Continuous Thrombin Generation Research Articles

Continuous thrombin generation in whole blood: New applications for assessing activators and inhibitors of coagulation

Hemostatic tests have been utilized to clarify the blood coagulation potential. The novel thrombin generation (TG) assay of this study provides explicit information and is the most physiologically-relevant hemostatic test ex vivo. We describe how this assay allows for TG under a number of relevant circumstances. First, whole blood (WB) from healthy individuals was analyzed ± 5 pM tissue factor (TF) and ± contact pathway inhibition. Without an exogenous initiator TG was decreased and delayed, but addition of 5 pM TF shortened the lag phase and increased peak thrombin. Additional experiments included fresh WB from a trauma patient analyzed for endogenous activity and TG from healthy donors subjected to TG antagonists which prolonged the lag phase whereas TG agonists consistently shortened the lag phase in a dose dependent manner. Lastly, platelet-poor plasma was reconstituted with packed red blood cells and TG was monitored in the presence and absence of both TF as an activator and PCPS as a phospholipid surface. Our data illustrate the potential that this continuous TG assay has in the evaluation of disorders relevant to blood coagulation and in the monitoring of treatments administered in response to these disorders.

Continuous Thrombin Generation in Whole Blood: New Applications

Background.Hemostatic tests have been utilized to clarify the blood coagulation potential of both healthy and diseased individuals. They include tests for whole blood and plasma clotting times, coagulation and fibrinolytic factors and, recently, thrombin generation (TG). TG assays provide explicit information and remain the most physiologically-relevant hemostatic tests ex vivo. We have currently been using a TG assay in whole blood, described by Ninivaggi et al. (2012), for the evaluation of several hemostatic agents and disorders of blood coagulation.Methods.Whole blood from either healthy donors or trauma patients was drawn into 3.2% sodium citrate [± 0.1 mg/mL corn trypsin inhibitor (CTI) prior to the assay; prevents contact pathway activation] or into 3.2% sodium citrate/0.1 mg/mL CTI. Selected agents were added to blood prior to recalcification and TG was monitored in a fluorogenic assay.Packed red blood cells (pRBC) from healthy donors were reconstituted with platelet-poor plasma (PPP) and treated with 0.1 mg/mL CTI. TG was monitored in the absence of any exogenous initiation, in the presence of 5 pM TF initiation and also in the presence of synthetic phospholipids.Results.Whole BloodA. Blood from 10 healthy donors was collected into both citrate (with CTI added prior to the assay) and CTI/citrate and TG was monitored ± 5 pM relipidated tissue factor (TF) initiation. In the absence of TF, blood collected into citrate led to a lag phase ~3-fold longer than that in the presence of TF and peak thrombin ~1.4-fold lower. Blood collected into CTI/citrate, in the absence of TF, led to a lag phase ~6.4-fold longer than that in the presence of TF and peak thrombin ~4.3-fold lower.B. Titrations of relipidated TF and FXIa into citrated blood containing CTI led to concentration-dependent changes in the duration of the lag phase.C. Several TG antagonists were evaluated in citrated blood containing CTI triggered with 5 pM TF.a. 0.5 U/mL unfractionated heparin prolonged the lag phase ~2-fold and suppressed the peak thrombin ~3-fold.b. Hemophilia A and B were "induced" via the addition of inhibitory antibodies to FVIII and FIX, respectively, and although the lag phase was not altered both additions led to significant TG suppression with peak thrombin dropping ~3-fold.c. Several thrombin and FXa inhibitors were evaluated at their pharmacologic concentrations. Fondaparinux slightly prolonged the lag phase, whereas dabigatran increased it by 2.6-fold. Rivaroxaban doubled the lag phase while bivalirudin was slightly less efficient. None of these inhibitors had a pronounced effect on the rate and peak value of TG.d. Activated protein C (APC) at 10 nM slightly prolonged the lag phase and suppressed the peak thrombin.D. Citrate blood from a trauma patient was tested for endogenous activity. In the presence of CTI and absence of an initiator, α-TF antibody did not alter TG, indicating the absence of functional TF. α-FXIa antibody prolonged the lag phase and suppressed TG and α-FIXa antibody completely abolished TG, indicating the presence of active FXIa and FIXa.Packed Red Blood Cells (pRBC)In the absence of an initiator, no TG was observed in pRBC/CTI reconstituted with PPP. The addition of 5 pM TF led to TG after ~8 min with a peak value slightly lower than in blood (described in A).The addition of phospholipids did not change the TG profile.Conclusions.This continuous whole blood TG assay requires 15 µL of blood for a triplicate analysis of a single condition and has the potential for the evaluation of TG in disorders relevant to blood coagulation and for the monitoring of treatments administered in response to these disorders. DisclosuresMann:Haematologic Technologies, Inc.: Employment, Equity Ownership; Baxter: Consultancy; Bayer: Consultancy; Biogen IDEC: Consultancy; CSL Behring: Consultancy; Merck: Consultancy; Pfizer: Consultancy; The Medicines Company: Consultancy; XO1: Consultancy; Vascular Solutions: Consultancy; Stago: Consultancy.

