Thrombin Generation Test Research Articles

Hemostasis in patients with primary myelofibrosis treated with ruxolitinib

Aim. To evaluate the effect of ruxolitinib therapy on hemostasis in patients with primary myelofibrosis (PMF).Materials and methods. 30 patients with PMF were examined, 16 of them received ruxolitinib (group 1) at the time of examination and 14 were untreated (group 2). The control group consisted of 30 healthy individuals. A complete blood count was performed, the platelets aggregation, the activity of Willebrand factor, factor VIII and natural anticoagulants were evaluated. In the thrombin generation test, the endogenous thrombin potential (ETP, nM×min) was determined; sensitivity to thrombomodulin and coagulation index were calculated. Kruskal-Wallis test with post hoc Dunn test was used to compare groups.Results. Hemoglobin and platelet count were lower in group 1 compared to group 2 and control. Platelet aggregation with collagen was lower in patients with PMF than in the control group, and lower in group 1 than in group 2: 2.2 (1.6; 5.7) % vs 41.6 (3.4; 64.8) %, p < 0.05. In patients in group 1, the activity of Willebrand factor – 150.0 (122.5; 195) % and factor VIII – 173 (148.5; 200) % was higher (p < 0.05) than in the control: 97 (84.8; 110) % and 104 (85; 130) % respectively. Antithrombin did not differ in the PMF and control group. Protein S was reduced in both groups with PMF: group 1 – 70 (58; 86,6) %, group 2 – 65 (43,6; 107,5) %, control – 102 (86; 109,0) %, p < 0.001. ETP and sensitivity to thrombomodulin in groups 1 and 2 were low, p < 0.001. The coagulation index tended to higher values than in the control: 5.3 (3.0; 11.4) – group 1; 3.2 (2.4; 7.3) – group 2; control – 1.9 (1.6; 2.2), p = 0.073.Conclusion. Ruxolitinib therapy leads to the failure of the platelet hemostasis, the procoagulant activity of plasma with an increase in the activity of Willebrand factor and factor VIII and a decrease in the activity of protein S.

Dysregulated neutrophil extracellular traps and haemostatic biomarkers as diagnostic tools and therapeutic targets in periprosthetic joint infection.

Bacterial infection activates neutrophils to release neutrophil extracellular traps (NETs) in bacterial biofilms of periprosthetic joint infections (PJIs). The aim of this study was to evaluate the increase in NET activation and release (NETosis) and haemostasis markers in the plasma of patients with PJI, to evaluate whether such plasma induces the activation of neutrophils, to ascertain whether increased NETosis is also mediated by reduced DNaseI activity, to explore novel therapeutic interventions for NETosis in PJI in vitro, and to evaluate the potential diagnostic use of these markers. We prospectively recruited 107 patients in the preoperative period of prosthetic surgery, 71 with a suspicion of PJI and 36 who underwent arthroplasty for non-septic indications as controls, and obtained citrated plasma. PJI was confirmed in 50 patients. We measured NET markers, inflammation markers, DNaseI activity, haemostatic markers, and the thrombin generation test (TGT). We analyzed the ability of plasma from confirmed PJI and controls to induce NETosis and to degrade in vitro-generated NETs, and explored the therapeutic restoration of the impairment to degrade NETs of PJI plasma with recombinant human DNaseI. Finally, we assessed the contribution of these markers to the diagnosis of PJI. Patients with confirmed PJI had significantly increased levels of NET markers (cfDNA (p < 0.001), calprotectin (p < 0.001), and neutrophil elastase (p = 0.022)) and inflammation markers (IL-6; p < 0.001) in plasma. Moreover, the plasma of patients with PJI induced significantly more neutrophil activation than the plasma of the controls (p < 0.001) independently of tumour necrosis factor alpha. Patients with PJI also had a reduced DNaseI activity in plasma (p < 0.001), leading to a significantly impaired degradation of NETs (p < 0.001). This could be therapeutically restored with recombinant human DNaseI to the level in the controls. We developed a model to improve the diagnosis of PJI with cfDNA, calprotectin, and the start tail of TGT as predictors, though cfDNA alone achieved a good prediction and is simpler to measure. We confirmed that patients with PJI have an increased level of NETosis in plasma. Their plasma both induced NET release and had an impaired ability to degrade NETs mediated by a reduced DNaseI activity. This can be therapeutically restored in vitro with the approved Dornase alfa, Pulmozyme, which may allow novel methods of treatment. A combination of NETs and haemostatic biomarkers could improve the diagnosis of PJI, especially those patients in whom this diagnosis is uncertain.

Heterozygous Prothrombin Mutation-Associated Thrombophilia.

