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The endogenous thrombin potential and the risk of venous thromboembolism
Thrombosis Research (2007) 121, 353–359
intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
The endogenous thrombin potential and the risk of venous thromboembolism Armando Tripodi ⁎, Ida Martinelli, Veena Chantarangkul, Tullia Battaglioli, Marigrazia Clerici, Pier Mannuccio Mannucci Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Internal Medicine and Medical Specialties, University and IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milan, Italy Received 22 December 2006; received in revised form 13 March 2007; accepted 26 April 2007 Available online 8 June 2007
KEYWORDS Laboratory testing; Thrombin generation; Thrombophilia; Screening
Abstract Background: The risk of venous thromboembolism (VTE) is increased by an excess of procoagulant or by a defect of anticoagulant proteins, with circumstantial risk factors playing a significant contribution. These conditions are directly linked to or are compatible with increased thrombin generation. Assuming that the more thrombin is generated the higher is the risk of VTE, an overall coagulation test monitoring ex vivo thrombin generation and reflecting the interaction of pro- and anticoagulant proteins would be useful to determine the risk of VTE. Patients and methods: This hypothesis was tested by measuring the endogenous thrombin potential (ETP) without or with thrombomodulin (TM) in plasmas from 403 individuals stratified according to their relative risk of VTE (no, low, intermediate, or high risk) according to whether or not they had congenital and/or circumstantial risk factors. Odds ratio (OR) and 95% confidence interval (CI), taken as a measure of the relative risk of having high levels of ETP, were calculated for the different categories relatively to the no-risk category. Results: When the ETP was measured with TM, ORs (95% CI) were 2.10 (1.23–3.60); 4.03 (2.18–7.45) and 4.96 (2.40–10.23) for the low-, intermediate and high-risk category. When ETP was measured without TM there was no gradient of OR values as function of the risk category. Conclusions: ETP measured with TM may help to distinguish individuals with different risk of VTE, however, the practical utility of measuring ETP in clinical practice remains to be evaluated in prospective studies. © 2007 Elsevier Ltd. All rights reserved.
⁎ Corresponding author. Via Pace 9, 20122-Milano, Italy. Tel.: +39 02 5503 5437; fax: +39 02 50320723. E-mail address: [email protected] (A. Tripodi). 0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.04.012
354
Introduction Venous thromboembolism (VTE) is a frequent disease with an estimated incidence of 1:1000 individuals per year in Western countries [1,2]. The pathogenesis of VTE is multifactorial and the main risk factors are genetic [3,4] or circumstantial [4]. The relative risk of VTE associated with each risk factor has been estimated for those which are more frequently found in thrombosis patients, such as factor V Leiden [5,6], the prothrombin G20210A [7] mutations or oral contraceptives use alone or in combination [8], whereas the estimate of the risk is less well established for the less common genetic risk factors such as antithrombin, protein C/S deficiencies, alone or in combination with circumstantial risk factors. Risk stratification may be important for the optimal management of patients after a first episode of VTE and a global assay reflecting the unbalance leading to hypercoagulability due to increased levels of procoagulant or decreased levels of anticoagulant proteins would be useful. The endogenous thrombin potential (ETP), which is defined as the area under the thrombin generation curve obtained upon activation of coagulation by small amounts of tissue factor as trigger and phospholipids as platelet substitutes [9] would be an appealing candidate assay. In this study we measured the levels of ETP in a large number of individuals selected for having well-established pre-defined conditions (genetic, circumstantial or both) known to increase the risk of VTE, with the goal to evaluate whether or not ETP changes were related to different degrees of thrombotic risk.
