Thrombin generation in patients after acute deep-vein thrombosis

Thrombin generation measurement may be of value for assessing the risk of venous thromboembolism, but its long term profile has not been assessed in patients. We evaluated thrombin generation by Calibrated Automated Thrombogram (CAT) in plasma during follow up of 104 consecutive patients after an acute episode of deep venous thrombosis. Blood was drawn three times over the course of 24 months. Thrombin generation was measured in absence and presence of thrombomodulin and compared to a reference range derived from thrombin generation curves in 137 healthy volunteers. Thrombin generation of patients showed significantly higher endogenous thrombin potential (ETP) and peak height compared to the reference population. Differences were more pronounced in assays triggered with 1 pM TF. Inhibition by thrombomodulin was attenuated in patients off anticoagulants as compared to the reference population (21% vs. 42.2%, p < 0.0001); inhibition in patients on anticoagulant treatment was less pronounced (9.7%, p < 0.0001). Protein C activity, protein S antigen as well as free protein S showed highly negative correlation with ETP in all patients. A significant negative relation was found between FVIII levels and thrombomodulin induced reduction of ETP and peak height. In conclusion, thrombin generation by CAT reflects changes in coagulation status in patients following a thromboembolic event and is most sensitive at CAT analysis triggered with 1 pM TF. A role for factor VIII as an important attributable cause of hypercoagulability is reflected by the reduced inhibitory effect of thrombomodulin at high factor VIII levels.     

Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma

The thrombin generation test is used to study coagulation in patients with haemorrhagic diseases or with high thrombotic risk. To our knowledge, this is the first study investigating the relative influence of coagulation factors on thrombin generation in plasma. The aim was to investigate the influence of coagulant factors, anticoagulant factors, and tissue factor (TF) on three parameters: endogenous thrombin potential (ETP), peak thrombin concentration, and lag time for the appearance of thrombin. At a low TF concentration, all factors except factor XI influenced thrombin generation. At a high TF concentration, only the factors of the extrinsic pathway exerted an influence. ETP and peak thrombin were linearly correlated to factor II concentration. Factor V and factor VII effects increased hyperbolically with factor concentration. The influence of factor X on thrombin generation depended on TF concentration. In the absence of factor VIII and factor IX, ETP fell to 60-70% of the normal when peak thrombin fell to 25-30% of the normal. Fibrinogen concentration influenced ETP and peak thrombin and decreasing fibrinogen levels shortened the lag time. As expected, decreasing antithrombin concentration caused dramatic increases in thrombin generation. Protein S prolonged the lag time, especially at a low TF concentration. No effect of protein C was observed, likely due to the absence of thrombomodulin. The thrombin generation test was more sensitive to factor deficiencies at low than at high TF concentration. ETP was not the most critical parameter for studying coagulation factor deficiencies. Instead, peak thrombin was the most sensitive parameter.     

Calibrated automated thrombin generation and modified thromboelastometry in haemophilia A

Introduction

Global coagulation tests may have a better relation with phenotype in haemophilia than traditional coagulation tests. These include the Calibrated Automated Thrombin generation assay (CAT) and modified thromboelastometry using low tissue factor triggering. Both have shown marked variability in thrombin generation and clot formation profiles respectively despite similar FVIII:C levels and have been suggested as means to monitor treatment. Data with modified thromboelastometry are largely limited to severe and moderate haemophiliacs. CAT measurements in haemophilia are generally performed at low TF concentrations (1 pM) because of a higher sensitivity for the intrinsic pathway at this concentration but is also sensitive for FVIII at higher concentrations (5 pM) and this has the advantage that inhibition of contact factor activation can be avoided. No formal comparison of both TF concentrations has been reported and the data on modified thromboelastometry in mild haemophilia are limited.

Methods

In this study we compared thrombin generation at 1 and 5 pM in 57 haemophilia patients without exposure to treatment and 41 patients after treatment. We also assessed the sensitivity of thromboelastometry for haemophilia A in 29 patients.