Comparison of Procoagulant and Proinflammatory Effects of Nuclear, Mitochondrial, and Bacterial DNA

Background: Sepsis is a syndrome in which infection triggers a systemic inflammatory and pro-coagulant response, with a prevalence of up to 1 case per 1000 and a mortality rate of up to 40%. Cell-free DNA (cfDNA) is elevated in sepsis, and correlates with mortality. This DNA may come from nuclear, mitochondrial, or bacterial sources. Nuclear and mitochondrial DNA may come from activated neutrophils which release neutrophil extracellular traps (NETs). CpG motifs on bacterial and mitochondrial DNA can stimulate inflammatory responses via TLR9, which is present on neutrophils, monocytes, and recently shown to be expressed on platelets. cfDNA can activate coagulation via the intrinsic pathway. cfDNA may thus play an important pathogenic role in sepsis. This study elucidates the relative effects of nuclear, mitochondrial, and bacterial DNA on inflammatory and pro-coagulant pathways.Methods: Mitochondrial DNA concentrations were measured by PCR using plasma samples from septic patients. Nuclear and mitochondrial DNA were purified from human embryonic kidney 293 cells, and bacterial DNA was from E. coli. Neutrophils from healthy donors were cultured with purified bacterial, mitochondrial, or nuclear DNA at 15µg/mL for 20h. IL-6 levels in the supernatants were measured by ELISA, and neutrophil viability was measured by flow cytometry for annexin-V binding and propidium iodide exclusion. The three types of DNA were added to either citrated normal human platelet-poor plasma or platelet-rich plasma, and continuous thrombin generation was measured (Technothrombin, Vienna, Austria). Light transmission aggregometry was performed in citrated platelet-rich plasma with co-treatment of a sub-threshold concentration of ADP and varying concentrations of DNA. Markers of platelet activation were measured by flow cytometry for P-selectin and activated integrin αIIbβ3. All reagents contained less than 0.06EU/mL of LPS by limulus amoebocyte lysate assay.Results: Cell-free mitochondrial DNA was elevated in plasma from septic patients compared to healthy controls. Bacterial, but not mitochondrial or nuclear, DNA increased neutrophil IL-6 secretion. Both mitochondrial and bacterial DNA increased neutrophil viability at 20h. At concentrations found in the plasma of critically-ill patients, mitochondrial, nuclear, and bacterial DNA increased thrombin generation in both platelet-poor plasma and platelet-rich plasma to a similar degree, and this effect was abolished by corn-trypsin inhibitor and reduced in FXII-depleted plasma, indicating dependence on the intrinsic pathway of coagulation. Independently of coagulation, nuclear DNA at high concentrations, such as may be seen in the NET micro-environment, was capable of causing aggregation of ADP pre-stimulated platelets in citrated plasma, which was accompanied by activation of integrin αIIbβ3and surface expression of P-selectin. This effect also occurred with synthetic phosphodiester oligonucleotides, and was abolished by DNase pre-digestion.Conclusions: cfDNA of bacterial origin can stimulate neutrophil IL-6 release, while both mitochondrial and bacterial DNA prolonged neutrophil viability. All types of DNA can activate coagulation via the contact pathway. DNA at high concentrations may be able to directly stimulate platelets. Total plasma cfDNA and cell-free mitochondrial DNA specifically are elevated in sepsis. Thus, nuclear, mitochondrial, and bacterial DNA may play distinct roles in the pathogenesis of sepsis. DisclosuresBhagirath:Pfizer: Research Funding.