Patients and methods Patients The study population was formed by symptomatic patients (i.e., individuals with previous VTE) and asymptomatic individuals (i.e., without previous VTE) seen at the Thrombosis Center for thrombophilia screening from September 2004 to July 2005, who were included in the study if they fitted into the pre-defined categories of VTE risk shown in Table 1. Patients had an objectively confirmed episode of VTE such as deep vein thrombosis with or without symptomatic pulmonary embolism. Deep vein thrombosis was diagnosed by compression ultrasonography or venography and pulmonary embolism by ventilation/perfusion scan or contrast computed tomography. Asymptomatic individuals were family members of patients with inherited
A. Tripodi et al. Table 1
Definition of VTE risk categories
Risk category No risk 1. Healthy subject (no VTE, no circumstantial risk factors, no thrombophilic abnormalities) Low risk 1. No VTE and • No thrombophilic abnormalities, OC, HRT or pregnancy • Heterozygous factor V Leiden or prothrombin G20210A • Protein C or protein S deficiency Intermediate risk 1. No VTE, heterozygous factor V Leiden, or prothrombin G20210A and • OC, HRT or pregnancy 2. Single, non-idiopathic VTE and • No thrombophilic abnormalities • Heterozygous factor V Leiden or prothrombin G20210A • Protein C or protein S deficiency High risk 1. VTE either Yes or No and • Antithrombin deficiency • Homozygous factor V Leiden or prothrombin G20210A • Combined thrombophilic abnormalities 2. Idiopathic VTE, either with or without thrombophilic abnormalities VTE, venous thromboembolism; OC, oral contraceptives, HRT, hormone replacement therapy. OC, HRT and pregnancy were present at the time of blood sampling. Thrombophilic abnormalities are: factor V Leiden, prothrombin G20210A, antithrombin-, protein C-, protein-S deficiency.
thrombophilia or healthy subjects, randomly selected from the whole population of partners or friend of thrombosis patients who volunteered to be investigated for thrombophilia in the same study period as patients. Circumstantial risk factors at the time of VTE such as surgery, trauma or prolonged immobilization (N 10 days), oral contraceptives use, pregnancy/puerperium were recorded. When these were absent, VTE was considered idiopathic. Individuals were excluded from the study if at the time of blood sampling they had one of such conditions known to affect thrombin generation as antithrombotic treatment (n = 198), antiphospholipid antibodies/lupus anticoagulants (n = 11), liver disease (n = 16) or recent thrombosis (in the last 3 months) (n = 9). No individuals had overt cancer or autoimmune disease. In asymptomatic individuals thrombosis was ruled out by means of a structured questionnaire, previously validated for the retrospective diagnosis of VTE [10]. Thrombophilia screening included the measurement of antithrombin, protein C and protein S and the search for factor V Leiden and prothrombin G20210A mutations and antiphospholipid antibodies/lupus
The endogenous thrombin potential and the risk of venous thromboembolism anticoagulants. Congenital deficiency of antithrombin, protein C or protein S was defined as activity levels below the lower limit of the normal range found in the same patient on two consecutive occasions 3 months apart, provided the same defect was found in at least one additional family member. The study population was stratified into four categories of increasing risk of VTE, defined according to data available from the literature (see below). All participants gave written informed consent and the study was approved by the Institutional Review Board of the Department of Medicine and Medical Specialties.
Risk categories Risk categories were defined according to the presence and type (idiopathic or not) of VTE, the presence and type of thrombophilic abnormality, the presence and type of circumstantial risk factor at the time of blood sampling (Table 1). The low-risk category included individuals with a relative risk of VTE up to 10-fold as estimated by previous studies [5,7,11–14]; the intermediate-risk category included those with a relative risk of VTE from 20to 30-fold [8,15–23] and the high-risk category those with a relative risk of VTE to more than 30fold [6,13,21,24,25].
Plasma preparation Blood was drawn by clean venepuncture and collected in vacuum tubes (Becton and Dickinson, Meylan, France) containing 105 mM trisodium citrate as anticoagulant in the proportion of 1/9 parts of anticoagulant/blood. Blood was centrifuged within 30 min at controlled room temperature for 20 min at 2880×g. Platelet-poor plasma was then harvested, quick frozen in liquid nitrogen and stored at − 70 °C until tested for ETP and thrombophilia screening which were performed no later than 6 months after blood collection.