Results and conclusion

We found that CAT discriminates well between normal individuals and haemophilia patients; also FVIII:C correlates well with the ETP/peak. We found no clear advantages of measurements at 1 compared to 5 pM but found increased variation over time at 1 pM. The sensitivity of modified thromboelastometry for haemophilia A was less than CAT with abnormal measurements largely limited to severe and moderate patients. Larger studies correlating both methods with clinical outcome are required.     

The value of 64-detector row computed tomography for the exclusion of pulmonary embolism

Identification of patients at high risk of recurrence after a first event of venous thromboembolism (VTE) remains difficult. Resistance to activated protein C (APC) is a known risk factor for VTE, but data on the risk of recurrence is controversial. We wanted to investigate whether APC resistance in the absence of factor V Leiden, determined with global coagulation test such as the thrombin generation assay, could be used as a marker for increased risk of recurrent VTE among women 18-65 years old after a first event of VTE. In a cohort of 243 women with a first event of VTE, plasma was collected after discontinuation of anticoagulant treatment and the patients were followed up for 46 months (median). Thrombin generation was measured via calibrated automated thrombography, at 1 pM and 10 pM of tissue factor (TF). In women without factor V Leiden (n=117), samples were analysed in the absence and in the presence of APC. Increase in ETP (endogenous thrombin potential) and peak height analysed in the presence of APC correlated significantly with higher risk of recurrence. At 1 pM, peak height correlated with increased risk of recurrence. In conclusion, high thrombin generation in the presence of APC, in women after a first event of VTE is indicative for an increased risk of a recurrence. We also found that thrombin generation at low TF (1 pM) is correlated with the risk of recurrence. Our data suggest that APC resistance in the absence of factor V Leiden is a risk factor for recurrent VTE.     

Microparticle-associated tissue factor activity correlates with plasma levels of bacterial lipopolysaccharides in meningococcal septic shock

Introduction

The plasma level of bacterial lipopolysaccharides (LPS) is associated with activation of the coagulation system, inhibition of fibrinolysis and the nature of the clinical presentation and outcome in patients with meningococcal disease. Tissue factor (TF)-bearing microparticles (MPs) appear to contribute to the pathogenesis of disseminated intravascular coagulation (DIC). The aim of this study was to investigate the relationship between MP-associated TF activity and the level of bacterial LPS in plasma from patients with meningococcal septic shock and meningitis.

Materials and methods

MPs isolated from citrated plasmas were assessed for TF-dependent activity with both a plasma-based thrombin generation assay (CAT) and whole blood-based thromboelastometry (ROTEM). The LPS level was measured using a chromogenic Limulus amebocyte lysate assay.

Results

MPs obtained from patients with meningococcal septic shock initiated significantly more efficient and TF-dependent thrombin generation in the CAT assay compared to MPs from patients with meningococcal meningitis. Differences in MP-associated TF activity between the septic shock patients and the meningitis patients were also evident when MPs were added to whole blood using ROTEM. The level of plasma LPS in patients with septic shock (range 2–2,100 EU/mL) was correlated with thrombogram parameters in the CAT assay; lagtime (r_s = − 0.84), time to peak (r_s = − 0.83), peak (r_s = 0.85) and ETP (r_s = 0.83).

Conclusions

MPs obtained from patients with meningococcal septic shock displayed more efficient TF-dependent thrombin generation and clot formation compared to MPs from meningitis patients. MP-associated TF activity was closely associated with plasma LPS levels in the septic shock group.     