Fluorogenic Peptide‐Based Substrates for Monitoring Thrombin Activity

The synthesis of a series of peptides containing C-terminal 7-amino-4-methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H-Gly-Gly-Arg-AMC, of which the water solubility and kinetic parameters (K(M) and k(cat)) are greatly improved over those of the substrate in current use in the TGT: Cbz-Gly-Gly-Arg-AMC. A series of N-terminally substituted Gly-Gly-Arg-AMC derivatives were synthesized, as well as implementation of structural changes at either the P(2) or P(3) position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3-triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the K(M) and k(cat) values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factor Xa (FXa) were quantified. An initial selection was made on basis of these data, and suitable substrates were further evaluated as substrates in the thrombin generation assay. Assessment of the acquired data showed that several substrates, including the SQ68 derivative Et-malonate-Gly-Arg-AMC and N-functionalized Gly-Gly-Arg-AMC derivatives, are suitable candidates for replacement of the substrate currently in use.

Development of a Continuous Thrombin Generation-Based Test to Measure Potency in Factor VIII Concentrates

Abstract 1206The potency assigned to a Factor (F) VIII-containing product should be predictive of its clinical effectiveness in treating Hemophilia A patients. Potency is assigned using assays that are calibrated against reference standards traceable to an international standard for FVIII concentrates. Therefore, FVIII products are labeled in International Units (IU). Most manufacturers are currently using either a one-stage clotting (OC) or 2-stage chromogenic substrate (CS) assays. However, for some FVIII products, discrepancies in potency values derived from these 2 assays can exceed 50 %. This presents a challenge to manufacturers, regulators and clinicians alike. Recently, a continuous thrombin generation test (TGT) has been introduced in clinical research. The TGT is physiologically more relevant; however, its utility in drug characterization is not yet established.In this report, we describe the development of a FVIII-TGT assay that is suitable for the measurement of FVIII activity in FVIII concentrates. We systematically optimized each of the TGT parameters to obtain the strongest response for a FVIII sample diluted in FVIII-deficient plasma to a concentration of 1 IU/mL. The relative response (ratio of levels of thrombin generation with and without FVIII), and the level of thrombin generation above instrument noise (signal-to-noise ratio) were used as criteria for optimization. In order to limit the variability of the assay, the final parameters were chosen such that small variations in the concentrations of reagents would have a minimal impact on assay outcome.Concentrations of FVIII-deficient plasma, tissue factor (TF), and corn trypsin inhibitor (CTI, inhibits contact activation) had strongest effects on the relative response to FVIII. Concentrations of procoagulant lipids, fluorogenic substrate and calcium ions were more important to achieve an optimal signal-to-noise ratio. Inhibition of contact activation mediated by the contact of plasma with plastic surfaces, control of temperature prior to the start of recording, pipetting technique and internal standards were important for assay reproducibility. Use of low levels of intrinsic factors IXa and XIa in conjunction with TF or omission of CTI allowed for the greatest sensitivity and linearity range of the assay.The performance and utility of the optimized FVIII-TGT were evaluated by measuring the potency of 6 plasma-derived and recombinant FVIII concentrates, and comparing the values to those by the CS assay and on the product label. Most of the products showed parallel dose-response curves between 0.01 and 0.3 IU/mL of plasma FVIII activity in the TGT-FVIII assay. One product had an activity of at least 20 % higher and another product 20 % lower than the labeled potency. The CS assay demonstrated a lack of parallelism in dose-responses for some products but identified the 2 products with the higher- and lower- than-labeled activities.In conclusion, the FVIII-TGT assay can be used to assign and compare the potency of FVIII concentrates. Additionally, the FVIII-TGT, being physiologically more relevant, may act as a reference in understanding the discrepancies in potency assignments in different FVIII concentrates. DISCLOSURE OF INTEREST:MJG, SAS, TKL, and MVO are employees of the U.S. FDA. The findings and conclusions in this presentation have not been formally disseminated by the FDA and should not be construed to represent any Agency determination or policy. Disclosures:No relevant conflicts of interest to declare.