Methods Antithrombin, protein C and protein S activity were measured with commercial methods (CoaMatic antithrombin, ProClot protein C and HemoSil protein S, Instrumentation Laboratory, Orangeburg, NY). Factor V Leiden and prothrombin mutations were searched with home-made methods as previously described [26,7]. Antiphospholipid antibodies/lupus anticoagulants were searched according to the criteria of the International Society on
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Thrombosis and Haemostasis [27]. ETP was measured according to Hemker and coworkers [9] as described in details by Chantarangkul et al. [28]. The test is based on the activation of coagulation in platelet-poor plasma after addition of human relipidated recombinant tissue factor (Recombiplastin, Instrumentation Laboratory) in the presence of the synthetic phospholipids 1,2-dioleoyl-sn-glycero3-phosphoserine (DOPS), 1,2-dioleoyl-sn-glycero-3phosphoetanolamine (DOPE) and 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) (Avanti Polar Lipids Inc., Alabaster, Alabama) in the proportion of 20/20/60 (M/M). The concentrations of tissue factor and phospholipids in the test system were 1 pM and 1.0 μM, respectively. Testing for ETP was also performed in the presence of soluble thrombomodulin (ICN Biomedicals, Aurora, Ohio) added in the reaction mixture at a final concentration of 4 nM. Continuous registration of the generated thrombin was achieved with a fluorogenic synthetic substrate (Z-Gly-Gly-Arg-AMC HCl, Bachem, Switzerland) added to the test system at a final concentration of 417 μM. The procedure was carried out with an automated fluorometer (Fluoroskan Ascent®, ThermoLabsystem, Helsinki, Finland). Readings from the fluorometer were automatically recorded and calculated by a dedicated software (Thrombinoscope™, Thrombinoscope BV, Maastricht, The Netherlands ), which displays thrombin generation curves [nM thrombin versus time (minute)] and calculates the area under the curve, defined as ETP and expressed as nM thrombin times minutes (nM · min). Thrombin generation is measured as a function of an internal calibrator for thrombin (Thrombin Calibrator, Thrombinoscope BV). ETP represents the plasmatic balance between the action of procoagulants and anticoagulants. To limit between-assay variability testing was performed by including on each working session a suitable number of plasma samples from the different categories of subjects under investigation.
Statistical analyses Continuous variables are expressed as medians and ranges and the non-parametric Mann–Whitney U test was used to test for differences of ETP values between groups. High levels of ETP were arbitrarily defined as an ETP value above the 75th percentile of the distribution of the values of the no-risk category. Odds ratios (ORs) and 95% confidence intervals (CI), taken as a measure of the relative risk of having high levels of ETP, were calculated for the low-, intermediate- and high-risk category compared with the no-risk category. Adjustment
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A. Tripodi et al.
Table 2
ETP (nM·min) values measured without or with thrombomodulin
Risk category
P value a
ETP without thrombomodulin
P value a
ETP with thrombomodulin
No risk N Median Min Max
145 1854 1132 3023
Low risk N Median Min Max
139 1956 806 4000
P b 0.05
139 1184 317 3536
NS
Intermediate risk N Median Min Max
72 2090 1326 3475
P b 0.001
72 1448 609 2867
P b 0.001
High risk N Median Min Max
47 2134 1298 3266
P b 0.001
47 1507 489 2451
P b 0.001
a
145 1223 226 2449
Statistical analysis refers to the no-risk category.
included age and sex and the statistical significance was set at p b 0.05. All analyses were performed with the SPSS version 13.0 software (Chicago, IL).
Results A total number of 403 patients or asymptomatic individuals [median age (range), 38 years (8–80)] were investigated. One-hundred and forty-five (56 males and 89 females) were included in the no-risk, 139 (52 males and 87 females) in the low-risk, 72 (19 males and 53 females) in the intermediate-risk and 47 (24 males and 23 females) in the high-risk category. The ETP values for the study population
Table 3
as measured without or with thrombomodulin are reported in Table 2. Median values ranged from 1854 (range, 1132–3023) nM · min in the no-risk to 2134 (1298–3226) nM · min in the high-risk category for the ETP measured without thrombomodulin and from 1223 (226–2449) nM · min to 1507 (489–2451) nM · min for the ETP with thrombomodulin. Median ETP levels measured without thrombomodulin for the low, intermediate and high risk were significantly higher than those for the no-risk category (Table 2). Median ETP levels with thrombomodulin for the intermediate and high risk were significantly higher than those for the no-risk category (Table 2). The relative risk (expressed as ORs) of having high ETP levels for the three risk categories is shown in Table 3. When ETP was measured without
Risk of VTE expressed by ETP levels measured with or without thrombomodulin
Risk category
ETP without thrombomodulin N (%)
No risk Low risk Intermediate risk High risk
36 58 36 24
a
(24.8) (41.7) (50.0) (51.1)
b
ETP with thrombomodulin
OR (95% CI)
N (%) a
OR b (95% CI)
1.00 2.65 (1.56–4.50) 3.26 (1.77–6.00) 2.73 (1.35–5.52)
35 (24.1) 50 (36.0) 40 (55.6) 29 (61.7)
1.00 2.10 (1.23–3.60) 4.03 (2.18–7.45) 4.96 (2.40–10.23)
ORs and 95% CI were calculated as a measure of the relative risk of having high levels of ETP in the different risk categories (low, intermediate and high risk) compared with the no-risk category. a Number (%) of individuals with high ETP (above the 75th percentile of the distribution of the values of the no-risk category). b Adjusted for age and sex.
