Platelet aggregation impacts thrombin generation assessed by calibrated automated thrombography

A calibrated automated thrombogram (CAT) is performed usually with human platelet-free plasma (PFP) but may be more relevant with platelet-rich plasma (PRP). In this case, platelets are not stimulated by subendothelial molecules like collagen. Our aim was to assess the consequence of strong (collagen) or weak (ADP) induction of platelet release and aggregation on thrombin generation. Platelet aggregation in PRP was triggered with 10 µg/mL collagen or 10 µM ADP using a lumi-aggregometer. Thrombin generation curves were monitored by CAT in different conditions: PRP, PRP with activated platelets (actPRP), aggregated PRP (agPRP), aggregated platelets resuspended in autologous PFP (resPRP), PFP and PFP obtained after aggregation (agPFP). We found a 3-fold shortening of the lag time and time to peak and a marked increase in velocity and thrombin peak without changes in endogenous thrombin potential (ETP) in agPRP with both agonists compared with PRP. The same holds true in agPFP but with a marked increase in ETP compared with PFP. Similar changes in the kinetics of thrombin generation were observed with actPRP-collagen and to a lesser extent in resPRP-collagen compared with PRP. By contrast, there were no modifications of the thrombin generation curves in actPRP-ADP. Alpha-2-macroglobin-thrombin complexes were unchanged in the different PRP conditions but were increased in PFP prepared from agPFP compared to control PFP. Platelet aggregation during activation by agonists other than thrombin did not increase thrombin generation but accelerated its kinetics mainly via platelet content release and platelet-derived extracellular vesicules formation. In diseases characterized by altered platelet granule content or release as well as altered platelet activation, a platelet aggregation step prior to CAT analysis may be clinically relevant to improve laboratory estimation of the bleeding/thrombotic balance.     

The effect of tissue factor concentration on calibrated automated thrombography in the presence of inhibitor bypass agents

Thrombin generation has been suggested as a method to monitor treatment with factor eight inhibitor bypassing activity (FEIBA) or recombinant FVIIa (rFVIIa). The sensitivity of the assay for individual coagulation factors is dependent on the tissue factor (TF) concentration. An inverse relation between the rFVIIa concentration needed to shorten the clotting time and TF concentration has been shown but the data on thrombin generation are inconsistent. Information on TF concentration in measurements with FEIBA is limited. We studied the influence of TF concentration (1 and 5 pM) on thrombin generation through spiking experiments with rFVIIa and/or FEIBA in the plasma of severe haemophilia A patients and after four and three treatment episodes, respectively, using the calibrated automated thrombin generation assay (CAT) in platelet poor plasma. Spiking with FEIBA showed a linear relation with the endogenous thrombin potential (ETP)/peak at 1 pM but substrate depletion at 5 pM. Spiking with rFVIIa showed a near linear dose-response relation with the ETP/peak at 1 pm but only a shortening of the initiation phase at 5 pM. Similar effects were present in post-treatment samples. FEIBA acted synergistically with rFVIIa. This suggest a role for CAT in monitoring inhibitor bypass treatment but low TF concentrations are required to avoid substrate depletion with FEIBA and to demonstrate the effect of rFVIIa.     

Effect of standardization and normalization on imprecision of calibrated automated thrombography: an international multicentre study

Calibrated automated thrombography (CAT) enables continuous measurement of thrombin generation (TG). Initial clinical studies using the CAT method showed large variability of normal values, indicating the necessity for a standardized CAT protocol. This international study assessed the intra- and inter-assay imprecision of CAT as well as the inter-centre variability of results in five European centres using locally available reagents and conditions (study 1) and a standardized protocol in which results were normalized (study 2). Samples with and without corn trypsin inhibitor from six healthy volunteers, two haemophilia patients and one protein C deficient patient were assayed. Study 1 confirmed that the use of different sources and concentrations of tissue factor (TF) and different phospholipid (PL) mixtures produced large variability in results. The second study demonstrated that, using the same source and concentration of TF, PL and the same test procedure, this variability could be significantly reduced. Normalization of results improved the inter-centre variability. The benefit of contact factor inhibition prior to TG measurement was confirmed. These results demonstrated that standardization of CAT reduces the variability of results to acceptable limits. Standardization and normalization should be considered in future clinical studies which apply TG testing to clinical decision making.     

