Thrombin Peak Height Research Articles

Enhanced thrombin and plasmin generation profiles in alpha-2-antiplasmin–deficient patients: Data from the Rare Bleeding disorders in the Netherlands study

Backgroundα2-Antiplasmin (A2AP) deficiency is a rare and often unidentified disorder characterized by increased fibrinolysis and subsequent bleeding. Global hemostasis assays may increase insight into the altered coagulation and fibrinolysis in these patients. ObjectivesTo explore thrombin and plasmin generation profiles in A2AP-deficient patients, corresponding A2AP activity levels and associated bleeding phenotypes. MethodsThe Nijmegen hemostasis assay was used to assess thrombin and plasmin generation in 23 A2AP-deficient patients (median age, 50 years; 70% women) from the cross-sectional Rare Bleeding disorders in the Netherlands study. Analyzed parameters included thrombin peak height, thrombin potential, fibrin lysis time, plasmin peak height, plasmin velocity index, and plasmin potential. These parameters were expressed as percentages of a reference obtained from 37 healthy controls (median age, 46 years; 57% women). The Nijmegen hemostasis assay data were correlated with A2AP activity levels and International Society on Thrombosis and Hemostasis Bleeding Assessment Tool scores using Pearson correlation coefficients. ResultsPatients’ A2AP activity levels ranged from 23% to 83% (reference range, 89%-122%). Plasmin generation increased, as evidenced by significantly shorter fibrin lysis times (73%; P < .001) and higher plasmin peak heights (203%; P < .001), plasmin velocity indices (302%; P < .001) and plasmin potentials (154%; P < .001) in A2AP-deficient patients than those in healthy controls. Moreover, significantly higher thrombin potentials (146%; P < .001) and thrombin peak heights (132%; P < .001) were observed. Enhanced plasmin generation parameters showed statistically significant correlations with lower A2AP activity levels and higher International Society on Thrombosis and Hemostasis Bleeding Assessment Tool scores. ConclusionA2AP-deficient patients exhibited augmented plasmin generation profiles that correlated with A2AP activity level and bleeding phenotype. Interestingly, increased thrombin generation profiles were also found in these patients.

Investigation of thrombin concentration at the time of clot formation in simultaneous thrombin and fibrin generation assays

Thrombin generation (TG) and fibrin clot formation represent the central process of blood coagulation. Up to 95% of thrombin is considered to be generated after the clot is formed. However, this was not investigated in depth. In this study, we conducted a quantitative analysis of the Thrombin at Clot Time (TCT) parameter in 5758 simultaneously recorded TG and clot formation assays using frozen plasma samples from commercial sources under various conditions of activation. These samples were supplemented with clotting factor concentrates, procoagulant lipid vesicles and a fluorogenic substrate and triggered with tissue factor (TF). We found that TCT is often close to a 10% of thrombin peak height (TPH) yet it can be larger or smaller depending on whether the sample has low or high TPH value. In general, the samples with high TPH are associated with elevated TCT. TCT appeared more sensitive to some procoagulant phenotypes than other commonly used parameters such as clotting time, TPH or Thrombin Production Rate (TPR). In a minority of cases, TCT were not predicted from TG parameters. For example, elevated TCT (above 15% of TPH) was associated with either very low or very high TPR values. We conclude that clotting and TG assays may provide complementary information about the plasma sample, and that the TCT parameter may serve as an additional marker for the procoagulant potential in plasma sample.

Endogenous thrombin potential and time-dependent thrombin generation parameters are independent risk factors for mortality in the general population