The endogenous thrombin potential and the risk of venous thromboembolism thrombomodulin, ORs (95% CI) adjusted for age and sex were 2.65 (1.56–4.50), 3.26 (1.77–6.00) and 2.73 (1.35–5.53) for the low-, intermediate- and high-risk category. The correspondent ORs when ETP was measured in the presence of thrombomodulin were 2.10 (1.23–3.60), 4.03 (2.18–7.45) and 4.96 (2.40–10.24). Ninety-five percent confidence intervals were relatively narrow in all instances and the null value was not included. When the ETP was measured without thrombomodulin there was no gradient of OR values as a function of the risk category; on the contrary, such a gradient was present when ETP was measured with thrombomodulin (Table 3). Of the other thrombogram parameters only peak heights displayed gradient of OR values which were however lower than those recorded for ETP (not shown).
Discussion The risk of VTE is increased by an excess of the procoagulant factors VIII, IX, XI, II or fibrinogen [7,29–36] or by a defect of the anticoagulant proteins antithrombin and protein C/S, with circumstantial risk factors playing a significant contribution (see Ref. [4] for review). The above conditions are directly linked to or are compatible with increased thrombin generation. The measurement of single coagulation proteins (anti- or procoagulant) provides indirect information on thrombin generation and the risk in individual patients. However, because of the many negative and positive feedback mechanisms which regulate thrombin formation, the assessment of the overall function of the coagulation system from the activity of its individual pro- or anticoagulant components presents with obvious limits. Furthermore, it is not yet precisely known how individual risk factors (genetic or circumstantial) interact with each other to determine the overall risk of VTE. All the above reasons may explain the variable clinical expressivity which is often observed in patients who carry the same coagulation abnormality [37] and suggest that an overall coagulation test reflecting the interaction of pro- and anticoagulants operating in plasma would be useful to determine the risk of VTE. Monitoring, tissue factor-induced thrombin generation is potentially useful if one assumes that the more thrombin is generated the higher is the risk of thrombosis. In this study we tested this hypothesis by measuring thrombin generation in plasmas from healthy individuals or patients carrying one or more factors associated with an increased risk of VTE. Healthy individuals and patients, selected on the basis of having inherited or circumstantial pro-
357
thrombotic conditions, alone or in combination, were stratified according to their relative risk of VTE as established by previous studies. Patients with more severe thrombophilic abnormalities, as antithrombin deficiency, homozygous factor V Leiden or prothrombin G20210A mutation, as well as those with previous idiopathic VTE were considered to be at higher risk than those with mild thrombophilic abnormalities due to heterozygous mutations or protein C/S deficiency or non-idiopathic VTE (see Table 1). Our findings show that thrombin generation increased from no-risk to low-, intermediateand high-risk individuals and patients. As expected, the whole amount of generated thrombin was considerably lower when the test was performed in the presence than in the absence of thrombomodulin, but the association between thrombin generation and the risk of VTE was higher (higher ORs) when the test was performed in the presence of thrombomodulin. This finding is not surprising, keeping with the concept that the protein C anticoagulant system needs to be fully activated by thrombomodulin in order to exert its anticoagulant activity [38]. Thrombomodulin is located on endothelial cells and much less in plasma. Therefore, ex vivo assays meant to assess thrombin generation should be designed to reflect as closely as possible conditions operating in vivo. In this respect, the addition of thrombomodulin is essential, but probably not sufficient to mimic in vivo conditions. Platelets and leucocytes are presumably needed, but the assay design (though possible) would be difficult in practice. These limitations notwithstanding our results in a cohort of individuals selected on the basis of their VTE risk support the concept that the more thrombin is generated the higher is the risk of VTE. Accordingly, individuals with high ETP levels measured in the presence of thrombomodulin may have nearly 5-fold increased risk of VTE compared to those with lower levels. Some limitations of this study must be addressed. First, the definition of risk categories is heterogeneous with respect to the reasons for referral, type of thrombophilia, circumstantial risk factors and previous VTE. However, the aim of this study was to evaluate the levels of a global test as ETP in individuals with different risk of VTE and not to assess the risk of first or recurrent VTE associated with ETP. Second, our study population is formed by highly selected individuals referred to the Thrombosis Center for thrombophilia screening, and therefore the results do not necessarily apply to the general population. Third, the selection criteria of our patient population do not allow any conclusion on the levels of ETP as predictor of the risk of VTE.
