Thrombin Generation In Samples Research Articles

Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

IntroductionHematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment.We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. MethodsThis is a single-centre, case-control, non-interventionist, prospective study.During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed:Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway.Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software.TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA).The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. ResultsThose patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests.Table 1 shows the results obtained.Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI.Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI.Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. ConclusionsUBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient.Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes.TFPI inhibition might emerge as a good therapy for some of these patients.Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field.This work was supported by Novo Nordisk Pharma S.A.Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LTlagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. [Display omitted] DisclosuresAlvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

Correlation between plasma clot properties, thrombin generation and whole blood fibrinolytic assays in patients presenting with STEMI

Abstract Background Impaired endogenous fibrinolysis is a novel risk factor for recurrent adverse cardiovascular events in acute coronary syndrome (ACS) patients. This is independent of conventional cardiovascular risk factors and unaffected by dual antiplatelet therapy (DAPT). The mechanism underlying impaired endogenous fibrinolysis in ACS patients is currently unclear. Aim To identify the relationship between whole blood fibrinolysis, plasma fibrinolysis and thrombin generation in samples from STEMI patients. Methods In a large, prospective, observational study of 500 patients presenting with ST-segment elevation myocardial infarction (STEMI), blood samples were taken on arrival, after DAPT loading, and before administration of heparin or PPCI. Non-anticoagulated venous whole blood was analysed using the point-of-care Global Thrombosis Test, which assesses the time taken for occlusive thrombus formation under high shear (occlusion time, OT) and time required for spontaneous restart of flow as a measure of endogenous fibrinolysis (lysis time, LT). Patients were divided into 4 groups based on quartiles (Q) of whole blood LT (Q1: LT<1500s, Q2:1501–3000s, Q3:3001–4500s, Q4:>4500s). Plasma samples (20 per quartile) were examined in a thrombin generation assay using 1pM tissue factor to initiate and using a turbidity assay to determine the plasma clot lysis time (CLT). Results Clinical characteristics of patients were similar in the four groups. The whole blood LT in the 4 groups were Q1: 1194 (1125–1329) s, Q2: 1859 (1634–2157) s, Q3: 3638 (3252–3962) s, Q4: 6000 (5523–6000) s. As LT increased, there was a trend towards longer plasma CLT (50% CLT Q2: 88.5 [73.5–102] vs. Q4: 100 [85–128.5] min, p=0.088). As a continuous variable, there was no significant relationship between whole blood LT and plasma CLT, or between endogenous thrombin potential (ETP) and either whole blood LT or plasma CLT. There was a significant negative correlation between OT and velocity index (r=−0.425, p=0.0138), ETP (r=−0.519, p=0.002), peak thrombin generation (r=−0.390, p=0.0247) and a positive correlation with lag-time (r=0.427, p=0.013). There was positive correlation between CLT and white cell count (WCC, r=0.388, p=0.026), C-reactive protein (CRP, r=0.477, p=0.005) and maximum absorbance (MA, r=0.530, p=0.002). MA correlated with WCC (r=0.436, p=0.011) and platelet count (r=0.357, p=0.042). There was a negative correlation between OT and WCC (r=−0.537, p=0.001) and CRP (r=−0.381, p=0.029). Conclusion In patients with STEMI, increased platelet reactivity (shorter OT) correlated with increased thrombin generation (higher ETP, peak thrombin generation, velocity index and reduced lag time), demonstrating the key role of thrombin in occlusive thrombus formation. Fibrinolysis in whole blood was poorly related to plasma CLT or thrombin generation, suggesting that cellular components such as platelets, erythrocytes and neutrophil extracellular traps may significantly influence endogenous fibrinolysis. Funding Acknowledgement Type of funding source: None

Antithrombin Reduction Corrected Thrombin Generation in Samples from Hemophilia A and B Patients with Inhibitors