Venous thromboembolism (VTE) is predisposed by thrombotic mutations in patients with hereditary thrombophilia. Although prothrombin deficiencies caused by homozygous or compound heterozygous mutations are associated with bleeding diathesis, rare cases have shown a correlation between heterozygous prothrombin mutations and thrombosis. We surveyed genetic variants involved in thrombosis and hemostasis in 347 patients with unprovoked VTE or having a positive family history of thrombosis. For patients identified with heterozygous prothrombin mutations, we conducted family investigations and performed a thrombin generation test (TGT) to elucidate the thrombotic risk. Novel mutants were expressed and subjected to functional assays to clarify the underlying thrombotic mechanisms. Heterozygous prothrombin mutations were identified in 3.5% of patients (12/347), including three novel mutations Phe382Ser, Phe382Leu, and Asp597Tyr found in one patient each, as well as previously reported Arg541Trp mutation in four patients and Arg596Gln mutation in five patients. A total of 42 mutation carriers were identified within the 12 pedigrees, among whom 64.3% (27/42) had experienced thrombotic events. TGT results demonstrated hypercoagulability for carriers of the five mutations, with Arg596Gln showing the highest thrombin generation potential followed by Arg541Trp. The Phe382-associated mutations severely impaired thrombomodulin-binding ability of thrombin, resulting in obviously reduced protein C (PC) activation. The Asp597Tyr mutation exhibited a mild reduction in both inactivation by antithrombin and PC activation reactions. The presence of heterozygous prothrombin mutations represents a potential genetic predisposition for VTE. All thrombosis-associated mutations potentiate coagulation activity by either conferring antithrombin resistance and/or impairing PC pathway activity.

Thrombin Generation Profile Using ST-Genesia after PEG-asparaginase in Pediatric Patients with Acute Lymphoblastic Leukemia.

Venous thromboembolism (VTE) etiology in children with acute lymphoblastic leukemia (ALL) is multifactorial. The use of global assays of hemostasis as a thrombin generation test (TGT) is useful to individualize VTE risk in adult patients. This prospective cohort study aimed to evaluate the usefulness of an automated TGT to evaluate VTE risk during ALL treatment in children. TGT (automated analyzer ST Genesia; ThromboScreen) and pro- and anticoagulant plasma proteins were analyzed during ALL treatment in pediatric patients following LAL-SEHOP-PETHEMA-2013 guidelines. Results were compared with a series of pediatric normal controls and evaluated according to pegylated asparaginase PEG-ASP administration and to VTE risk factors. The study included 67 patients: males n = 35, B-ALL (n = 60). None had a VTE during the evaluated period. Compared to healthy controls, the normalized endogenous thrombin potential (N-ETP) ratio in patients was higher and ETP inhibition (ETP-inh) was lower, especially after PEG-ASP administration. Plasmatic protein C and protein S levels decreased after PEG-ASP administration, but antithrombin mean level did not. A bivariant analysis showed that ETP-inh was lower in patients >10 years old (p = 0.05) and in those with non-O blood type (p = 0.005). A linear mixed model also showed a higher TGT prothrombotic profile in patients with inherited thrombophilia. TGT could be a biomarker of a high VTE risk in ALL pediatric patients. Non-O blood group and inherited thrombophilia were associated with a significantly higher thrombotic profile, and an increased profile was also observed after administration of PEG-ASP.

Long-term anticoagulant therapy in the aspect of secondary prevention of recurrent pulmonary embolism

Aim. To determine differences in laboratory coagulation parameters using routine and integral monitoring methods in patients depending on anticoagulant therapy &gt;12 months after pulmonary embolism (PE) with a high recurrence risk.Material and methods. The study included 72 patients who, according to indications, received long-term anticoagulant therapy &gt;12 months due to a high risk of recurrent thromboembolism. During the follow-up period (15-20 months from the first pulmonary embolism episode), the plasma coagulation parameters were studied using a conventional panel and D-dimer assessment, as well as a thrombin generation test and thrombodynamics. Patients adherent to prolonged anticoagulation were included in group 1. During the collection period, 8 patients independently stopped taking anticoagulants, despite the high risk of recurrent pulmonary embolism (group 2).Results. There were no significant differences in coagulation parameters and D-dimer levels between the studied groups of patients. In the group of patients continuing to take anticoagulants, there was an increase in clot growth delay parameters in comparison with patients of group 2 according to the thrombodynamics results (p=0,046) and the thrombin generation test in platelet-poor plasma in comparison with reference values (p=0,001).Conclusion. Long-term anticoagulant therapy effectively prevents recurrent venous thromboembolism. In patients after primary pulmonary embolism who refuse anticoagulation, the clot growth delay values are shortened, which indicates an increased risk of recurrent venous thromboembolism. The most stable plasma coagulation parameters are observed in patients taking direct oral anticoagulants.