Thrombin generation determined by calibrated automated thrombography (CAT) in pediatric patients with congenital heart disease

INTRODUCTION: Thrombin generation was studied in pediatric patients with congenital heart disease (CHD) undergoing cardiac surgery using the calibrated automated thrombography (CAT) in terms of the lag time until the onset of thrombin formation, time to thrombin peak maximum (TTP), endogenous thrombin potential (ETP), and thrombin peak height. The possible suitability to determine the coagulation status of these patients was investigated. MATERIALS AND METHODS: CAT data of 40 patients with CHD (age range from newborn to 18 years) were compared to data using standard coagulation parameters such as prothrombin (FII), antithrombin (AT), tissue factor pathway inhibitor (TFPI), prothrombin fragment 1.2 (F 1.2), thrombin-antithrombin (TAT), activated partial thromboplastin time (aPTT), and prothrombin time (PT). RESULTS: A significant positive correlation was seen between ETP and FII (p<0.01; r=0.369), as well as between peak height and F II (p<0.01; r=0.483). A significant negative correlation was seen between ETP and TFPI values (p<0.05; r=-0.225) while no significant correlation was seen between peak height and TFPI. A significant negative correlation was seen between F 1.2 generation and ETP (p<0.05; r=-0.254) and between F 1.2 generation and peak height (p<0.05; r=-0.236). No correlation was seen between AT and ETP or peak. CONCLUSIONS: Our data indicate that CAT is a good global test reflecting procoagulatory and inhibitory factors of the hemostatic system in pediatric patients with CHD.     

Blood coagulation kinetics: high throughput method for real-time reaction monitoring

A high throughput 384-well plate assay of blood function in 60 microl reactions with the fluorogenic thrombin substrate, boc-VPR-MCA, allowed for real-time monitoring of coagulation under a diverse set of reaction conditions. Using recalcified, citrated whole blood diluted 3-fold with corn trypsin inhibitor (to block Factor XIIa), addition of 0 to 13.8 pM of tissue factor (TF) reduced the time of maximal rate of thrombin production T(max) from 45 min to 11 min. Over this range of TF,T(max) was reduced from 35 min to 6 min by co-addition of 10 nM convulxin to activate platelets via GPVI. The maximal rate of thrombin production at T(max) was not a function of exogenously-added TF,Va, or reVIIa, but increased 30% with added convulxin. Addition of 0.07 to 0.7 pM TF along with convulxin produced small, but detectable reductions in T(max). Addition of up to 0.67 nM reVIIa reduced T(max) by up to 53% in the range of 0.7 to 7 pM TF. Interestingly, platelet factor 4 (2.7 microM) caused a prolongation of T(max) from 45 min to 78 min at 0 TF, while protamine (1.8 microM) reduced T(max) to 30 min at 0 TF. Finally, combinatorial reaction studies with exogenously-added ADP, histamine, fMLP, indomethacin, anti-CD18, and fibrinogen revealed no unusual synergies amongst the agents, but demonstrated a striking procoagulant activity of added fibrinogen, due to protease contaminants in the "purified" fibrinogen. This high throughput approach allowed automated profiling of blood (50 reactions/ml of blood) to generate large data sets for testing cellular-proteomic kinetic models, screening drug interactions, and potentially monitoring subtle changes in the functional phenotype of a patient blood sample.     

Circulating levels of inflammatory cytokines (IL-1 beta and TNF-alpha), resistance to activated protein C, thrombin and fibrin generation in uncomplicated pregnancies

We studied 33 women during normal uneventful pregnancies and with no history of previous adverse pregnancy events for markers of activated coagulation and thrombin activity including prothrombin fragment 1.2(PF1.2), thrombin- antithrombin (TAT), and soluble fibrin polymer (SFP). In addition, we measured potential thrombin generation through the addition of thromboplastin to patient plasma in the presence of a thrombin-specific chromogenic substrate determined serially over a period of time--Endogenous Thrombin Potential assay (ETP). This assay was performed with plasma treated and untreated with activated protein C (APC). The fibrinolytic system was assessed by measurement of thrombin activatable fibrinolysis inhibitor (TAFI). These findings were correlated with the levels of pro-inflammatory cytokines, interleukine-1 beta and tumor necrosis factor-alpha. Our data supports previous reports that indicate that resistance to activated protein C and coagulation activation markers are commonly increased in the later 2/3rds of pregnancy. There are no differences in thrombin generation potential, as determined by the ETP assay without the addition of APC, in the three trimesters. However, the thrombin reserve (TR), the ETP result without APC divided by the ETP result with the addition of APC, is increased above the reference range in the 2nd and 3rd trimesters. Patients with increased TR and resistance to APC had increased levels of TNF-alpha. Increased proinflammatory cytokines are reportedly associated with changes in the APC system with a decrease in the ability to generate APC. A sub-group of pregnancies with APC resistance had increased levels of TNF-alpha and may be important in the risk for adverse pregnancy outcomes.     