BackgroundThrombin generation (TG) is used as a global test of coagulation and is an indicator of thrombosis and bleeding risk. Until now, data on the association of TG and mortality are inconclusive. ObjectivesWe investigated the association between TG and mortality in the prospective Moli-sani cohort (n = 21 920). MethodsTG was measured using calibrated automated thrombinography using PPP-Reagent Low. Lag time (LT), endogenous thrombin potential (ETP), peak height, time-to-peak (TTP), and velocity index were quantified. The association of TG and mortality was studied by Cox regression and adjusted for sex, age, body mass index, smoking, contraceptives, and medical history (cardiovascular diseases, hypertension, hypercholesterolemia, diabetes, and cancer). ResultsLT and TTP were 4.1 ± 1.0 minutes and 6.6 ± 1.5 minutes, on average. The peak height was 364 ± 88 nM, velocity index was 163 ± 63 nM/min, and ETP was 1721 ± 411 nM·min. ETP was negatively associated with all-cause mortality (hazard ratio [HR], 0.86; 95% CI, 0.81-0.92; P < .001). Subjects in the lowest quintile of the ETP (ETPQ1) had a 1.3-fold higher mortality rate. Additionally, a high TTP/LT ratio was negatively associated with mortality (HR, 0.71; 95% CI, 0.57-0.89; P = .003). Individuals in quintile 1 of the TTP/LT ratio had a 1.4-fold higher mortality rate compared with the remainder of the cohort. Subjects that were both in ETPQ1 and TTP/LTQ1 had a 1.8-fold higher mortality rate, regardless of whether they reported history of cardiovascular disease at baseline (HR, 1.61 [CI: 1.07-2.42]) or not (HR, 1.89 [CI: 1.51-2.36]). ConclusionLow ETP and TTP/LT ratios are independent risk factors for all-cause mortality in the general population.

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.

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.

A Short Thrombin Generation Time-to-Peak/Lag Time Ratio Is an Independent Risk Factor for Mortality in the General Population

Background Thrombin generation (TG) is used to detect small changes in coagulation indicating a bleeding or prothrombotic phenotype. Until now, large prospective studies are missing to study the association of TG parameters with the occurrence of future cardiovascular events and mortality. In this study, we investigated the prospective value of TG measured in baseline plasma samples of a large cohort (n=20,991) with overall mortality (1,098; 5.3%) with a median follow-up of 8.2 years. Methods Thrombin generation was measured by calibrated automated thrombinography (CAT) using PPP Reagent low. The lag time (LT), endogenous thrombin potential (ETP), peak height, time-to-peak (TTP) and velocity index (VI) were quantified. Subjects on anticoagulant therapy were excluded from analysis, because this affects the measurement of TG. Cox regression was used to analyze differences in survival in subjects according to their variations in the TG parameters. The reported results are obtained from a crude model considering only single TG parameters standardized for their standard deviation, and from a final multivariable model further adjusted for sex, age, BMI, smoking, contraceptive use, antiplatelet medication and medical history (hypertension, hypercholesterolemia, diabetes, history of cardiovascular diseases or cancer). Results The TG lag time at 1 pM tissue factor was 4.1 min ± 1.0 on average, and the peak height was 363 nM ± 89. The time-to-peak was 6.6 min ± 1.6 on average, the velocity index was 163 nM/min ± 63 and the endogenous thrombin potential was 1717 nM·min ± 415. The SD-standardized lag time was significantly associated with mortality risk in the crude model (HR LT=1.09; CI:1.03-1.16; p=0.002) and after adjustment for all covariates, including medical history (HR LT=1.09; CI: 1.03-1.15; p=0.002). In contrast, a short time-to-peak was significantly associated with reduced mortality risk (HR TTP=0.92; CI: 0.86-0.98; p=0.012). As the opposite associations of LT and TTP with mortality could have a synergistic effect on the mortality risk, we combined the variables into the novel TTP/LT-ratio. The mean TTP/LT ratio was 1.61 ± 0.14. The hazard ratio for mortality decreased by 40% for each standard deviation increment of the TTP/LT-ratio in the crude model (HR ratio=0.606; CI:0.562-0.654; p&amp;lt;0.001), and by 18% in the fully adjusted model (HR ratio=0.824; CI: 0.766-0.887; p&amp;lt;0.001). The TTP/LT-ratio variable was divided in quintiles for further analysis: Quintile 1 included subjects up to a TTP/LT-ratio of 1.50, quintile 2 ranged from 1.50 to 1.58, quintile 3 ranged from 1.58 to 1.62, quintile 4 ranged from 1.62 to 1.70, and quintile 5 consisted of subjects with a TTP/LT-ratio above 1.70. Subjects in TTP/LT-ratio quintile 1 had an increased mortality risk (HR ratio_Q1vsQ5 =2.96; CI: 2.42-3.63; p&amp;lt;0.001), even after full adjusting (HR ratio_Q1vsQ5 =1.46; CI: 1.18-1.79; p&amp;lt;0.001; Figures 1 and 2). Moreover, subjects in TTP/LT-ratio quintile 2, have a similar statistically significant, although less pronounced increase risk for mortality in the crude model (HR ratio_Q1vsQ5 =2.17; CI: 1.18-2.68; p&amp;lt;0.001) and the fully adjusted model (HR ratio_T2vsT5 =1.29; CI: 1.04-1.60; p=0.019; Figure 1). Conclusion A prolonged TG lag time and a shortened time-to-peak in the general population and within the normal ranges are risk factors for mortality. The combination of a long LT and a short TTP results in a low TTP/LT-ratio, which was identified as an independent risk factor for increased mortality risk in the general population.