358 In conclusion our study shows that ETP measured in the presence of thrombomodulin may help to distinguish patients with different risk of VTE, although it does not clearly distinguish intermediate- from high-risk categories as shown by the similar estimates (4.03 versus 4.96) and by the overlap of the confidence intervals. This likely reflects the limitation concerning the definition of risk categories due to the limited data available in the literature. The advantages of measuring thrombin generation in plasma using the global test ETP instead of testing for single anti- or procoagulant proteins are obvious, but its utility in clinical practice remains to be evaluated in prospective studies.
A. Tripodi et al.
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intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
The endogenous thrombin potential and the risk of venous thromboembolism Armando Tripodi ⁎, Ida Martinelli, Veena Chantarangkul, Tullia Battaglioli, Marigrazia Clerici, Pier Mannuccio Mannucci Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Internal Medicine and Medical Specialties, University and IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milan, Italy Received 22 December 2006; received in revised form 13 March 2007; accepted 26 April 2007 Available online 8 June 2007
KEYWORDS Laboratory testing; Thrombin generation; Thrombophilia; Screening
Abstract Background: The risk of venous thromboembolism (VTE) is increased by an excess of procoagulant or by a defect of anticoagulant proteins, with circumstantial risk factors playing a significant contribution. These conditions are directly linked to or are compatible with increased thrombin generation. Assuming that the more thrombin is generated the higher is the risk of VTE, an overall coagulation test monitoring ex vivo thrombin generation and reflecting the interaction of pro- and anticoagulant proteins would be useful to determine the risk of VTE. Patients and methods: This hypothesis was tested by measuring the endogenous thrombin potential (ETP) without or with thrombomodulin (TM) in plasmas from 403 individuals stratified according to their relative risk of VTE (no, low, intermediate, or high risk) according to whether or not they had congenital and/or circumstantial risk factors. Odds ratio (OR) and 95% confidence interval (CI), taken as a measure of the relative risk of having high levels of ETP, were calculated for the different categories relatively to the no-risk category. Results: When the ETP was measured with TM, ORs (95% CI) were 2.10 (1.23–3.60); 4.03 (2.18–7.45) and 4.96 (2.40–10.23) for the low-, intermediate and high-risk category. When ETP was measured without TM there was no gradient of OR values as function of the risk category. Conclusions: ETP measured with TM may help to distinguish individuals with different risk of VTE, however, the practical utility of measuring ETP in clinical practice remains to be evaluated in prospective studies. © 2007 Elsevier Ltd. All rights reserved.
⁎ Corresponding author. Via Pace 9, 20122-Milano, Italy. Tel.: +39 02 5503 5437; fax: +39 02 50320723. E-mail address: [email protected] (A. Tripodi). 0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.04.012
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Introduction Venous thromboembolism (VTE) is a frequent disease with an estimated incidence of 1:1000 individuals per year in Western countries [1,2]. The pathogenesis of VTE is multifactorial and the main risk factors are genetic [3,4] or circumstantial [4]. The relative risk of VTE associated with each risk factor has been estimated for those which are more frequently found in thrombosis patients, such as factor V Leiden [5,6], the prothrombin G20210A [7] mutations or oral contraceptives use alone or in combination [8], whereas the estimate of the risk is less well established for the less common genetic risk factors such as antithrombin, protein C/S deficiencies, alone or in combination with circumstantial risk factors. Risk stratification may be important for the optimal management of patients after a first episode of VTE and a global assay reflecting the unbalance leading to hypercoagulability due to increased levels of procoagulant or decreased levels of anticoagulant proteins would be useful. The endogenous thrombin potential (ETP), which is defined as the area under the thrombin generation curve obtained upon activation of coagulation by small amounts of tissue factor as trigger and phospholipids as platelet substitutes [9] would be an appealing candidate assay. In this study we measured the levels of ETP in a large number of individuals selected for having well-established pre-defined conditions (genetic, circumstantial or both) known to increase the risk of VTE, with the goal to evaluate whether or not ETP changes were related to different degrees of thrombotic risk.