Exchange transfusion activates coagulation and alters the coagulation profile in newborn infants

Exchange transfusion (ET) with adult blood is a standard procedure for neonates with severe hyperbilirubinemia. How ET affects newborn coagulation system remains, however, largely unknown. Thus, we prospectively evaluated the effect of ET on thrombin formation and coagulation profile in 18 newborns (22 ETs). Prothrombin fragment F1+2 and thrombin-antithrombin complexes increased considerably during ET while platelets were significantly reduced. Protein C increased less (p < 0.001) and factor VIIIc more (p < 0.001) than expected based on their levels in the infused blood. Further, in vitro thrombin generation initiated by 5 pM tissue factor was analysed. Before the first ET, newborn endogenous thrombin potential (ETP) and thrombin peak remained at approximately 60% of adult control plasma levels, but the lag time to thrombin burst in newborn plasma was approximately 45% shorter than the lag time in adult plasma. At the end of the first ET, the thrombin burst still started approximately 35% earlier in newborn than adult plasma, whereas ETP and thrombin peak were increased to > 90% of adult levels. ETP and peak remained elevated at adult levels until the beginning of the second ET. APC-induced reductions in newborn ETP remained unaltered throughout the first ET. The reductions of ETP by APC were less pronounced in newborn than adult plasma (p < 0.0001). We conclude that ET is associated with multiple procoagulant changes and increased in vivo thrombin formation. This ET-induced procoagulant challenge may be of clinical significance in sick newborns already prone to bleeding and thrombotic complications.     

The defective down regulation of fibrinolysis in haemophilia A can be restored by increasing the TAFI plasma concentration.

TAFI (thrombin activatable fibrinolysis inhibitor) down regulates fibrinolysis after activation by relatively high concentrations of thrombin generated during coagulation via thrombin mediated factor XI activation and subsequent activation of the intrinsic pathway. It is this secondary burst of thrombin that is severely diminished in haemophilia A, a deficiency of coagulation factor VIII. We therefore investigated the role of TAFI in haemophilia A by measuring the clot lysis times of tissue factor induced fibrin formation and tPA mediated fibrinolysis. In haemophilia A plasma clot lysis times were normal at relatively high tissue factor concentrations but severely decreased at moderate to low tissue factor concentrations, indicating that the thrombin generation via the extrinsic pathway was insufficient to activate TAFI. Addition of factor VIII, TAFI or thrombomodulin restored the clot lysis times at low tissue factor concentrations. This confirms the hypothesis that the bleeding disorder in haemophilia A is not merely a defect in the initial clot formation but is in fact a triple defect: reduced thrombin formation via the extrinsic pathway at low tissue factor concentrations, a reduced secondary burst of thrombin generation via the intrinsic pathway and a defective down regulation of the fibrinolytic system by the intrinsic pathway.     

Influence of recombinant hirudin and unfractionated heparin on thrombin and factor Xa generation in extrinsic and intrinsic activated systems.