Blood Coagulation and Platelet Function Are Impaired By the ARP2 p.R258G Variant in Mice

Introduction: Venous thromboembolism (VTE) displays significant heritability, yet the genes explaining this heritability are not completely characterized. We previously identified that tissue factor pathway inhibitor heterozygosity ( Tfpi+/−) on the homozygous F5L background ( F5L/L) produces a lethal perinatal thrombotic phenotype. Using a sensitized ENU mutagenesis screen, we discovered an autosomal dominant missense mutation in the Actr2 gene (p.R258G, Actr2G) that suppresses thrombosis and restores survival to F5L/LTfpi+/− mice. Inheritance of the Actr2Gmutation is associated with nearly complete suppression of the lethal F5L/LTfpi+/- phenotype. Actr2 codes for the ARP2 protein, an essential component of the ARP2/3 complex, which mediates actin cytoskeletal rearrangements during platelet activation. We hypothesized that the Actr2G variant affects blood coagulation and platelet function to reduce thrombosis. Methods: To investigate potential effects of the Actr2G mutation on the coagulation cascade, prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma-dependent thrombin generation assays (TGA) were used to evaluate clot formation. A mouse tail clip assay was used to measure bleeding time and quantify blood loss. Finally, platelets were assessed for functional defects using platelet spreading and aggregometry assays. Results: Actr2 +/Gplasma had significantly prolonged PT and aPTT compared to wild type controls (N&amp;gt;17, p&amp;lt;0.03). There was also a significant reduction in Actr2+/G thrombin peak height compared to wildtypes (N=10; p=0.03). Despite the delayed time to clot formation, Actr2+/G mice did not exhibit a bleeding phenotype and showed no significant differences in blood loss from tail clip assays. Activated Actr2+/G platelets exhibited reduced surface area and adhesion (N=5; p&amp;lt;0.02), as well as significant defects in collagen induced whole blood aggregation (N=10; p&amp;lt;0.02). Discussion: The results of the PT, aPTT and TGA indicate reduced thrombin generation potential, suggesting a defect in the common pathway of coagulation. This hypocoaguability may be due to a decrease in circulating procoagulant factors, or an increase in endogenous thrombin inhibitors. Both the significantly decreased aggregation and defective platelet spreading in the Actr2+/G mice indicate that Actr2+/G platelets are less responsive to agonists, a protective mechanism that contributes to overall thrombosis suppression. Conclusion: In summary, our studies demonstrate the pleiotropic effects that the Actr2G mutation exerts in suppressing thrombosis. The combination of altered plasma coagulation and platelet reactivity likely contribute to suppression of the F5L/LTfpi+/− phenotype. Defects in these actin-mediated processes precipitated by Actr2+/G are the likely root of the F5L/LTfpi+/- phenotype rescue, demonstrating the power of this gene in maintaining hemostatic balance. However, the combination of these thrombosis suppressing phenotypes is not significant enough to prolong the bleeding time in mice with the Actr2G mutation. Thus, our results suggest that targeting actin proteins such as ARP2 for novel antithrombotic drug therapies may have clinical applicability with limited effects on bleeding.