A thrombin-driven neural net diagnoses the antiphospholipid syndrome without the need for interruption of anticoagulation

AbstractThrombosis is an important manifestation of the antiphospholipid syndrome (APS). The thrombin generation (TG) test is a global hemostasis assay, and increased TG is associated with thrombosis. APS is currently diagnosed based on clinical and laboratory criteria, the latter defined as anti-cardiolipin, anti–β2-glycoprotein I antibodies, or lupus anticoagulant (LA). APS testing is often performed after a thrombotic episode and subsequent administration of anticoagulation, which might hamper the interpretation of clotting assays used for LA testing. We set out to develop an artificial neural network (NN) that can diagnose APS in patients who underwent vitamin K antagonist (VKA) treatment, based on TG test results. Five NNs were trained to diagnose APS in 48 VKA-treated patients with APS and 64 VKA-treated controls, using TG and thrombin dynamics parameters as inputs. The 2 best-performing NNs were selected (accuracy, 96%; sensitivity, 96%-98%; and specificity, 95%-97%) and further validated in an independent cohort of VKA-anticoagulated patients with APS (n = 33) and controls (n = 62). Independent clinical validation favored 1 of the 2 selected NNs, with a sensitivity of 88% and a specificity of 94% for the diagnosis of APS. In conclusion, the combined use of TG and NN methodology allowed for us to develop an NN that diagnoses APS with an accuracy of 92% in individuals with VKA anticoagulation (n = 95). After further clinical validation, the NN could serve as a screening and diagnostic tool for patients with thrombosis, especially because there is no need to interrupt anticoagulant therapy.

A novel GATA1 variant p.G229D causing the defect of procoagulant platelet formation

IntroductionGATA1 is one of the master transcription factors in hematopoietic lineages development which is crucial for megakaryocytic differentiation and maturation. Previous studies have shown that distinct GATA1 variants are associated with varying severities of macrothrombocytopenia and platelet dysfunction. ObjectiveTo determine the underlying pathological mechanisms of a novel GATA1 variant (c. 686G > A, p. G229D) in a patient with recurrent traumatic muscle hematomas. MethodsComprehensive phenotypic analysis of the patient platelets was performed. Procoagulant platelet formation and function were detected using flow cytometry assay and thrombin generation test (TGT), respectively. The ANO6 expression was measured by qPCR and western blot. The intracellular supramaximal calcium flux was detected by Fluo-5N fluorescent assay. ResultsThe patient displayed mild macrothrombocytopenia with defects of platelet granules, aggregation, and integrin αIIbβ3 activation. The percentage of the procoagulant platelet formation of the patient upon the stimulation of thrombin plus collagen was lower than that of the healthy controls (40.9 % vs 49.0 % ± 5.1 %). The patient platelets exhibited a marked reduction of thrombin generation in platelet rich plasma TGT compared to the healthy controls (peak value: ∼70 % of the healthy controls; the endogenous thrombin potential: ∼40 % of the healthy controls). The expression of ANO6 and intracellular calcium flux were impaired, which together with abnormal granules of the patient platelets might contribute to defect of procoagulant platelet function. ConclusionsThe G229D variant could lead to a novel platelet phenotype characterized by defective procoagulant platelet formation and function, which extended the range of GATA1 variants associated platelet disorders.

Laboratory evaluation of the hormonal agents effects on the plasma hemostasis system in women of reproductive age

Background: With the evolution of hormonal contraception, such as implementation of low dose agents, new regiments and administration routes of contraceptives, the risk of thrombotic complications persists, although is decreasing. The search for an available test for hemostasis assessment, which would allow for the prediction of thrombotic complications in high risk patients, remains relevant. Global tests for hemostasis assessment, such as thrombin generation test and thrombodynamics test, are attracting the specialists' attention due to the possibility of a quick integrative assessment of plasma hemostasis, especially during choice and tailoring of an optimal version of hormonal therapy.&#x0D; Aim: To assess the value of the integral thrombodynamics test in the multifactorial assessment of hemostasis system in women of reproductive age using hormonal contraceptives.&#x0D; Materials and methods: This observational prospective comparative study included 408 women aged 18 to 49 years followed from 2018 to 2022 in The Nikiforov Russian Center of Emergency and Radiation Medicine and Centre of Miscarriage Prevention and Treatment of the Maternity Hospital No. 1, St. Petersburg. From these, 208 women (mean age, 38.0 ± 7.0 years) were taking hormonal contraceptives (HC+ group), and 200 women (mean age, 37.5 ± 9.2 years) were in the control group (HC-). In the HC+ group, 163 women were taking combination oral contraceptives, 24 had an intrauterine levonorgestrel-releasing system, 8 used vaginal rings, and 13 were using progestin only oral contraceptives. Multifactorial clinical and laboratory assessment included taking past history, measurement of pro- and anticoagulation blood parameters, fibrinolysis, and endothelial function parameters (automatic coagulometer ACL TOP 500, Instrumentation Laboratory, USA). The integral assessment of the hemostasis system was performed with Thrombodynamics Registrator T-2 (Hemacor, Russia).&#x0D; Results: The groups were similar as per their age, chronic venous insufficiency and smoking. The HC+ group had significantly higher proportion of women with cardiovascular disorders (p = 0.0037), obesity (p = 0.0004), and headache (p 0.0001), compared to the HC- group. The thrombodynamics test showed a significantly higher rate of clot formation in the women taking hormonal contraception, compared to that in the HC- group (36.2 [30.1; 43.6] and 30.3 [28.0; 33.6] mcm/min, respectively, р 0.001). Hypercoagulation identified by the thrombodynamics test was associated with slowing down of XIIа-dependent fibrinolysis (6 [5; 8] min in the HC- and 12.8 [8; 16] min in the HC+ group, p 0,001) and higher levels of endothelial dysfunction markers (FVIII, 113 [85; 156] and 150 [107; 180]%, p = 0.015; vFW, 98 [85; 133] and 146 [95; 168]%, respectively, p = 0.003). The analysis of plasma hemostasis parameters depending on the presence of thromboembolic risk factors has shown that higher number of the risk factors is associated with higher velocity parameters in the thrombodynamics test in the range of chronometric hypercoagulation.&#x0D; Conclusion: The integral thrombodynamics test for the assessment of plasma hemostasis allows for identification of hemostasis dysfunction in women taking hormonal contraceptives. To prevent the risk of thromboembolic complications, it is desirable to assess blood coagulation system parameters before administration of hormonal agents.