Thrombin generation before and after multicomponent blood collection

Apheresis technology has made tremendous progress up to the development of automated blood component collection, which offers increased efficiency in donor blood use, but the concern about the contact of donor blood with artificial surfaces remains. Activation of the hemostatic system is a major issue in this context and is controversial. The aim of this study was to estimate the effect of apheresis on continuous thrombin generation (TG), representing a new tool to examine the overall function of the plasmatic clotting system. Twenty-six voluntary blood donors, fulfilling the law requirement for apheresis donation, participated in the study. Two units of platelets (6 x 10(11)) and 1 unit of red cells (250 mL; hematocrit level, 80%) were collected using two types of cell separators (Amicus, Fenwal, Inc.; and Trima Accel, Gambro BCT). Each donor underwent collection on both apheresis systems with at least 8 weeks in between. Samples of blood were collected before, immediately after, and 48 hours after apheresis. TG was measured using a slow fluorogenic substrate by means of calibrated automated thrombography (CAT). CAT data changed only slightly, and no significant changes were seen before, immediately after, and 48 hours after apheresis (p > 0.05). The variables did not differ significantly between the two different apheresis systems (p > 0.05). Using a CAT-based technique, no change in variables of continuous TG were observed, suggesting that multicomponent blood collection did not lead to severe alterations in the hemostatic system of the donors.

Pediatric patients with congenital heart disease: thrombin generation measured by calibrated automated thrombography

The aim of this study was to investigate the possible suitability of the calibrated automated thrombography to determine the coagulation status of pediatric patients with congenital heart disease. Thrombin generation was measured in 60 patients with congenital heart disease using the calibrated automated thrombography and compared to data using standard coagulation parameters such as prothrombin, antithrombin, tissue factor pathway inhibitor, prothrombin fragment 1.2 (F 1.2), and activated partial thromboplastin time. A significant positive correlation was observed between prothrombin and the endogenous thrombin potential (P < 0.01; r = 0.295) as well as between prothrombin and peak height (P < 0.01; r = 0.581). A significant negative correlation was seen between tissue factor pathway inhibitor and endogenous thrombin potential (P < 0.01; r = -0.480) and between tissue factor pathway inhibitor and peak height (P < 0.01; r = -0.234). No statistically significant correlation was found between antithrombin and parameters of continuous thrombin generation. Significant correlation was seen neither between activated partial thromboplastin time and F1.2 nor between activated partial thromboplastin time and prothrombin. The data presented here indicate that calibrated automated thrombography measurements determine thrombin generation more accurately and therefore reflect better the coagulation status of pediatric patients with congenital heart disease then standard global coagulation assays such as activated partial thromboplastin time.

Calibrated automated thrombin generation in normal uncomplicated pregnancy

Pregnancy is associated with substantial changes in the haemostatic system and a six-fold higher incidence of venous thromboembolism. Conventional global tests, such as prothrombin time and activated partial thromboplastin time, do not definitely detect this hypercoagulable condition. We investigated whether the changes in haemostatic system during pregnancy are reflected in the calibrated automated thrombography (CAT). Thrombin generation was measured in platelet-poor plasma (PPP) of 150 healthy pregnant women without any pregnancy associated diseases by means of CAT. In addition, prothrombin (FII), antithrombin (AT), protein S, protein C, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and prothrombin fragments 1+2 (F1+2) were measured. Endogenous thrombin potential (ETP) and peak of thrombin generation increased significantly with gestational weeks, while lag time and time to peak remained unchanged. A significant increase of PAI-1, TFPI, F1+2 and TAT as well as a significant decrease of free protein S, protein S antigen, and protein S activity was observed. Levels of AT and protein C remained stable during pregnancy. Division of population in trimester of pregnancy and analysis of differences between the trimesters showed rather similar results. Our study shows that endogenous thrombin potential does increase with duration of normal uncomplicated pregnancy. Whether parameters of continuous thrombin generation will correlate with thrombembolic disease remains to be shown.