Patients with moderate hemophilia A and B with a severe bleeding phenotype have an increased burden of disease

BackgroundPatients with moderate hemophilia express varying bleeding phenotypes. ObjectivesTo assess the burden of disease in patients with moderate hemophilia and a mild or severe phenotype incorporating the thrombin generation profile. MethodsThis sub-study of the 6th Hemophilia in the Netherlands study, analyzed data of adults with moderate hemophilia A or B. Patient characteristics and information on bleeding tendency, joint status, and quality of life were obtained from electronic patient files and self-reported questionnaires. A severe bleeding phenotype was defined as an annual bleeding rate ≥5, an annual joint bleeding rate ≥3, and/or the use of secondary/tertiary prophylaxis, and a mild phenotype vice versa. TG was measured with the Nijmegen Hemostasis Assay. ResultsThis study included 116 patients: 21% had a severe phenotype of whom 46% used prophylaxis. Patients with a severe phenotype treated on demand reported a higher median annual bleeding rate (7), annual joint bleeding rate (3), and more frequently an impaired joint (77%) than patients with a severe phenotype on prophylaxis (2; 0; 70%) or patients with a mild phenotype (0; 0; 47%). Furthermore, patients with a severe phenotype treated on demand experienced a more decreased quality of life. Despite similar factor activity levels, patients with a severe phenotype had a lower thrombin peak height and thrombin potential (0.7%; 0.06%) than patients with a mild phenotype (21.3%; 46.8%). ConclusionPatients with moderate hemophilia and a severe phenotype treated on demand displayed a high burden of disease as well as a low thrombin generation profile advocating them toward more intensive prophylactic treatment.

Comparative Thrombin Generation in Animal Plasma: Sensitivity to Human Factor XIa and Tissue Factor.

Preclinical evaluation of drugs in animals helps researchers to select potentially informative clinical laboratory markers for human trials. To assess the utility of animal thrombin generation (TG) assay, we studied the sensitivity of animal plasmas to triggers of TG, human Tissue Factor (TF), and Activated Factor XI (FXIa). Pooled human, mouse, rat, guinea pig, rabbit, bovine, sheep, and goat plasmas were used in this study. TF- or FXIa-triggered TG and clotting were measured via fluorescence and optical density, respectively. Thrombin peak height (TPH) and time (TPT), clot time (CT), and fibrin clot density (FCD) were all analyzed. The trigger low and high sensitivity borders (LSB and HSB) for each assay parameter were defined as TF and FXIa concentrations, providing 20 and 80% of the maximal parameter value, unless the baseline (no trigger) value exceeded 20% of the maximal, in which case, LSB was derived from 120% of baseline value. Normal human samples demonstrated lower TPH HSB than most of the animal samples for both TF and FXIa. Animal samples, except mice, demonstrated lower TPT LSB for FXIa versus humans. Most rodent and rabbit samples produced baseline TG in the absence of TG triggers that were consistent with the pre-activation of blood coagulation. FCD was not sensitive to both TF and FXIa in either of the plasmas. Animal plasmas have widely variable sensitivities to human TF and FXIa, which suggests that optimization of trigger concentration is required prior to test use, and this complicates the extrapolation of animal model results to humans.

Elevated thrombin generation levels in the left atrial appendage in patients with atrial fibrillation

BackgroundAtrial Fibrillation (AF) is the most common sustained tachi-arrhythmia. Thrombus formation in the left atrial appendage (LAA) increases the risk of stroke and systemic embolism in patients with AF. ObjectivesThe aim of this study was to compare thrombin generation in the LAA to the LA among patients with AF. MethodsA cross-sectional study of consecutive patients with AF undergoing pulmonary veins catheter ablation. Blood samples from the femoral vein (FV), right atrium (RA), left atrium (LA), and LAA were collected during the catheter ablation procedures. Thrombin generation was assessed by a Calibrated Automated Thrombogram. The LAA-calibrated automated thrombogram parameters were compared with the RA, LA, and FV. ResultsA total of 47 consecutive patients were enrolled in the study. The endogenous thrombin potential and peak height were significantly higher in the LAA compared with the LA, the mean differences and 95% CI between the LA and LAA were -378.9 (-680.5, -77.2) (nM∗min) and -66.7 (-119.6, -13.8) (nM) in the endogenous thrombin potential and peak height respectively. ConclusionIn patients with AF undergoing catheter ablation, the LAA demonstrated increased thrombin generation compared with the LA. This finding might contribute to the understanding of why the LAA is more predisposed to thrombus formation than the LA. Clinical Trials RegistrationNCT03795883

In patients with hemophilia, a decreased thrombin generation profile is associated with a severe bleeding phenotype