Patients and methods Patients The study population was formed by symptomatic patients (i.e., individuals with previous VTE) and asymptomatic individuals (i.e., without previous VTE) seen at the Thrombosis Center for thrombophilia screening from September 2004 to July 2005, who were included in the study if they fitted into the pre-defined categories of VTE risk shown in Table 1. Patients had an objectively confirmed episode of VTE such as deep vein thrombosis with or without symptomatic pulmonary embolism. Deep vein thrombosis was diagnosed by compression ultrasonography or venography and pulmonary embolism by ventilation/perfusion scan or contrast computed tomography. Asymptomatic individuals were family members of patients with inherited
A. Tripodi et al. Table 1
Definition of VTE risk categories
Risk category No risk 1. Healthy subject (no VTE, no circumstantial risk factors, no thrombophilic abnormalities) Low risk 1. No VTE and • No thrombophilic abnormalities, OC, HRT or pregnancy • Heterozygous factor V Leiden or prothrombin G20210A • Protein C or protein S deficiency Intermediate risk 1. No VTE, heterozygous factor V Leiden, or prothrombin G20210A and • OC, HRT or pregnancy 2. Single, non-idiopathic VTE and • No thrombophilic abnormalities • Heterozygous factor V Leiden or prothrombin G20210A • Protein C or protein S deficiency High risk 1. VTE either Yes or No and • Antithrombin deficiency • Homozygous factor V Leiden or prothrombin G20210A • Combined thrombophilic abnormalities 2. Idiopathic VTE, either with or without thrombophilic abnormalities VTE, venous thromboembolism; OC, oral contraceptives, HRT, hormone replacement therapy. OC, HRT and pregnancy were present at the time of blood sampling. Thrombophilic abnormalities are: factor V Leiden, prothrombin G20210A, antithrombin-, protein C-, protein-S deficiency.
thrombophilia or healthy subjects, randomly selected from the whole population of partners or friend of thrombosis patients who volunteered to be investigated for thrombophilia in the same study period as patients. Circumstantial risk factors at the time of VTE such as surgery, trauma or prolonged immobilization (N 10 days), oral contraceptives use, pregnancy/puerperium were recorded. When these were absent, VTE was considered idiopathic. Individuals were excluded from the study if at the time of blood sampling they had one of such conditions known to affect thrombin generation as antithrombotic treatment (n = 198), antiphospholipid antibodies/lupus anticoagulants (n = 11), liver disease (n = 16) or recent thrombosis (in the last 3 months) (n = 9). No individuals had overt cancer or autoimmune disease. In asymptomatic individuals thrombosis was ruled out by means of a structured questionnaire, previously validated for the retrospective diagnosis of VTE [10]. Thrombophilia screening included the measurement of antithrombin, protein C and protein S and the search for factor V Leiden and prothrombin G20210A mutations and antiphospholipid antibodies/lupus
The endogenous thrombin potential and the risk of venous thromboembolism anticoagulants. Congenital deficiency of antithrombin, protein C or protein S was defined as activity levels below the lower limit of the normal range found in the same patient on two consecutive occasions 3 months apart, provided the same defect was found in at least one additional family member. The study population was stratified into four categories of increasing risk of VTE, defined according to data available from the literature (see below). All participants gave written informed consent and the study was approved by the Institutional Review Board of the Department of Medicine and Medical Specialties.
Risk categories Risk categories were defined according to the presence and type (idiopathic or not) of VTE, the presence and type of thrombophilic abnormality, the presence and type of circumstantial risk factor at the time of blood sampling (Table 1). The low-risk category included individuals with a relative risk of VTE up to 10-fold as estimated by previous studies [5,7,11–14]; the intermediate-risk category included those with a relative risk of VTE from 20to 30-fold [8,15–23] and the high-risk category those with a relative risk of VTE to more than 30fold [6,13,21,24,25].
Plasma preparation Blood was drawn by clean venepuncture and collected in vacuum tubes (Becton and Dickinson, Meylan, France) containing 105 mM trisodium citrate as anticoagulant in the proportion of 1/9 parts of anticoagulant/blood. Blood was centrifuged within 30 min at controlled room temperature for 20 min at 2880×g. Platelet-poor plasma was then harvested, quick frozen in liquid nitrogen and stored at − 70 °C until tested for ETP and thrombophilia screening which were performed no later than 6 months after blood collection.