Evaluation of Innovative Laboratory Tests to Predict a Thrombotic Phenotype in a Family with Dysfibrinogenemia and a Novel FGG Mutation

Background: Hypodysfibrinogenemia is a rare hereditary fibrinogen disorder characterized by quantitative and qualitative fibrinogen defects. These fibrinogen defects can cause thrombotic and hemorrhagic phenotypes. Unfortunately, predicting the phenotype in a specific patient is often not possible with routine coagulation tests. Aims: To characterize the phenotype and the genetic profile of a family with hypodysfibrinogenemia and to investigate the ability of innovative tests to predict bleeding and/or thrombotic phenotypes in asymptomatic family members. Methods: The proband, a 60-year-old woman with both bleeding and thrombotic complications who is currently on DOAC treatment, and two daughters were referred to our Hemophilia Treatment Center (HTC) for phenotypical and genotypical analysis of a congenital fibrinogen disorder (CFD). Extensive laboratory testing was done, as well as DNA-sequence analysis and molecular modelling. Thrombin generation and microfluidic testing were also performed to investigate their ability in phenotype prediction. Results: Fibrinogen activity and antigen levels led to the diagnosis of dysfibrinogenemia in the proband and hypodysfibrinogenemia in both daughters. In all three cases, the same heterozygous missense mutation in the FGG gene was uncovered. This likely pathogenic mutation leads to the p.(Tyr375Cys) amino acid change. Molecular modeling predicted possible conformational changes or covalent dimerization of the fibrinogen molecule. Thrombin generation was elevated in one daughter. Microfluidic testing showed enhanced fibrin formation in both daughters, regardless of the coagulation trigger. Conclusion: We described a family with hypodysfibrinogenemia in whom a novel heterozygous missense mutation in the FGG gene was found, possibly leading to conformational changes or covalent dimerization of the fibrinogen molecule. Furthermore we showed that microfluidic testing and thrombin generation can indicate a thrombotic phenotype in these patients, not detected with routine coagulation tests.

Reference intervals and biological variation in parameters of the thrombin generation test in healthy individuals.

Establish the referenceintervals (RIs) and analyze biological variability (BV) to introduce the thrombin generation test (TGT) into clinical practice. To determine the RIs parameters of TGT, we analyzed platelet-poor plasma (PPP) (n = 123), rich (PRP) (n = 76), and microparticle-mediated TGT (MP-TGT) (n = 32) in healthy participants. For the BV study, we collected samples from five participants over 5 weeks. A nested analysis of variance (ANOVA) was performed to evaluate the BV results. The between-individual variation (CVG ), within-individual variation (CVI ), analytical variation (CVA ) for TGT on PPP for all parameters were from 5.5% to 17.3%, 5.4% to 17.7%, and 2.6% to 5.3%, respectively. For PRP, the CVG , CVI , and CVA were ranged from 3.0% to 23.7%, 8.4% to 23.0%, and 4.1% to 6.9%, respectively. The index of individuality (II) ranged from 0.3 to 3.1 for PPP and from 0.3 to 4.5 for PRP. The reference change value (RCV) for PPP was from 19.8% to 50.1%, while for PRP, it was 27.2% to 66.5%. We recommend using the RIs for the parameters ETP (nM/min): 1101.6-2332.1 and Peak (nM): 163.5-381.3 for PPP and ETP (nM/min): 1088.5-2634.9; Peak (nM): 72.6-210.7 for PRP. The resulting MP-TGT are highly dependent on age require a larger sample. For TGT on PPP and PRP the RIs developed on our population for Peak and ETP parameters can be used. Time parameters: Lagtime and ttPeak, min with II < 0.6, require monitoring over time with RCV calculation.