International Normalized Ratio (INR), coagulation factor activities and calibrated automated thrombin generation – influence of 24 h storage at ambient temperature

International Normalized Ratio (INR) measurements are used to monitor oral anticoagulation therapy with coumarins. Single coagulation factor activities and calibrated automated thrombin (CAT) generation are considered as more advanced methods for evaluating overall haemostatic capacity. The aims were to assess the variability of INR, coagulation factor activities, and CAT, during 24 h of storage of blood samples at ambient temperature. A total of 24 patients on stable coumarin treatment were followed prospectively for 6 weeks. INR was analyzed at 0, 6 and 24 h after blood sampling and 1-stage clotting activity of coagulation factors II, VII, IX, and X as well as CAT generation was recorded after 0 and 24 h respectively. Statistical analyses included Bland–Altman plot, 95% limits of agreement, and a variability test using a mixed effect model. The level of INR remained statistically unchanged from 0 to 6 and 24 h of storage. Coagulation factor activities and CAT revealed no significant difference induced by 24 h of storage, although the limits of agreement were wide. Patients’ individual INR, coagulation factor activities, and CAT generation were not significantly influenced by 24 h storage of blood samples, but for the CAT generation analyses a trend toward time dependency was detected.     

Tissue factor pathway inhibitor is the main determinant of thrombin generation in haemophilic patients

The thrombin generation (TG) assay evaluates haemostatic balance, which is influenced by the levels of many coagulation factors and inhibitors. Our objective was to identify the determinant factors of TG in haemophilia A (HA) and haemophilia B (HB) patients and to compare them to those in healthy controls. Coagulation factor and inhibitor levels, and TG, were measured in platelet‐poor plasma from 40 patients with HA, 32 patients with HB and 40 healthy subjects. Data were analysed using multiple regression models. In HA patients, factor VIII was a positive determinant of endogenous thrombin potential (ETP) and peak, whereas tissue factor pathway inhibitor (TFPI) and factor V were negative determinants of ETP and peak. In HB patients, FIX was a positive determinant of ETP and peak, FVII being a positive determinant of peak. Antithrombin and protein S (PS) were negative determinants of ETP while FX was a negative determinant of peak. Above all, in HB patients, TFPI was a negative determinant of ETP and peak. In healthy subjects, FVIII was a positive determinant of ETP and peak, whereas FX and protein S were negative determinants of these parameters. TFPI was not a negative determinant of either peak or ETP. In haemophilic patients, the determinant factors of TG are all implicated in FXa generation and inhibition, the crucial determinant factor being TFPI whatever the type of haemophilia, A or B. These findings contribute to the rationale that recently place TFPI as a target for innovative therapies of haemophilia.     

Marked elevation of thrombin generation in patients with elevated FVIII:C and venous thromboembolism

Elevated plasma factor VIII coagulant activity (FVIII:C, > 150 IU/dl) is a risk factor for venous thromboembolism (VTE). We hypothesized that increased FVIII:C may exert a prothrombotic effect by increasing basal thrombin generation. To test this hypothesis we have measured prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complex (TAT) in three groups: (i) patients with objectively confirmed VTE and elevated FVIII:C; (ii) patients with VTE and no detectable thrombophilia; and (iii) healthy age- and sex-matched control subjects. In the group of patients with elevated FVIII:C, TAT and F1 + 2 levels were increased in 85% and 78% of individuals respectively. This frequency of coagulation activation is dramatically higher than that reported for other recognized constitutional thrombophilias. In the group of patients with VTE but no proven thrombophilia, increased thrombin generation was present in 30% of individuals. Basal thrombin generation was significantly higher in patients with elevated FVIII:C compared with individuals with VTE but no documented thrombophilia (median TAT = 8.65 microg/l versus 2.95 microg/l, median F1 + 2 = 1.5 nmol/l versus 0.87 nmol/l; P < 0.0001, P < 0.001). Overall FVIII:C levels were strongly correlated with levels of thrombin generation (r= 0.5, P < 0001). The clinical significance of such markedly increased F1 + 2 and TAT levels in patients with high FVIII:C levels remains unclear.     

The measurement and application of thrombin generation

It is the capacity to generate thrombin, and the enzymatic work that thrombin does, that determines blood coagulability. Therefore, measurement of the enzymatic work potential of thrombin provides a method for quantifying the composite effect of the multiple factors that determine coagulation capacity. The application of measurement of thrombin generation to clinical decision making has been hampered by numerous technical difficulties and pitfalls, many of which have now been overcome. Technical advances now permit rapid, reproducible measurement. A review of clinical studies performed to date indicates the need to appreciate the precise methodology used in each case and the need to consider standardisation in future studies. Applying thrombin generation measurement to clinical decision making will require up-to-date estimates of risks relating to the disorder and the intended therapeutic intervention. Ultimately management studies will be required if the clinical utility of measurement of thrombin generation is to be proven.     