Methods Antithrombin, protein C and protein S activity were measured with commercial methods (CoaMatic antithrombin, ProClot protein C and HemoSil protein S, Instrumentation Laboratory, Orangeburg, NY). Factor V Leiden and prothrombin mutations were searched with home-made methods as previously described [26,7]. Antiphospholipid antibodies/lupus anticoagulants were searched according to the criteria of the International Society on
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Thrombosis and Haemostasis [27]. ETP was measured according to Hemker and coworkers [9] as described in details by Chantarangkul et al. [28]. The test is based on the activation of coagulation in platelet-poor plasma after addition of human relipidated recombinant tissue factor (Recombiplastin, Instrumentation Laboratory) in the presence of the synthetic phospholipids 1,2-dioleoyl-sn-glycero3-phosphoserine (DOPS), 1,2-dioleoyl-sn-glycero-3phosphoetanolamine (DOPE) and 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) (Avanti Polar Lipids Inc., Alabaster, Alabama) in the proportion of 20/20/60 (M/M). The concentrations of tissue factor and phospholipids in the test system were 1 pM and 1.0 μM, respectively. Testing for ETP was also performed in the presence of soluble thrombomodulin (ICN Biomedicals, Aurora, Ohio) added in the reaction mixture at a final concentration of 4 nM. Continuous registration of the generated thrombin was achieved with a fluorogenic synthetic substrate (Z-Gly-Gly-Arg-AMC HCl, Bachem, Switzerland) added to the test system at a final concentration of 417 μM. The procedure was carried out with an automated fluorometer (Fluoroskan Ascent®, ThermoLabsystem, Helsinki, Finland). Readings from the fluorometer were automatically recorded and calculated by a dedicated software (Thrombinoscope™, Thrombinoscope BV, Maastricht, The Netherlands ), which displays thrombin generation curves [nM thrombin versus time (minute)] and calculates the area under the curve, defined as ETP and expressed as nM thrombin times minutes (nM · min). Thrombin generation is measured as a function of an internal calibrator for thrombin (Thrombin Calibrator, Thrombinoscope BV). ETP represents the plasmatic balance between the action of procoagulants and anticoagulants. To limit between-assay variability testing was performed by including on each working session a suitable number of plasma samples from the different categories of subjects under investigation.
Statistical analyses Continuous variables are expressed as medians and ranges and the non-parametric Mann–Whitney U test was used to test for differences of ETP values between groups. High levels of ETP were arbitrarily defined as an ETP value above the 75th percentile of the distribution of the values of the no-risk category. Odds ratios (ORs) and 95% confidence intervals (CI), taken as a measure of the relative risk of having high levels of ETP, were calculated for the low-, intermediate- and high-risk category compared with the no-risk category. Adjustment
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A. Tripodi et al.
Table 2
ETP (nM·min) values measured without or with thrombomodulin
Risk category
P value a
ETP without thrombomodulin
P value a
ETP with thrombomodulin
No risk N Median Min Max
145 1854 1132 3023
Low risk N Median Min Max
139 1956 806 4000
P b 0.05
139 1184 317 3536
NS
Intermediate risk N Median Min Max
72 2090 1326 3475
P b 0.001
72 1448 609 2867
P b 0.001
High risk N Median Min Max
47 2134 1298 3266
P b 0.001
47 1507 489 2451
P b 0.001
a
145 1223 226 2449
Statistical analysis refers to the no-risk category.
included age and sex and the statistical significance was set at p b 0.05. All analyses were performed with the SPSS version 13.0 software (Chicago, IL).