BackgroundHeterogeneity in clinical bleeding phenotype has been observed in hemophilia patients with similar FVIII or FIX activity levels. Thrombin generation and plasmin generation, as a global hemostasis assay, may contribute to a better prediction of which patients are at an increased risk of bleeding. ObjectivesThe objective of this study was to describe the association between clinical bleeding phenotype and thrombin generation and plasmin generation profiles in patients with hemophilia. MethodsThe Nijmegen Hemostasis Assay, which simultaneously measures thrombin and plasmin generation, was performed in plasma samples of patients with hemophilia participating in the sixth Hemophilia in the Netherlands study (HiN6). Patients receiving prophylaxis underwent a washout period. A severe clinical bleeding phenotype was defined as a self-reported annual bleeding rate of ≥5, a self-reported annual joint bleeding rate of ≥3, or the use of secondary/tertiary prophylaxis. ResultsIn total, 446 patients, with a median age of 44 years, were included in this substudy. Thrombin generation and plasmin generation parameters differed between patients with hemophilia and healthy individuals. The median thrombin peak height was 1.0 nM, 25.9 nM, 47.1 nM, and 143.9 nM in patients with severe, moderate, and mild hemophilia and healthy individuals, respectively. A severe bleeding phenotype was observed in patients with a thrombin peak height of <49% and a thrombin potential of <72% compared to healthy individuals, and was independent of the hemophilia severity. The median thrombin peak height was 0.70% in patients with a severe clinical bleeding phenotype and 30.3% in patients with a mild clinical bleeding phenotype. The median thrombin potentials for these patients were 0.06% and 59.3%, respectively. ConclusionA decreased thrombin generation profile is associated with a severe clinical bleeding phenotype in patients with hemophilia. Thrombin generation in combination with bleeding severity may be a better tool to personalize prophylactic replacement therapy irrespective of hemophilia severity.

Assessment of DOAC in GEriatrics (Adage Study): Rivaroxaban/Apixaban Concentrations and Thrombin Generation Profiles in NVAF Very Elderly Patients.

Although a growing number of very elderly patients with atrial fibrillation (AF), multiple conditions, and polypharmacy receive direct oral anticoagulants (DOACs), few studies specifically investigated both apixaban/rivaroxaban pharmacokinetics and pharmacodynamics in such patients. To investigate: (1) DOAC concentration-time profiles; (2) thrombin generation (TG); and (3) clinical outcomes 6 months after inclusion in very elderly AF in-patients receiving rivaroxaban or apixaban. Adage-NCT02464488 was an academic prospective exploratory multicenter study, enrolling AF in-patients aged ≥80 years, receiving DOAC for at least 4 days. Each patient had one to five blood samples at different time points over 20 days. DOAC concentrations were determined using chromogenic assays. TG was investigated using ST-Genesia (STG-ThromboScreen, STG-DrugScreen). We included 215 patients (women 71.1%, mean age: 87 ± 4 years), 104 rivaroxaban and 111 apixaban, and 79.5% receiving reduced-dose regimen. We observed important inter-individual variabilities (coefficient of variation) whatever the regimen, at C max [49-46%] and C min [75-61%] in 15 mg rivaroxaban and 2.5 mg apixaban patients, respectively. The dose regimen was associated with C max and C min plasma concentrations in apixaban (p = 0.0058 and p = 0.0222, respectively), but not in rivaroxaban samples (multivariate analysis). Moreover, substantial variability of thrombin peak height (STG-ThromboScreen) was noticed at a given plasma concentration for both xabans, suggesting an impact of the underlying coagulation status on TG in elderly in-patients. After 6-month follow-up, major bleeding/thromboembolic event/death rates were 6.7%/1.0%/17.3% in rivaroxaban and 5.4%/3.6%/18.9% in apixaban patients, respectively. Our study provides original data in very elderly patients receiving DOAC in a real-life setting, showing great inter-individual variability in plasma concentrations and TG parameters. Further research is needed to understand the potential clinical impact of these findings.