The effect of red blood cells on thrombin generation

We have developed an assay of thrombin generation in clotting whole blood. Because our goal was to study patients with red blood cell diseases that are associated with thrombosis, the initial evaluation of this assay analysed the effect of the haematocrit and haemolysate on the total amount of thrombin activity generated and the maximal concentration of thrombin achieved. Both of these parameters were proportional to the haematocrit throughout a wide range of clinically relevant cell concentrations. Red cell lysate also augmented thrombin generation. Because this effect was removed by filtration of the lysate, we propose that it was related to red cell microparticles.     

Impact of different inhibitor reactivities with commercial factor VIII concentrates on thrombin generation

In order to describe the haemostatic role of a variation in inhibitor reactivity with different factor VIII (FVIII) concentrates, we have compared inhibitor titres against a panel of FVIII concentrates and correlated titre with the capacity to inhibit thrombin generation. Three plasma-derived concentrates were tested in vitro in mixing experiments with inhibitor plasmas from 11 patients with severe haemophilia A: Fanhdi, which contains von Willebrand factor (VWF) with a final ratio of approximately 1:1 (VWF IU per IU FVIII:C); Haemate-P with a ratio of 2.5:1 and Hemofil-M containing only trace amounts of VWF. In addition, the recombinant FVIII concentrate Kogenate Bayer containing no VWF was included. Inhibitor titres and the capacity to generate thrombin were measured. A statistically significant difference in measured titres was found with the highest titres recorded against Hemofil-M. The inhibitor titres needed to inhibit 50% maximum thrombin generation were the lowest for Kogenate Bayer and the highest and similar for Fanhdi and Haemate-P with intermediate titres needed for inhibition of Hemofil-M. In this study, the thrombin generation assay provides additional indications for the role of VWF in the treatment of patients with inhibitors. The VWF-containing concentrates Fanhdi and Haemate-P, added to FVIII-deficient plasma with the presence of inhibitor, generate more thrombin than do the purified concentrates Hemofil-M and Kogenate Bayer.     

The effects of chemotherapeutic agents on the regulation of thrombin on cell surfaces

Thromboembolic disorders are common in cancer patients. Two major contributing factors are central venous catheters for drug delivery and the use of l-aparaginase, which decreases the plasma antithrombin level, but the causes of the hypercoagulable state in these patients are not fully understood. In this study, the T24/83 cell line was used as a model to investigate the effects of chemotherapeutic agents on cell surface thrombin regulation. Plasma thrombin generation and prothrombin consumption was increased in most of the treated cells, particularly vincristine- and adriamycin-treated cells (P < 0.05), compared with controls. However, no free thrombin generation or prothrombin consumption was observed in factor VII (FVII)-depleted plasma. No significant differences in the levels of thrombin-alpha2-macroglobulin (IIa-alpha2M) and thrombin-anti-thrombin (TAT) were observed between controls and any of the treatments, except for vincristine- and adriamycin-treated cells, which showed a significant difference in TAT production (P < 0.05). Also, there was an upregulation in tissue factor (TF) mRNA expression in etoposide-, methotrexate- and vincristine-treated monolayers compared with controls, as well as an upregulation in TF protein production in vincristine-treated cells. The data suggests that thrombin generation occurs via the extrinsic (TF-dependent) coagulation pathway on cell surfaces and that some chemotherapeutic agents are able to upregulate TF mRNA and protein expression in T24/83 cells.     

Cyclic strain delays the expression of tissue factor induced by thrombin in human umbilical vein endothelial cells

Most studies of tissue factor (TF) expression in endothelial cells (EC) are performed under stationary culture conditions. The purpose of this study was to determine the influence of mechanical stimuli such as cyclic strain (CS) on the expression of TF in EC exposed to thrombin (Thr). Human umbilical vein endothelial cells (HUVEC) were exposed to 4 U·mL⁻¹ Thr in the presence or absence of 10% average CS at 60 cycles·min⁻¹ and then TF expression was measured. TF messenger RNA (mRNA) expression peaked at 2 hours in HUVEC exposed to Thr, but at 4 hours in HUVEC exposed to both Thr + CS. TF expression was inhibited by p38 and extracellular signal-regulated protein kinase (ERK) inhibitors. For both Thr or Thr + CS stimuli, p38 and ERK activity peaked at 5 minutes (p < 0.05). Nuclear factor-kappa B levels remained high in the Thr group but not in the Thr + CS group, while Egr-1 levels were elevated in the Thr + CS group. We demonstrated CS-delayed, Thr-induced TF mRNA expression in HUVEC, which may be modulated by p38 and ERK inhibitors.     