Thrombin generation before and after multicomponent blood collection

The development of apheresis technology has increased efficiency in donor blood use by collecting specific blood components in several combinations. The question of donor safety raised by the contact of donor blood with foreign, only in part biocompatible surfaces remains. The aim of this study was to estimate the effect of multicomponent blood collection on thrombin generation performing an overall function test of coagulation. 26 blood donors were included. Per apheresis two units of platelets and one unit of RBCs were collected by two cell separators (Amicus and Trima Accel). Each donor underwent the procedure on both apheresis systems. Samples were collected before, immediately after, and 48 hours after apheresis. Thrombin generation was measured by means of calibrated automated thrombography (CAT). CAT-data changed only slightly and no significant changes were seen before, immediately after, and 48 hours after apheresis. The parameters did not differ significantly between the two different apheresis devices. No change in parameters of continuous thrombin generation occurred, suggesting that apheresis did not lead to severe alterations in the haemostatic system.

Continuous thrombin generation for monitoring phenprocoumon anticoagulation

Continuous thrombin generation for monitoring phenprocoumon anticoagulation

Caution in the interpretation of continuous thrombin generation assays: a rebuttal

Caution in the interpretation of continuous thrombin generation assays: a rebuttal

Activation of clotting factors XI and IX in patients with acute myocardial infarction.

In acute coronary events, plaque rupture and the subsequent formation of the catalytic tissue factor-factor VIIa complex is considered to initiate coagulation. It is unknown whether clotting factors XI and IX are activated in acute coronary events. Therefore, we prospectively investigated the activation of clotting factors XI and IX as well as activation of the contact system and the common pathway in 50 patients with acute myocardial infarction (AMI), in 50 patients with unstable angina pectoris (UAP), and in 50 patients with stable angina pectoris (SAP). Factor XIa-C1 inhibitor complexes, which reflect acute activation of factor XI, were detected in 24% of the patients with AMI, 8% of the patients with UAP, and 4% of the patients with SAP (P<0.05), whereas factor XIa-alpha(1)-antitrypsin complexes, which reflect chronic activation, were observed equally in all 3 study groups. Factor IX peptide levels were significantly higher in the patients with AMI and UAP compared with the patients with SAP (P<0.01). No differences regarding markers of the common pathway were demonstrated. Fibrinopeptide A levels were elevated in patients with AMI compared with patients with UAP and those with SAP (P<0.01). Factor XIIa- or kallikrein-C1 inhibitor complexes were not increased. In conclusion, this is the first demonstration of the activation of clotting factors XI and IX in patients with acute coronary syndromes. Because these clotting factors are considered to be important for continuous thrombin generation and clot stability, their activation might have clinical and therapeutic consequences.

Reactivation of the Coagulation System: Rationale for Long-Term Antithrombotic Treatment

Coronary artery thrombosis superimposed on a disrupted atherosclerotic plaque has emerged as the pivotal pathophysiologic event in acute coronary syndromes (i.e., unstable angina, myocardial infarction, and sudden death). The various clinical manifestations depend on the extent and duration of thrombus deposition, which are determined by several local and systemic thrombogenic risk factors. The thrombotic response to plaque disruption involves both platelet activation and thrombin generation. Accordingly, combined treatment with aspirin and heparin has proved more efficacious than either treatment alone in the risk reduction of serious cardiac events in patients with unstable angina or non-Q-wave infarction. However, withdrawal of heparin is, even after prolonged treatment, associated with an increased short-term risk of serious cardiac events relative to the risk in patients given only aspirin. Furthermore, the long-term relative event rate seems not to be influenced by administration of heparin or direct antithrombins in the acute phase. Both transient hypercoagulability associated with heparin withdrawal and continuous thrombin generation over a longer term related to the underlying disease may explain the rebound in clinical events. Longer duration of combined antiplatelet and anticoagulant treatments, e.g., until healing of the culprit lesion or even until stabilization of vulnerable, yet nondisrupted plaques, may improve long-term clinical outcome.