Prognostic value of an automated thrombin generation assay in COVID-19 patients entering hospital: A multicentric, prospective observational study

IntroductionThe prognostic significance of the thrombin generation assay (TGA) with a thrombomodulin (TM) challenge in patients entering hospital with severe COVID-19 is uncertain. MethodsWe prospectively evaluated an automated TGA (aTGA) using the ST-ThromboScreen® assay and ST-Genesia® analyser in 179 patients with severe COVID-19 during their admission to 2 university hospitals. The primary outcome was early survival at Day 28 (D28). Secondary outcomes were late survival at Day 90 (D90), later transfer to an intensive care unit (ICU), and occurrence of any thrombotic complications during hospitalisation. ResultsAmong the 174 patients, 50 were initially admitted to ICUs. Forty-two were transferred to ICUs before D28. Fourteen patients, all in ICUs, died before D28, and 20 before D90, all but 1 in ICUs. None of the aTGA-derived results were associated with vital status either at D28 or D90. Nine patients had a thrombotic event with no association with the aTGA results. Later transfer to the ICU was associated with higher velocity index, thrombin peak height and endogenous thrombin potential (ETP) values of the aTGA performed with TM, and mainly with a lower TM-induced decrease in ETP (odds ratio 15.5 (2.15–132), p = 0.009). ConclusionsaTGA, a global assay supposed to evidence coagulopathy, could predict neither early or late survival, nor thrombotic events, in hospitalised COVID-19 patients. Its clinical justification in that setting is thus unlikely. A relative resistance of the ETP to TM was associated with later transfer to the ICU and deserves further investigation.

Sex-dependent balance between thrombin and plasmin generation in the presence of thrombomodulin.

Assessing simultaneous generation of thrombin (TG) and plasmin (PG) is an approach to evaluate the balance between coagulation and fibrinolysis with sensitivity to predict endogenous thrombin and plasmin generation. The addition of thrombomodulin (TM), provides the essential component for thrombin activation of protein C and thrombin-activatable fibrinolysis inhibitor. However, the influence of sex on the balance between TG and PG with and without TM addition has not been investigated to date. To investigate the possible sex-based differences in TG and PG in the presence and absence of TM. Simultaneous TG and PG were measured in plasma samples obtained from 17 males and 17 females upon tissue factor and tissue plasminogen activator addition. Thrombin- and plasmin-specific fluorogenic substrates Z-Gly-Gly-Arg-AMC and Boc-Glu-Lys-Lys-AMC were used in the study. Thrombin and plasmin peak height (TPH and PPH) and production rate (TPR and PPR) values were determined. To evaluate the balance between TG and PG, the ratios between TPH and PPH (TPH/PPH) and TPR and PPR (TPR/PPR) were calculated. TPH between males and females demonstrated significant difference regardless of TM addition. TPR demonstrated differences between males and females only upon TM addition, while PG parameters was not dependent on the sex of the donor. TM significantly lowered TPH/PPH in males, and enhanced TPR/PPR in females. Thus, TPH/PPH and TPR/PPR significantly differed between men and women. Our results indicate that TM may act differently in males and females by shifting the underlying TG/PG balance to fibrinolysis in males and to coagulation in females.

Translational insights into mechanisms underlying residual venous obstruction and the role of factor XI, P-selectin and GPVI in recurrent venous thromboembolism

BackgroundResidual venous obstruction (RVO) after deep vein thrombosis (DVT) is considered a risk factor of recurrent venous thromboembolism (VTE), arterial events and post-thrombotic syndrome (PTS). We hypothesized thrombo-inflammatory markers might be associated with RVO and clinical outcomes. Materials and methodsIn a DVT cohort with routine RVO-assessment and 5-year follow-up, patients were invited for blood withdrawal after stopping anticoagulants. Thrombin generation potential, coagulation enzyme:inhibitor complexes, soluble platelet markers and clinical markers were measured in platelet-poor plasma. Associations were represented as odds ratio (OR) or hazard ratio (HR) per standard deviation. ResultsPatients with RVO (102/306, 33 %) had higher rates of PTS (24 vs. 12 %, p = 0.008), but similar rates of recurrence (16 vs. 15 %, p = 0.91) and arterial events (7 vs. 4 %, p = 0.26). RVO was associated with thrombin peak height (OR 1.40 [1.04–1.88]), endogenous thrombin potential (ETP, OR 1.35 [1.02–1.79]), and CRP (OR 1.74 [1.10–2.75]). Recurrent VTE was associated with ETP (HR 1.36 [1.03–1.81]), FXIa:C1-inhibitor (HR 1.34 [1.04–1.72]), thrombin:antithrombin (HR 1.36 [1.16–1.59]), soluble P-selectin (HR 2.30 [1.69–3.11]), soluble glycoprotein VI (sGPVI, HR 1.30 [1.01–1.69]), D-dimer (HR 1.56 [1.31–1.86]), and factor VIII (HR 1.44 [1.15–1.82]). Arterial events were associated with sGPVI (HR 1.80 [1.25–2.59]). PTS was not associated with any marker. ConclusionsOur findings indicate RVO was associated with thrombo-inflammation, but this did not predict clinical outcomes in this setting. Importantly, we found recurrent VTE was associated with ongoing coagulation and platelet activation in patients well beyond the acute phase of DVT. Furthermore, sGPVI indicated an increased risk of arterial events, highlighting the role of platelets in arterial thrombosis following DVT.