Endothelial dysfunction as predictor of atherosclerosis during a translatitudinal sea voyage in the Arctic: prospective study

INTRODUCTION. Human labor activity in the Arctic takes place in severe climatic conditions associated with cooling, atmospheric pressure drops, high humidity, photoperiodicity, low oxygen content in the air. Endothelial dysfunction is an early pathophysiological sign and an independent predictor of poor prognosis in most cardiovascular diseases.&#x0D; OBJECTIVE.Тo analyze the dynamics of the level of metalloproteinase-9 and thrombin activity as predictors of the development of atherosclerosis in sailors during a translatitudinal sea voyage in the Arctic.&#x0D; MATERIALS AND METHODS. A prospective clinical and laboratory study of crew members was performed during the integrated marine research expedition “Transarctic-2019”. A clinical and laboratory study of the level of metalloprotease-9 (enzyme-linked immunosorbent assay), coagulation analysis of the parameters of the thrombin generation test, as well as ultrasonic dopplerography of the thickness of the intima-media complex were carried out. Statistical data processing was carried out using the methods of descriptive and analytical statistics in the StataCorp Stata 14.2 program. The study protocol was approved by the local ethical committee of the SSMU (protocol No. 03/5 dated May 27, 2015) and supported by the state task reg. No. NIOKTR 121030300111-7.&#x0D; RESULTS. A statistically significant increase in the level of MMP-9 in sailors after the expedition was noted, the relationship between the level of MMP-9, indicators of the thrombin generation test (lag time, min (p=0.0190), tPeak (p=0.0177), Peak (p= 0.0217) and KIM thickening.&#x0D; DISCUSSION. MMP-9 levels may be a predictor of early atherosclerosis and atherosclerotic plaque instability, as well as a risk factor for future adverse cardiovascular events. The relationship of MMP-9 with the kinetics of thrombin (one of the regulators of the antithrombotic state of the endothelium) during a translatitudinal voyage in sailors indicates the formation of readiness for prothrombotic events.&#x0D; CONCLUSION. Thus, in Arctic conditions, under conditions of chronic oxidative stress, which contribute to the progression of the development of endothelial dysfunction in the presence of external risk factors, the likelihood of progression of atherosclerotic vascular lesions increases, and, as a result, the development of vascular events.

Global tests in patients with Ph-negative myeloproliferative neoplasms

Introduction: A frequent clinical manifestation of Ph-negative myeloproliferative neoplasms (MPN) is the development of thrombosis. To identify the state of hypercoagulation it is relevant and promising to introduce global tests for evaluating the hemostasis — the thrombin generation test (TGT) and thromboelastography (TEG). Aim: to evaluate the parameters of thrombin generation and thromboelastography in patients with Ph-negative MPN. Material and methods. In total, 62 patients with MNP were included in the study: 27 with polycythemia vera (PV), 14 with essential thrombocythemia (ET) and 21 with primary myelofibrosis (PMF). The control group included 55 practically healthy individuals, comparable in gender and age (19 people in the study of TEG, 36 people in the study of TGT). The TEG was performed using a “TEG 5000” thromboelastograph. TGT was measured with Calibrated Automated Thrombinography. Results: Ly30 and Ly60 in TEG in patients were significantly lower (0.35 (0.20–0.48), 0.00 (0.00–0.40) and 0.00 (0.00 — 0.43) and 3.15 (2.45–3.60), 1.25 (0.10–3.58) and 0.60 (0.00–3.05) respectively), than in the control (1.60 (1.05–2.75) and 6.20 (4.15–8.30), respectively), which indicates the ineffectiveness of fibrinolysis. The values of MA and G in patients with ET and PV significantly exceeded the control (69.15 (67.98–70.78) mm and 65.20 (59.65–63.83) mm versus 62.00 (57.75–6.75) mm and 11.20 (10.60–12.15) din/cm2 and 9.40 (7.40–11.60) din/cm2 versus 8.20 (6.85–8.75) din/cm2, respectively. The most pronounced change in sensitivity to TM was observed in patients with ET (27.94 (17.35–43.58) % and 13.29 (-3.48–23.60) %, respectively; p &lt; 0.05). A significant decrease in ETP was observed in patients with PV and PMF. Conclusion. The study of hemostasis in patients with MPN using TEG and TGT revealed the presence of multidirectional changes associated with the disease. The TEG showed that an increase in the time required for the onset of fibrin formation is combined with increased clot strength and inhibited fibrinolysis, which are risk factors for the development of thromboembolic complications. The study of TGT determined a decrease in the quantitative characteristics of thrombin generation and at the same time the failure of the anticoagulant system of protein C, leading to the development of hypercoagulation.

