A surgery-related bleeding occurred in 12 patients during 21 (12%) surgeries. Thrombin generation was significantly reduced or delayed in patients who reported surgery related-bleeding. Among the 34 (68%) surgeries performed without haemostatic treatment in patients with three impaired TGA parameters, a surgery-related bleeding was reported in 44% of cases (15 surgeries out of 34). The present study confirmed that TGA is an interesting laboratory test in FXI deficiency, for determining the bleeding risk and guiding the haemostatic management of surgeries, while taking into account the surgical bleeding risk and the history of bleeding.

Cerebrovascular events in patients with isolated anti-phosphatidyl-serine/prothrombin antibodies

The interest of extra-criteria antiphospholipid antibodies is growing, especially in patients negative for conventional antibodies. In this study we aimed to assess the clinical utility of anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) testing in patients negative for Beta2-Glycoprotein 1(β2GPI)-dependent tests, for identifying antiphospholipid syndrome (APS) patients that developed cerebrovascular events (CVE). When screening APS patients attending our center, out of 119 aPS/PT IgG/IgM-positive patients, thus patients negative for aβ2GPI and aCL, 42 patients (35%) tested negative for β2GPI-dependent tests and were tested with thrombin generation assay (TGA). Ten patients (24%), with isolated aPS/PT IgG/IgM, had a history of CVE. Lupus anticoagulant (LA)-positive test was more frequently observed in patients with CVE (8/22 vs. 2/20; p = 0.045). Out of the 10 patients who experienced CVE, 3 patients were aPS/PT IgG positive (all LA positive), and 8 patients were aPS/PT IgM positive (6/8 LA positive). One patient was positive for both aPS/PT IgG and IgM. LA-positive patients had only high titers of aPS/PT IgG/IgM, all of them being ≥ 80 U/ml, while the 2 LA-negative patients were aPS/PT IgM positive with medium titers [40–60 U/ml]. LA-positive patients had significantly altered TGA profile when compared to those who were LA negative, considering all TGA parameters. LA-positive patients had significantly higher tLag (8.4 ± 3.3 min vs. 6.6 ± 1.8 min; p = 0.046), higher tPeak (14 ± 4.3 min vs. 11 ± 2.7 min; p = 0.015) and lower Peak (207 ± 152 nM vs. 356.3 ± 104.7 nM; p < 0.001) and lower AUC (2109.7 ± 1006.9 nM vs. 2772.5 ± 776.8 nM; p = 0.033). The use of aPS/PT might be of help in identifying patients with CVE and APS, as also confirmed by TGA testing.

Coagulation Kinetics in Adolescent Females Initiating Combined Oral Contraceptives

Objectives: The objective of this pilot study was to evaluate the effect of combined oral contraceptives (COC) on rotational thromboelastometry (ROTEM®) and Technothrombin®TGA (thrombin generation assay) coagulation kinetics, as a surrogate marker of thrombotic risk, in adolescent females. The epidemiology of risk factors associated with contraception-related venous thromboembolism (VTE) in adolescents is not well defined. Estrogen contained in COC produces activated protein C resistance thus increasing the risk of thrombosis. The risk of COC-related VTE is significantly impacted by duration of COC use. The highest risk of COC-related VTE is during the first 6 to 12 months in first-time users. Furthermore, the risk of contraception-related VTE is higher for obese women as compared with non-obese women. Recent evidence found that endogenous thrombin potential (ETP) increased during COC initiation, but have not followed users longitudinally.Methods: This longitudinal cohort included adolescents, ages 12-21 years, initiating COC containing 35 mcg ethinyl estradiol/0.25 mg norgestimate. Balanced recruitment was utilized to strive for equal numbers of obese and non-obese subjects and varied gynecologic age (i.e. years from menarche). Exclusion criteria included personal/family history of thrombophilia or venous thromboembolism, personal history of bleeding diatheses, contraindication to COC, tobacco use, hemoglobin < 10 g/dL, and anti-platelet or anticoagulant medication use. Citrated whole blood samples were collected at baseline (pre-COC), 1, 3, 6, and 12 months for coagulation studies (TGAÒ and ROTEMÒ) and FSH suppression to document COC adherence. TGA parameters included: ETP and peak thrombin production. ROTEMÒkinetic parameters evaluated included: clotting time (CT); viscosity amplitude at 10 & 20 minutes (A10 & A20); and maximum clot firmness (MCF). Changes in parameters were explored over 6 months of COC exposure for coagulation kinetics and 12 months for ETP.Results: Interim analyses included 15 subjects out of a planned 30 total (86.7% White; mean age 16.8 ± 2.8 years; mean BMI 25.1 kg/m2, BMI range 16.2-43.3 kg/m2, 33.3% obese). Post-COC, mean ETP was elevated above baseline (3176.8 nM*min) as early as 1 month and was highest at 6 months (3869.6 & 3890.8 nM*min; p<0.05) (Figure 1A).Mean peak thrombin production was increased from baseline to 6 months (179.5 vs 224.7 nM; p<0.05). From baseline to 3 months, there was a significant (p<0.05) increase in mean A10 (48.0 vs. 52.1 mm), A20 (53.8 vs. 58.6 mm), and MCF (54.0 vs. 58.6 mm) (Figures 1B,1C).There was a significant decrease in mean CT from baseline to 6 months (252.6 vs. 210.6 seconds; p<0.05).Conclusions: After COC initiation, adolescents demonstrated hypercoagulable alterations in TGA and all ROTEM® kinetic parameters. These alterations were evident as early as 1 month but continued to increase over the next 5 months. Continued study will assess if hypercoagulable alterations persist at 12 months. Further study will explore the impact of obesity status at COC initiation on thrombotic risk, as assessed by these global coagulation assays. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An in vitro pharmacodynamic spiking study of befovacimab, a tissue factor pathway inhibitor monoclonal antibody, in blood samples from patients with severe FVIII deficiency.

Tissue factor pathway inhibitor (TFPI) is an endogenous protein that inhibits the extrinsic (tissue factor) pathway and negatively regulates thrombin production during coagulation. Inhibiting TFPI may become a useful target for haemophilia drug development to allow greater thrombin generation without use of the intrinsic (contact) pathway. The in vitro effects of befovacimab, a humanized TFPI neutralizing antibody, were studied in whole blood and plasma samples from patients with severe FVIII deficiency. Blood and plasma obtained from participants was supplemented in vitro with befovacimab (0.5, 1, 5, 10 and 100nM) or recombinant factor VIII (rFVIII) 5-, 10- and 40% and analysed using rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay. The in vitro coagulation effects of befovacimab were compared to samples supplemented with rFVIII. Befovacimab induced consistent pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in α-angle; induced reductions in dPT clotting time; and improvements in TGA parameters (reduced lag time and increased thrombin generation parameters). There was a modest concentration-dependent response generally from 0.5- to 10nM, after which, the pharmacodynamic effect plateaued through the 100nM concentration. Befovacimab concentrations of 5 to 10nM showed pro-coagulant activity comparable to blood samples supplemented with rFVIII 10-40%. Befovacimab has modest dose-response effects from 0.5 to 10nM with minimal improvement with higher concentrations. In vitro befovacimab blood concentrations of 5 to 10nM had pro-coagulant effects similar to blood supplemented with rFVIII 10- to 40%.