Development of a Quantitative Systems Pharmacology Model to Explore Hemostatic Equivalency of Antithrombin Lowering

Fitusiran is an investigational siRNA therapeutic that targets antithrombin (AT) to rebalance hemostasis in people with hemophilia A (PwHA) or B (PwHB), irrespective of inhibitor status. Phase 3 clinical studies have demonstrated that fitusiran prophylaxis significantly reduced bleeding events compared to on-demand or prophylaxis treatment with factor concentrates or bypassing agents (Srivastava et al. Blood 2021; Young et al. Blood 2021; Kenet et al. ISTH 2022). To better understand the hemostatic equivalency of fitusiran prophylaxis (i.e., AT lowering), a quantitative systems pharmacology (QSP) model was developed to investigate thrombin generation in PwHA and PwHB in the context of AT lowering. QSP modeling is a mechanistic approach that integrates clinical and nonclinical data of the coagulation pathway to simulate the results of multiple in vitro coagulation assays, including the thrombin generation assay (TGA) and to mechanistically understand the readouts of these assays. The QSP model builds on prior published models of the coagulation cascade (Attarwala et al. ISTH 2017; Sridharan et al. Blood 2017), updated to represent reported steady-state levels of coagulation factors and accessory proteins in the plasma of healthy individuals and PwHA and PwHB (Figure 1). In addition, the model was extended to describe the impact of α-2-macroglobulin, a key regulator of thrombin, with increased influence at reduced AT levels that is critical to understand the impact of fitusiran on thrombin generation (Kattula et al. Blood 2020). After representing coagulation dynamics from plasma from healthy individuals, PwHA, and PwHB, the QSP model was applied to simulate TGA and relevant metrics, including thrombin peak height (Figure 1). TGA simulations were performed across different percentages of AT and FVIII. These simulations reproduced experimental TGA data from AT- and FVIII-double depleted plasma that was spiked with different levels of AT and/or FVIII. For these in vitro experiments, Factor VIII (FVIII) and AT-double depleted plasma was generated from congenital FVIII-deficient plasma by immunodepleting AT using an anti-AT antibody and recombinant AT protein was spiked back in to achieve different levels of AT. The QSP simulations also reproduced the reported formation of α-2-macroglobulin complexes with thrombin in each condition. Subsequently, the model was validated using both internal and published TGA and activated partial thromboplastin time (aPTT) datasets that were not used during model development. Next, a large (n=1000) virtual population (VP) of severe PwHA (<1% FVIII) was generated based on calibration of the model to individual patient data from completed fitusiran clinical studies. This analysis considered individual patient pharmacokinetic and AT time courses to describe thrombin peak height associated with different levels of AT reduction from fitusiran prophylaxis within a representative population of severe PwHA. The target therapeutic AT range of fitusiran prophylaxis treatment at steady state is 15-35%. Based on this model, the peak thrombin generated at 15-35% AT was similar to peak thrombin observed at 10-20 IU/kg FVIII in PwHA. In summary, by considering the impact of α-2-macroglobulin, the QSP model provides a mechanistic representation of thrombin generation under conditions of AT lowering, consistent with internal TGA data from donor-derived spiked plasma and clinical TGA data from both PwHA and PwHB. The VP analysis of fitusiran prophylaxis provided hemostatic equivalency with FVIII in a representative population of severe PwHA, with a targeted therapeutic range of AT of 15-35% resulting in a thrombin peak profile which was comparable to 10-20 IU/kg FVIII. 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