Descriptive and Epidemiological Study of Joint Damage in Non-Severe Hemophilia: Update of the Damage Study of the Mediterranean Group

Introduction: There is growing evidence indicating joint damage (JD) in non-severe hemophilia (nSH) patients. Although age and baseline factor (F) VIII/FIX levels have been proposed as good predictors of JD in mild hemophilia (MiH), data are scarce and in moderate hemophilia (MoH) are lacking. Besides, global hemostatic capacity (GHC) in this population and its contribution to JD has not been assessed. Methods: We designed a cross-sectional, observational and multicenter study carried out in 12 Spanish hospitals (Mediterranean Group). We evaluated JD in nSH patients and its correlation with age, baseline FVIII/FIX and GHC. JD was defined as a HEAD-US score &amp;gt;0. Baseline FVIII (chromogenic assay), FIX (clotting assay) and GHC (assessed by thrombin generation test, TGT, Genesia ®, Stago) were evaluated in a central laboratory. Results were expressed as median (25 th-75 th percentiles). Categorical variables were expressed with percentage. A p value&amp;lt;0.05 was considered statistically significant. Results: A total of 98 nSH patients were included: 10 (10.2%) MoH (9 HA: age= 35.6 [21.7-45.0] years old (YO); FVIII = 4.0 [4.0-5.0] IU/dL; 1 HB: FIX= 2 IU/dL) and 88 MiH (84 HA: age= 38.6 [19.0-52.1 YO]; FVIII= 15.5 [11.0-24.0] IU/dL; 4 HB: FIX= 30.0 [16.8-35.4] IU/dL). Target joints were observed in patients with MiH (7/88: 8.6% of the MiH patients). HEAD-US score was obtained in 73 patients (74.5% of the total participating patients): nine MoH patients presented a HEAD-US= 1 (0-6) (age: 32.7 [21.7-42.9] YO) while 64 MiH patients presented a HEAD-US= 0 (0-3) (age: 37.9 [17.8-48.7] YO) (comparison between MoH and MiH patients: HEAD-US: p=0.427, age: p= 0.614). Only 55.5% (5/9) MoH patients and 45.3% (29/64) MiH patients, presented JD (p=0.725). Baseline FVIII/FIX was 13.0 (7.0-20.0) IU/dL and 16.0 (11.0-22.5) IU/dL in patients with or without JD respectively (p= 0.311). These mild difference of number of patients with JD and degree of JD between MiH and MoH were corroborated by not detecting correlation between baseline FVIII/FIX levels and JD (r= -0.095; p= 0.426) (Figure 1). JD increased with age (r= 0.380; p= 0.001) reinforcing previous findings. GHC correlated with baseline FVIII/FIX (Table 1) but not with the degree of JD, suggesting that thrombin generation, with the used conditions, might not be helpful to predict JD in this population. Conclusions: Around 50% of patients with nSH presented JD. This JD increased with age, supporting previous data. Although there was a correlation between baseline FVIII/FIX levels and thrombin generation, none of the TGT parameters were predictive of the degree of JD, enhancing that other variables might be influencing the development of JD in nSH patients. Of note, MiH individuals may develop severe JD due to presence of target joints. In summary, our results highlight the importance of evaluating JD in nSH in order to determine whether new protocols are required for the diagnosis, prevention, and treatment of JD in nSH patients.

The Procoagulant Phospholipid Dependent Clotting Time and the Initiation Phase of Thrombin Generation Test Are Mandatory for the Evaluation of the Risk for Severe Early Onset Preeclampsia

Bakground: Preeclampsia occurs in up to 7% of pregnancies in Western Countries and is a leading cause of maternal and fetal morbidity and mortality. Classification of preeclampsia is based on the timing of symptoms' onset and the severity of the clinical status. Accordingly, early and late preeclampsia are characterized by symptom onset before or after 32-34 weeks of gestation respectively. Pathogenesis of preeclampsia is characterized by abnormal placental vascular development with impaired invasion of the uterine spiral arteries by trophoblasts, resulting in fetal complications and maternal endothelial dysfunction. Intact antithrombotic properties of the endothelial cells and efficient regulation of thrombin generation and platelet activation at the microenvironment of the uterus are necessary conditions for successful trophoblast invasion and development of feto-maternal circulation during normal pregnancy. , Prompt identification of pregnant women at risk of preeclampsia worsening could help to anticipated therapeutic intervention and improvement of the clinical outcome. Aim: The ROADMAP-Early Onset Preeclampsia study (ROADMAP-EOP) aiming to respond to this unmet need assessed the clinical relevance of biomarkers of hypercoagulability and endothelial activation for prompt identification of pregnant women at high risk of preeclampsia. Materials and Methods: An observational single center retrospective case-control study was conducted in pregnant women diagnosed with preeclampsia from July 2020 to July 2021. Pregnant women were retrospectively enrolled upon EOP diagnosis and stratified in mild EOP group (n=34) and severe EOP group (n=15 according to the criteria of the International Society for the Study of Hypertension in Pregnancy ). Eligible women were included in the study before initiation of any treatment for preeclampsia. The control group (n=35) consisted of women with uncomplicated pregnancy. All women were assessed with thromboelastometry, Calibrated Automated Thrombo-gram®, tissue factor activity (TFa), Procoagulant phospholipid dependent clotting time (Procoag-PPL), Proteins S, TFPI, D-dimer, antithrombin, thrombomodulin, fibrinogen prothrombin time and activated partial thromboplastin time. Primary and secondary end points were severe EOP and EOP respectively. Principal component analysis (PCA) was performed to check for associations among the biomarkers and the clinical out-come. Results: There were no significant differences regarding age, gravidity, gestational age and the body mass index (BMI) between EOP groups and normal pregnancy as well as within the EOP groups. Platelet counts were similar in healthy pregnant women, and mild EOP group, and significantly lower in severe EOP (p&amp;lt;0.05). No other statistically significant differences were found between-groups. The PCA showed that the repartition of data allowed to discriminate the patients with EOP from healthy pregnant women as well as patients with mild EPO from those with severe EOP. When biomarkers of hypercoagulability were considered according to the severity of EOP in PCA analysis the lag-time and PPL clotting time were independently associated with the risk of severe EOP (Table 1and Figure 1)). The increase of the lag-time by 1 min was associated with a 20.2-fold increase of the risk of severe EOP. The marginal effects curve shows that for a lag-time longer than 2.6 min, the probability of severe EOP was higher than 50% .Longer Procoag-PPL clotting time was associated with lower risk of severe EOP. The marginal effects curve, showed that for Procoag-PPL clotting time longer than 58.75 sec the probability of severe EOP decreased by 50%. These data suggest that a biological score composed of the Procoag-PPL® clotting time and thrombin generation (Throbmogram-Thrombinoscope® PPP-reagent 1 pM-TF as-say) could be a useful tool to identify pregnant women with high probability of EOP Conclusion: the Procoag-PPL® clotting time and thrombin generation assay reflect cellular derived hypercoagulability and allow accurate screening of pregnant women for the prompt identification of those at risk of EOP and the evaluation of its severity. This concept has to be validated in a large prospective cohort trial. . An external validation of the proposed methodology needs to be performed in a large prospective observational study.