Using biochemically defined conditions on a fast kinetic centrifugal analyzer the effect of recombinant hirudin (rH) and unfractionated heparin (UH) on thrombin and factor Xa generation was investigated. Diluted fibrinogen deficient human plasma was incubated with increasing concentrations of the anticoagulants and protease generation was initiated either by extrinsic (EA; thromboplastin/calcium chloride) or intrinsic (IA; ellagic acid/cephaloplastin/calcium chloride) activation of the coagulation process. Generation of thrombin or factor Xa was measured continuously by amidolytic assays using the specific chromogenic substrates Spectrozyme TH and Spectrozyme FXa. By means of calibration curves for thrombin and factor Xa the IC50 values for the inhibition of the proteases were calculated. It was found that rH and UH were nearly equally effective in inhibiting both the thrombin and factor Xa formation after IA, whereas in EA system rH produced a stronger inhibition on thrombin generation than UH, which in general showed a more pronounced effect after intrinsic than after extrinsic activation. The results suggest that, with regards to thrombin and factor Xa generation, rH does not exhibit a much higher activity than UH. This may be an expression that thrombin-mediated positive feedback-reactions are not influenced by rH as strongly as expected when using a highly specific and selective thrombin inhibitor. Furthermore, it can be concluded that protease generation assays may be useful in the characterization of anticoagulants/antithrombotics.     

Platelets and phospholipids in tissue factor-initiated thrombin generation

The influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 microM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 microM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 x 10(8)/ml to 0.5 x 10(8)/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient's blood was accelerated from approximately 10 min to 5 min when the platelet concentration increased from 0.05 x 10(8)/ml to 0.11 x 10(8)/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 x 10(8)/ml (range 0.5-3.1 x 10(8)/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 x 10(8)/ml to 3.1 x 10(8)/ml (4.0+/-0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 x 10(8)/ml.     

Age-dependency of thrombin generation measured by means of calibrated automated thrombography (CAT)

Many coagulation parameters, such as PT or aPTT, show age-dependency. In this study we investigated if the generation of thrombin, possibly better reflecting overall haemostasis, shows an age-dependency. Thrombin generation was measured in platelet poor plasma of 121 children and 86 adults at different ages by means of calibrated automated thrombography (CAT). Correlation analysis shows that endogenous thrombin potential (ETP) (r = 0.702), lag time (r = -0.266), peak (r = 0.533) and time to peak (r = -0.214) are significantly correlated with age (p < 0.01). 'Younger' (age limit 35 years) and 'older' adults were compared with groups of children and adolescent aged between 0.5 and 6 years, 7 and 11 years and 12 to 17 years by means of the Mann-Whitney-U-Test. ETP values of all children and adolescents were significantly lower than those of adults. In the group of the youngest children, additionally shorter lag times and times to peak and lower peak levels differed significantly from those of adults. In the group of 7- to 11-year-old children, lag times were significantly longer than those of both groups of adults, while lower peaks and longer times to peak differed only from the group of the 'older' adults. In the group of the 12- to 17-year-olds, the values of ETP were lower than those of adults. In addition, both adult groups differed significantly in all studied parameters. Our results show an age-dependency of thrombin generation even beyond the juvenile period. If thrombin generation measurement is to be used as a routine method, age has to be considered. Assuming that thrombin potential is an indicator for the risk of thrombosis, our findings are in accordance with the observation of an increased incidence of thrombembolic disease with higher age.     

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.     

Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state

The risk for thrombosis is significantly increased in systemic lupus erythematosus (SLE), affecting both venous and arterial vessels. Activated platelets are known to participate in thrombus formation and growth. A general feature of activated cells is the shedding of microparticles (MP) which support coagulation by exposure of negatively charged phospholipids and possibly tissue factor (TF). In this work we characterized circulating MP in patients with SLE and their relationship with a procoagulant state. Thirty patients with SLE (aged 15-72 years, mean age 38 years) and 20 healthy controls (aged 22-54 years, mean age 34 years) were studied; patients fulfilled 4 revised criteria for SLE. The number and cellular source of circulating MP were determined by flow cytometry using double labeling with specific monoclonal antibodies and annexin V. Thrombin generation was measured as the endogenous thrombin potential (ETP) without the addition of exogenous phospholipids and TF; under these conditions the generation of thrombin depended directly on the number of MP present in plasma. Thrombin anti-thrombin (TAT) and plasmin-antiplasmin (PAP) complexes were measured by ELISA. Compared to the controls, circulating MP were significantly elevated in the patient group (1218 +/- 136 vs 653 +/- 74 x 10(3)/ml plasma, p: 0.0007). In both groups, most of these MP were of platelet origin (927 +/- 131 vs 517 +/- 72 x 10(3)/ml plasma, p:0.009 ). ETP was higher among patients as compared to the controls (804 +/- 64 vs 631 +/- 37 nM thrombin, p: 0.025). Plasma levels ofTAT in patients and controls were 3.4 +/- 0.8 and 1.4 +/- 0.5 microg/L, respectively (p:0.04), and of PAP complexes were 62.5 +/- 14 and 24.05 +/- 2.5 microg/ml, respectively (p: 0.014). The number of platelet-derived MP correlated significantly with thrombin generation (r: 0.42; p: 0.038) and TAT levels (r: 0.40; p: 0.035). We did not find an association of circulating MP with disease activity nor with the presence of antiphospholipid antibodies. The increased number of circulating platelet-derived microparticles and their association with high ETP and activation of the coagulation system suggest that these microparticles play an important role in the pathogenesis of the prothrombotic state in SLE patients.     

Atorvastatin reduces thrombin generation and expression of tissue factor, P-selectin and GPIIIa on platelet-derived microparticles in patients with peripheral arterial occlusive disease

We investigated the effects of statin treatment on platelet-derived microparticles (PMPs) and thrombin generation in atherothrombotic disease. Nineteen patients with peripheral arterial occlusive disease were randomised to eight weeks of treatment with atorvastatin or placebo in a cross-over fashion. Expression of GPIIIa (CD61), P-selectin (CD62P), tissue factor (TF, CD142) and phosphatidylserine (PS; annexin-V or lactadherin binding) was assessed on PMPs. Thrombin generation in vivo was assessed by measurement of prothrombin fragment 1+2 in plasma (F1+2) and ex vivo by using the calibrated automated thrombogram (CAT). During atorvastatin treatment, expression of TF, P-selectin and GPIIIa was significantly reduced vs. placebo (p<0.001 for all). No effect on annexin-V or lactadherin binding was seen. Thrombin generation was significantly reduced during atorvastatin as assessed by both the CAT assay (p<0.001) and by measurements of F1+2 (p<0.01). Subsequent in vitro experiments showed that when TF on microparticles (MPs) was blocked by antibodies, the initiation of thrombin generation was slightly but significantly delayed. Blocking PS on MPs using annexin-V or lactadherin resulted in almost complete inhibition of thrombin generation. In conclusion, atorvastatin reduces thrombin generation and expression of TF, GPIIIa and P-selectin on PMPs in patients with peripheral vascular disease. Microparticle-bound TF slightly enhances initiation of thrombin generation whereas negatively charged surfaces provided by MPs or lipoproteins could reinforce thrombin generation. Statins may inhibit initiation of thrombin generation partly through a microparticle dependent mechanism but the main effect is probably through reduction of lipoprotein levels.     

Acute release of tissue factor pathway inhibitor after in vivo thrombin generation in baboons

Tissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.     

The technique of measuring thrombin generation with fluorogenic substrates: 1. Necessity of adequate calibration