Background: Severe hemophilia A and B patients with inhibitors experience serious musculoskeletal hemorrhage as well as high risk of limb and life threatening bleeds. However, lack of effect of FVIII or FIX substitution therapy and short functional half-life of by-passing agents, leave these patients with very limited bleed preventive treatment options. ALN-AT3 (Alnylam Pharmaceuticals, Cambridge, MA, USA), a subcutaneously administered investigational RNAi therapeutic targeting reduction of antithrombin for potential treatment of hemophilia is currently in phase 1 clinical development in hemophilia A and B patients without inhibitors. Initial data from that ongoing study in 12 patients suggest an AT KD dependent correction of thrombin generation. This study aims to assess changes in peak thrombin generation in samples from patients with severe hemophilia A and B with inhibitors following in vitro reduction of antithrombin.Materials and methods: Citrated plasma samples were obtained from patients with severe hemophilia A and B with high responding inhibitors. Samples were spiked in vitro with isotype specific control IgG or a monoclonal antibody (Haemtech Inc, Essex Junction, VT, USA) targeting antithrombin knockdown of 50% and 90%. Dynamic formation of thrombin was measured by calibrated automated thrombin generation using 1pM tissue factor PPP reagent and 4μM phospholipid (Thrombinoscope, Maastricht, The Nederlands). The primary effect measure was peak thrombin (nM). Data were tested by a 1-way ANOVA and p<0.05 was considered statistically significant.Results: A total of 12 inhibitor hemophilia samples were investigated; 9 hemophilia A and 3 hemophilia B. All the control samples demonstrated a profound defect in thrombin generation with a median peak thrombin of 19.9 nM (range 6.7 - 42.4). Patients with severe hemophilia A and inhibitors had a median peak thrombin generation of 19.7 nM (range 6.7 - 42.4), whereas patients with severe hemophilia B and inhibitors had a median peak thrombin generation of 19.2nM (range 19.4 - 38.1). An AT reduction dependent improvement in peak thrombin generation was observed in all 12 tested plasma samples (Figure 1). In the first 12 subjects, peak thrombin generation was increased up to 363% from a mean of 22nM (control) to 39 nM (50% AT reduction) and 80nM (90% AT reduction) (p<0.05); levels comparable to thrombin generation observed in healthy male volunteers and in hemophilia patients treated with ALN-AT3.Conclusions: These in vitro data suggest that reduction of AT is a promising approach for restoring hemostatic balance and correcting thrombin generation in hemophilia patients with inhibitors. Furthermore, the present laboratory data compare well with clinical data generated with ALN-AT3 administered to patients with hemophilia A or B. Thus, both laboratory and emerging clinical data suggest that targeting antithrombin could be a promising approach for restoring hemostatic balance in hemophilia. The potential for low volume subcutaneous administration, infrequent dosing, and applicability to persons with hemophilia who have inhibitors, make ALN-AT3 a particularly encouraging investigational therapy. [Display omitted] DisclosuresKenet:Bayer, Novo Nordisk: Other: Advisory Boards, Speakers Bureau; Opko Biologics: Consultancy, Other: Advisory Boards; BPL; Baxelta: Research Funding; Pfizer: Honoraria. Off Label Use: ALN-AT3 is an investigational RNAi therapeutic targeting the endogenous anticoagulant antithrombin.. Chowdary:Sobi: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees. Sehgal:Alnylam Pharmaceuticals: Employment, Equity Ownership. Akinc:Alnylam Pharmaceuticals: Employment, Equity Ownership. Sorensen:Alnylam Pharmaceuticals: Employment, Equity Ownership.

Thrombin generation and fibrin clot formation under hypothermic conditions: An in vitro evaluation of tissue factor initiated whole blood coagulation

BackgroundDespite trauma-induced hypothermic coagulopathy being familiar in the clinical setting, empirical experimentation concerning this phenomenon is lacking. In this study, we investigated the effects of hypothermia on thrombin generation, clot formation, and global hemostatic functions in an in vitro environment using a whole blood model and thromboelastography, which can recapitulate hypothermia. MethodsBlood was collected from healthy individuals through venipuncture and treated with corn trypsin inhibitor, to block the contact pathway. Coagulation was initiated with 5pM tissue factor at temperatures 37°C, 32°C, and 27°C. Reactions were quenched over time, with soluble and insoluble components analyzed for thrombin generation, fibrinogen consumption, factor (f)XIII activation, and fibrin deposition. Global coagulation potential was evaluated through thromboelastography. ResultsData showed that thrombin generation in samples at 37°C and 32°C had comparable rates, whereas 27°C had a much lower rate (39.2 ± 1.1 and 43 ± 2.4 nM/min vs 28.6 ± 4.4 nM/min, respectively). Fibrinogen consumption and fXIII activation were highest at 37°C, followed by 32°C and 27°C. Fibrin formation as seen through clot weights also followed this trend. Thromboelastography data showed that clot formation was fastest in samples at 37°C and lowest at 27°C. Maximum clot strength was similar for each temperature. Also, percent lysis of clots was highest at 37°C followed by 32°C and then 27°C. ConclusionsInduced hypothermic conditions directly affect the rate of thrombin generation and clot formation, whereas global clot stability remains intact.

Relationship between antithrombin and thrombin levels in plasma and serum.

Antithrombin levels were measured following the addition of varying amounts of thrombin to defibrinated plasma and following the intravenous injection of thrombin into rats. In the in vitro studies the decrease in antithrombin was proportional to the thrombin added. The addition of 30–40 u of thrombin to 1 ml of plasma caused a 50% decrease in antithrombin. Similar results were obtained in the in vivo studies. Six-hundred units of thrombin injected into heparinized rats caused a 40% reduction in plasma antithrombin. Thrombin generation in samples of recalcified plasma was measured according to the method of Pitney and Dacey ( J. Clin. Path. 6:9, 1953) and the values compared with changes in antithrombin. The amount of thrombin generated when full strength plasma was recalcified was only a fraction of the amount potentially available according to estimations by the two-stage method. When plasma was diluted with one to two volumes of saline before activation the amount of thrombin generated was increased two- to threefold. The antithrombin level of the serum from the diluted samples was correspondingly decreased.

Coagulation defect in horse plasma.

Summary1. The delayed thrombin generation in samples of horse plasma was made normal by 1) addition of normal human plasma, or 2) addition of reabsorbed heated human serum, or 3) addition of washed human or horse platelets, or 4) freezing of the horse plasma with its normal content of platelets. 2. These results indicate that the clotting defect of the horse plasma investigated is caused by lack of a factor similar to the Hageman factor. 3. Blood platelets, after washing or freezing, apparently substitute for the effect of the lacking plasma factor. 4. The coagulation defects in horse plasma apparently reflect various types of hemophilioid deficiencies.