PB0292 Thrombotic Risk Evaluation in Pediatric Patients with Acute Lymphoblastic Leukemia Using Thrombin Generation Test

PB0292 Thrombotic Risk Evaluation in Pediatric Patients with Acute Lymphoblastic Leukemia Using Thrombin Generation Test

Genetic Analysis of Hereditary Coagulation Factor V Deficiency in Two Chinese Families Caused by Compound Heterozygous Mutations.

This study aims to provide a preliminary discussion of the molecular basis of FV deficiency caused by compound heterozygous mutations in two Chinese families. Relative coagulation index was measured by the one-stage clotting method and the FV:Ag was measured by ELISA. All exons and flanking regions of the F5 gene were amplified by PCR and directly sequenced. ClustalX-2.1-win was used to analyze the conservation of mutations. The online software was used to predict the pathogenicity of mutations. PyMOL was used to analyze the variation in the spatial structure of the FV protein before and after mutations. Calibrated automated thrombogram was used to analyze the function of the mutant protein. Phenotyping suggested that both probands had a simultaneous decrease in FV:C and FV:Ag. Their genetic tests showed that proband A had a missense mutation p.Ser111Ile in exon 3 and a polymorphism p.Arg2222Gly in exon 25. At the same time, the proband B had a missense mutation p.Asp96His in exon 3 and a frame-shift mutation p.Pro798Leufs*13 in exon 13. Meanwhile, the p.Ser111Ile is conserved among homologous species. The bioinformatics and protein model analysis revealed that p.Ser111Ile and p.Pro798Leufs*13 were pathogenic and could affect the structure of the FV protein. The thrombin generation test revealed that the clotting function of proband A and B had been affected. These four mutations may be responsible for the reduction of FV levels in two Chinese families. Moreover, the p.Ser111Ile mutation is a novel pathogenic variant that has not been reported.

Predicting risks of prothrombotic readiness under COVID-19 using genetic testing

COVID-19 poses a significant hazard as regards decompensation of underlying chronic diseases, specific damage to the cardiovascular system, and a high risk of negative health outcomes such as thrombotic events. The coronavirus infection pathogenesis is rather complicated and has not been studied yet; this is largely due to peculiar features of the virus and the initial state of homeostasis in a patient. In this study, our aim was to analyze molecular-genetic markers of homeostasis in patients with the new coronavirus infection COVID-19 as a prognostic trigger of developing pro-thrombotic readiness. Hospitalized patients with COVID-19 were chosen as study objects. We performed molecular-genetic analysis of basic genes significant for homeostasis including several factors such as V (rs6025), II (rs1799963), I (rs1800790), VII (rs6046), XIII A1 (rs5985)), IGN A2 (rs1126643), IGN B3 (rs5918), and PAI-1 (rs1799889). The thrombinemia severity was identified by thrombin generation tests using the Ceveron®alpha automated coagulation analyzer with TGA-module. Allelic variants of PAI-1, prothrombin (FII), and fibrinogen (FI) determined high thrombinemia as per the thrombin kinetics test (endogenous thrombin potential (AUC), peak thrombin concentration (peak-thrombin), time necessary to reach thrombin peak (tPeak), levels of fibrinogen and D-dimer) in COVID-19 patients during the entire hospitalization. We established that elevated thrombin generation becoming apparent through elevated levels of endogenous thrombin potential (AUC) might be a prognostic indicator of the pro-thrombotic state in patients with genetic polymorphisms of PAI-I and fibrinogen. The study results indicate that pro-thrombotic readiness is determined genetically in case COVID-19 patients have allelic variants in PAI-I, prothrombin (factor II) and fibrinogen (factor I) genes.