In fluorogenic thrombin generation (TG) measurement the concentrations of thrombin are obtained from the course of fluorescence intensity. Because of fluorescence quenching, in one series of normal plasmas (n = 60), the rate of fluorescence increase at fixed thrombin activity was 70 +/- 13% of that in buffer, in another (n = 139) 75 +/- 8%. Using a calibration factor (CF) measured in buffer therefore underestimates thrombin concentrations in plasma and introduces a source of error. A fixed CF also neglects the 25-35% increase of CF during the experiment and thus distorts the form of the TG curve so that the ETP cannot be determined. Continuous individual calibration (CIC), in which CF is determined continuously in a parallel sample, avoids such systematic errors but adds random error because the thrombin course is calculated from two different measurements. We determined the intra-individual coefficients of variation (CV) of the peak-height and ETP as obtained with CIC to those obtained with a fixed CF measured in buffer. With the fixed CF, the CVs varied between 18% and 49%; with CIC they lowered to 4-7% (n = 5 x 12), i.e. in a range allowing clinical application. It is shown that CIC can be discarded for the measurement of peak thrombin values and replaced by comparison to a reference plasma only if quenching is not a systematic confounder. This was shown to be the case in the set of 139 normal plasmas but not in the set used for determining the intraindividual CVs, a difference that may depend upon preanalytical conditions.     

Use of calibrated automated thrombinography +/- thrombomodulin to recognise the prothrombotic phenotype

There is currently no validated method to detect a prothrombotic phenotype. The question remains, can tissue factor (TF) induced thrombin generation (TG), as measured with the calibrated automated thrombinography (CAT) technique, according to Hemker et al., recognise a prothrombotic state either as such, or when the activated protein C (APC)-system is boosted with thrombomodulin (TM)? We determined the normal range of CAT-TG +/- TM in a group of 71 healthy blood donors, in 11 healthy women using oral contraceptives (OC), and in 89 patients with a history of venous thromboembolism (VTE), divided into a group of 50 in which a prothrombotic risk factor could be found (VTEprf+) and 39 others (VTEprf-). The endogenous thrombin potential (ETP) in the OC, VTEprf+ and VTEprf- group was significantly higher than for the controls. In the presence of TM, the differences were significantly higher than in its absence. The VTEprf+ group had a higher ETP, +/- TM than the VTEprf-group. In conclusion, TG, measured with the CAT technique in the presence of TM is capable of detecting the prothrombotic phenotype with a high sensitivity of 0.93 (95% confidence limits 0.82-0.99).     

The role of platelets and recombinant factor VIIa on thrombin generation, platelet activation and clot formation

In the present study we assessed the effect of platelet counts and rFVIIa on thrombin generation, platelet activation and clot formation after tissue factor pathway activation in human plasma aiming to investigate the mechanism by which rFVIIa induces haemostasis in patients with severe thrombocytopenia. Plasma samples with platelet counts from 5 x 10(9)/l to 150 x 10(9)/l were spiked with rFVIIa (1 micro g/ml) or buffer. Clotting was initiated in the presence of diluted thromboplastin. Thrombin generation was assessed using the Thrombogram-Thrombinoscope trade mark assay. The kinetics of platelet activation was assessed using flow cytometry to measure the expression the P-selectin on platelet membrane of washed platelets suspended in defibrinated homologous PPP. Thromboelastography was used to evaluate the effect of platelets and rFVIIa on the kinetics of clot formation and clot's firmness. In the presence of low platelet counts the endogenous thrombin potential (ETP) and the maximum concentration of generated thrombin (Cmax) were reduced by 60%-70%. The lag-time of thrombin generation and the time required to reach the Cmax (Tmax) were prolonged, the velocity of platelet activation was decreased and thrombus formation was delayed. Recombinant FVIIa accelerated thrombin generation and platelet activation but it did not significantly modify ETP or Cmax. Recombinant FVIIa enhanced platelet activation in a TF and thrombin dependent manner since its effect on the studied parameters was abolished when TF was omitted or when hirudin was added into the experimental system respectively. Recombinant FVIIa normalized the velocity of clot formation but it did not modify clot firmness, which depended mainly on platelets' count. In conclusion, in experimental conditions simulating severe thrombocytopenia rFVIIa in the presence of low amounts of TF, accelerates thrombin generation, without increasing the maximum amount of generated thrombin, thus leading in enhanced platelet activation and rapid clot formation.