Markers of platelet activation and thrombin generation

The laboratory assessment of platelet biology and coagulation has traditionally focused on abnormalities, congenital or acquired, that provide insights for understanding hemostatic defects, unexplained bleeding, and pharmacologic titration of compounds designed to attenuate thrombotic potential. Because atherosclerotic vascular disease is associated with thrombotic events and there exists a clear link between atherosclerosis, inflammation, and thrombosis, the development of markers that have pathobiologic, prognostic, and treatment-related relevance has taken on a role of considerable importance.     

Evaluation of the overall haemostatic effect of recombinant factor VIIa by measuring thrombin generation and stability of fibrin clots

Summary. It has been reported that thrombin generation test (TGT) may be a useful tool to monitor recombinant factor VIIa (rFVIIa). However, TGT does not reflect the stability of fibrin clot and its resistance to fibrinolysis which are crucial. Using whole‐blood thromboelastography (TEG) and tissue plasminogen activator (tPA), we developed an in‐vitro model to assess fibrin clot stability. Fibrin fibres were thicker in haemophiliacs compared with controls (P < 0.0001). After addition of rFVIIa 90 μg kg^?1, the diameter of fibrin fibres was dramatically decreased (P = 0.006). TEG‐tPA assay showed a dose‐dependent improvement of clot stability in the presence of rFVIIa. These data demonstrate a significant correlation between fibrin clot structure and its stability (P = 0.001). We also showed a correlation between thrombin generating capacity and clot resistance to fibrinolysis. Despite this overall correlation, a relatively large spreading around a general trend was observed, suggesting that the two assays bring complementary information on the haemostatic effect of rFVIIa.     

Surface 12-lead electrocardiographic findings and plasma markers of thrombin activity and generation in patients with myocardial ischemia at rest

Background: Myocardial ischemia at rest is typically associated with atherosclerotic coronary artery disease, atheromatous plaque rupture, and intracoronary thrombosis. In areas of advanced disease and vascular injury, the extent of thrombus is influenced largely by a delicate balance of procoagulant factors, favoring thrombus initiation, growth, and development, and anticoagulant factors, attempting to limit potentially flow-limiting coronary thrombosis. Thrombin, a 308 amino acid serine protease, is considered the most potent procoagulant factor in the setting of acute vessel wall injury, playing an essential role in the conversion of fibrinogen to fibrin, accelerating the prothrombinase complex, activating platelets, and stabilizing fibrin polymers. The purpose of this study was to determine the relationship between electrocardiographic abnormalities and markers of thrombin activity and generation among patients with unstable angina and non-Q-wave myocardial infarction.Methods and Results: In a study of 36 patients (59.1 ± 11.0 years) with myocardial ischemia at rest participating in the Thrombolysis in Myocardial Ischemia (TIMI) IIIB trial, thrombin activity in plasma, as determined by fibrinopeptide A (FPA), prothrombin fragment 1.2 (F 1.2), and thrombin-antithrombin III complexes (TAT) concentrations, were found to be increased significantly when compared with healthy volunteers (p < 0.004). Thrombin generation was also increased modestly compared with age-matched patients with stable coronary artery disease undergoing elective cardiac catheterization. Given that the surface 12-lead electrocardiogram (ECG) is frequently abnormal in patients with ischemic chest pain at rest and represents a readily available, first-line diagnostic test for assessing disease activity and treatment response, we investigated whether ECG abnormalities and thrombin activity/ generation in plasma were correlated. Twenty-six patients (72%) had ECG changes compatible with myocardial ischemia at the time of study entry, including 18 (50%) with newly inverted T waves (or pseudonormalization), 14 (39%) with reversible ST-segment depression, and 4 (11%) with transient (<30 minutes) ST-segment elevation. Within the predefined ECG groups there were no differences in plasma thrombin activity between patients with and those without confirmed abnormalities. Similarly, there were no differences in either plasma thrombin activity or generation between the predefined ECG groups.Conclusion: Although ECG abnormalities supporting the presence of myocardial ischemia occur commonly in patients with chest pain at rest, they do not correlate closely with markers of thrombin activity and generation in plasma. The diagnostic and prognostic capabilities of these diagnostic tools, considered either alone or together, require further investigation.Presented in part at the American College of Cardiology 42nd annual Scientific Session, Anaheim, CA, March 14–18, 1993.