Прогнозирование риска развития протромбогенной готовности при инфекции COVID-19 с использованиемгенетического тестирования

COVID-19 poses a significant hazard as regards decompensation of underlying chronic diseases, specific damage to the cardiovascular system, and a high risk of negative health outcomes such as thrombotic events. The coronavirus infection pathogenesis is rather complicated and has not been studied yet; this is largely due to peculiar features of the virus and the initial state of homeostasis in a patient. In this study, our aim was to analyze molecular-genetic markers of homeostasis in patients with the new coronavirus infection COVID-19 as a prognostic trigger of developing pro-thrombotic readiness. Hospitalized patients with COVID-19 were chosen as study objects. We performed molecular-genetic analysis of basic genes significant for homeostasis including several factors such as V (rs6025), II (rs1799963), I (rs1800790), VII (rs6046), XIII A1 (rs5985)), IGN A2 (rs1126643), IGN B3 (rs5918), and PAI-1 (rs1799889). The thrombinemia severity was identified by thrombin generation tests using the Ceveron®alpha automated coagulation analyzer with TGA-module. Allelic variants of PAI-1, prothrombin (FII), and fibrinogen (FI) determined high thrombinemia as per the thrombin kinetics test (endogenous thrombin potential (AUC), peak thrombin concentration (peak-thrombin), time necessary to reach thrombin peak (tPeak), levels of fibrinogen and D-dimer) in COVID-19 patients during the entire hospitalization. We established that elevated thrombin generation becoming apparent through elevated levels of endogenous thrombin potential (AUC) might be a prognostic indicator of the pro-thrombotic state in patients with genetic polymorphisms of PAI-I and fibrinogen. The study results indicate that pro-thrombotic readiness is determined genetically in case COVID-19 patients have allelic variants in PAI-I, prothrombin (factor II) and fibrinogen (factor I) genes.

Presence and activity of Fibrinogen like protein 2 in platelets.

Fibrinogen-like protein 2 (FGL2) is a serine protease capable of converting prothrombin into thrombin (i.e., prothrombinase-like activity) while bypassing the classic coagulation cascade. It has been reported to be expressed by mononuclear blood cells and endothelial cells. There are multiple reports that FGL2 supports tumor development and metastasis. However, in the blood, the origin and functional significance of FGL2 has not been established. To determine if FGL2, a malignancy related enzyme, is present in platelets. Peripheral blood samples were collected in K2 EDTA tubes. Blood cells and platelets were separated and thoroughly washed to produce plasma-free samples. Procoagulant activity was measured in the cell lysates using a thrombin generation test or an adjusted prothrombin time (PT) test in plasma deficient of factor X. The findings were further supported by confocal microscopy, immunoprecipitation, flow cytometry, enzyme-linked immunosorbent assays and specific inhibition assays. FGL2 protein was readily detected in platelets. Also, despite being expressed by lymphocytes, FGL2 prothrombinase-like activity was solely detected in platelet samples, but not in white blood cell samples. Quiescent platelets were shown to contain the FGL2 protein in an active form. Upon activation, platelets secreted the active FGL2 into the milieu. Active FGL2 is found in platelets. This suggests another role for the involvement of platelets in malignancies.

A case-report of the unprovoked thrombotic event in a patient with thymoma and severe FVII deficiency

BackgroundFactor VII deficiency is a rare bleeding disorder caused by a deficiency of clotting factor VII. However, there have been some case reports of venous thrombosis in patients with factor VII deficiency, especially underlying the prothrombotic risk factors exposure. Patients with factor VII deficiency require special considerations before undergoing surgery to minimize the risk of bleeding or thrombogenesis.Case presentationHere, we described a patient with early-stage thymoma and severe factor VII deficiency who experienced an unprovoked thrombotic episode before thymectomy and a fatal thrombotic event after surgery. By adopting gene screening, a reported homozygous F7 mutation (p.His408Gln) and a novel heterozygous PROS1 mutation (p.Pro147Ala) were identified. The former resulted in severe factor VII deficiency but did not protect against thrombosis, and the latter was correlated with normal expression and cofactor activities of protein S through the thrombin generation test. The perioperative infusion of recombinant factor VII concentrate and the absence of antithrombotic prophylaxis may collectively contribute to her fatal thrombotic event after surgery.ConclusionsFor the patients with severe factor VII deficiency undergoing surgery, uniform replacement therapy may not be recommended, and antithrombotic prophylaxis should be used in the case with thrombotic history to minimize the risk of bleeding and thrombogenesis.