A comprehensive method to explore inhibitory kinetics and mechanisms of an anticoagulant peptide derived from Crassostrea gigas

A comprehensive method was applied to evaluate the anticoagulant activity of a novel anticoagulant peptide (NAESLRK) derived from oyster (Crassostrea gigas). The anticoagulant peptide drastically reduced the extrinsic clotting activity and also impaired the intrinsic clotting activity slightly. Consistent with clotting data, the thrombin peak height was reduced to 84.7 nmol/L from 123.4 nmol/L, and thrombin generation time was delayed to 4.67 min from 4.42 min when the extrinsic trigger was applied. The inhibitory kinetics of FXIa, FIXa, FXa, FIIa, and APC in a purified component system rationally explained the reduction of extrinsic clotting activity and impairment of thrombin generation. Besides the inhibition of FXa and FIIa activity, the activation processes of FX and FII by intrinsic/extrinsic tenase complex and prothrombinase were also damaged. The anticoagulant activity in the plasma system was the result of comprehensive inhibition of various factors. The research provided a novel method for anticoagulant evaluation and inhibitory mechanism of bioactive peptides from food products.

Combining factor VIII levels and thrombin/plasmin generation: A population pharmacokinetic-pharmacodynamic model for patients with haemophilia A.

AimsProphylactic treatment of haemophilia A patients with factor VIII (FVIII) concentrate focuses on maintaining a minimal trough FVIII activity level to prevent bleeding. However, due to differences in bleeding tendency, the pharmacokinetic (PK)‐guided dosing approach may be suboptimal. An alternative approach could be the addition of haemostatic pharmacodynamic (PD) parameters, reflecting a patient's unique haemostatic balance. Our aim was to develop a population PK/PD model, based on FVIII activity levels and Nijmegen Haemostasis Assay (NHA) patterns, a global haemostatic assay that measures thrombin/plasmin generation simultaneously.MethodsPK/PD measurements were collected from 30 patients treated with standard half‐life FVIII concentrate. The relationship between FVIII activity levels and the thrombin/plasmin generation parameters (thrombin potential, thrombin peak height and plasmin peak height), were described by sigmoidal E max functions.ResultsThe obtained EC50 value was smallest for the normalized thrombin potential (11.6 IU/dL), followed by normalized thrombin peak height (56.6 IU/dL) and normalized plasmin peak height (593 IU/dL), demonstrating that normalized thrombin potential showed 50% of the maximal effect at lower FVIII activity levels. Substantial inter‐individual variability in the PD parameters, such as EC50 of thrombin potential (86.9%) was observed, indicating that, despite similar FVIII activity levels, haemostatic capacity varies significantly between patients.ConclusionThese data suggest that dosing based on patients' individual PK/PD parameters may be beneficial over dosing solely on individual PK parameters. This model could be used as proof‐of‐principle to examine the application of PK/PD‐guided dosing. However, the relation between the PD parameters and bleeding has to be better defined.

Thrombin generation in children using ThromboScreen reagent kit with ST Genesia—A pilot study

Thrombin generation assays assess overall coagulation system and are widely used in research; however, they still need standardization and clinical validation. The new ST Genesia is a benchtop, automated analyzer that normalizes each thrombin generation parameter using a reference plasma. The ThromboScreen reagent kit has two triggers, one of which contains thrombomodulin to assess the effect of the protein C pathway. This study aimed to make a pilot approach to the ThromboScreen reference range in children and evaluate the impact of sex, age, and pro- and anticoagulant plasma proteins on thrombin generation parameters. This study included 55 healthy children from the following age groups: 1-6years (n=14), 7-11years (n=15), and 12-17years (n=26). Children younger than 1year were excluded from the study. We measured thrombin generation using ThromboScreen, coagulation routine and test, pro- and anticoagulant proteins. Age did not influence ThromboScreen results. Males showed significantly lower endogenous thrombin potential and peak height values than females. The strongest determinants of endogenous thrombin potential were von Willebrand factor parameters, whereas for endogenous thrombin potential inhibition, the strongest determinants were protein C and protein S. No statistically significant differences were found betweengroups on temporal parameters. For the ThromboScreen reagent kit, it may not be necessary to subdivide reference ranges according to age for children (>1year).