Results A total number of 403 patients or asymptomatic individuals [median age (range), 38 years (8–80)] were investigated. One-hundred and forty-five (56 males and 89 females) were included in the no-risk, 139 (52 males and 87 females) in the low-risk, 72 (19 males and 53 females) in the intermediate-risk and 47 (24 males and 23 females) in the high-risk category. The ETP values for the study population
Table 3
as measured without or with thrombomodulin are reported in Table 2. Median values ranged from 1854 (range, 1132–3023) nM · min in the no-risk to 2134 (1298–3226) nM · min in the high-risk category for the ETP measured without thrombomodulin and from 1223 (226–2449) nM · min to 1507 (489–2451) nM · min for the ETP with thrombomodulin. Median ETP levels measured without thrombomodulin for the low, intermediate and high risk were significantly higher than those for the no-risk category (Table 2). Median ETP levels with thrombomodulin for the intermediate and high risk were significantly higher than those for the no-risk category (Table 2). The relative risk (expressed as ORs) of having high ETP levels for the three risk categories is shown in Table 3. When ETP was measured without
Risk of VTE expressed by ETP levels measured with or without thrombomodulin
Risk category
ETP without thrombomodulin N (%)
No risk Low risk Intermediate risk High risk
36 58 36 24
a
(24.8) (41.7) (50.0) (51.1)
b
ETP with thrombomodulin
OR (95% CI)
N (%) a
OR b (95% CI)
1.00 2.65 (1.56–4.50) 3.26 (1.77–6.00) 2.73 (1.35–5.52)
35 (24.1) 50 (36.0) 40 (55.6) 29 (61.7)
1.00 2.10 (1.23–3.60) 4.03 (2.18–7.45) 4.96 (2.40–10.23)
ORs and 95% CI were calculated as a measure of the relative risk of having high levels of ETP in the different risk categories (low, intermediate and high risk) compared with the no-risk category. a Number (%) of individuals with high ETP (above the 75th percentile of the distribution of the values of the no-risk category). b Adjusted for age and sex.
The endogenous thrombin potential and the risk of venous thromboembolism thrombomodulin, ORs (95% CI) adjusted for age and sex were 2.65 (1.56–4.50), 3.26 (1.77–6.00) and 2.73 (1.35–5.53) for the low-, intermediate- and high-risk category. The correspondent ORs when ETP was measured in the presence of thrombomodulin were 2.10 (1.23–3.60), 4.03 (2.18–7.45) and 4.96 (2.40–10.24). Ninety-five percent confidence intervals were relatively narrow in all instances and the null value was not included. When the ETP was measured without thrombomodulin there was no gradient of OR values as a function of the risk category; on the contrary, such a gradient was present when ETP was measured with thrombomodulin (Table 3). Of the other thrombogram parameters only peak heights displayed gradient of OR values which were however lower than those recorded for ETP (not shown).
Discussion The risk of VTE is increased by an excess of the procoagulant factors VIII, IX, XI, II or fibrinogen [7,29–36] or by a defect of the anticoagulant proteins antithrombin and protein C/S, with circumstantial risk factors playing a significant contribution (see Ref. [4] for review). The above conditions are directly linked to or are compatible with increased thrombin generation. The measurement of single coagulation proteins (anti- or procoagulant) provides indirect information on thrombin generation and the risk in individual patients. However, because of the many negative and positive feedback mechanisms which regulate thrombin formation, the assessment of the overall function of the coagulation system from the activity of its individual pro- or anticoagulant components presents with obvious limits. Furthermore, it is not yet precisely known how individual risk factors (genetic or circumstantial) interact with each other to determine the overall risk of VTE. All the above reasons may explain the variable clinical expressivity which is often observed in patients who carry the same coagulation abnormality [37] and suggest that an overall coagulation test reflecting the interaction of pro- and anticoagulants operating in plasma would be useful to determine the risk of VTE. Monitoring, tissue factor-induced thrombin generation is potentially useful if one assumes that the more thrombin is generated the higher is the risk of thrombosis. In this study we tested this hypothesis by measuring thrombin generation in plasmas from healthy individuals or patients carrying one or more factors associated with an increased risk of VTE. Healthy individuals and patients, selected on the basis of having inherited or circumstantial pro-
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thrombotic conditions, alone or in combination, were stratified according to their relative risk of VTE as established by previous studies. Patients with more severe thrombophilic abnormalities, as antithrombin deficiency, homozygous factor V Leiden or prothrombin G20210A mutation, as well as those with previous idiopathic VTE were considered to be at higher risk than those with mild thrombophilic abnormalities due to heterozygous mutations or protein C/S deficiency or non-idiopathic VTE (see Table 1). Our findings show that thrombin generation increased from no-risk to low-, intermediateand high-risk individuals and patients. As expected, the whole amount of generated thrombin was considerably lower when the test was performed in the presence than in the absence of thrombomodulin, but the association between thrombin generation and the risk of VTE was higher (higher ORs) when the test was performed in the presence of thrombomodulin. This finding is not surprising, keeping with the concept that the protein C anticoagulant system needs to be fully activated by thrombomodulin in order to exert its anticoagulant activity [38]. Thrombomodulin is located on endothelial cells and much less in plasma. Therefore, ex vivo assays meant to assess thrombin generation should be designed to reflect as closely as possible conditions operating in vivo. In this respect, the addition of thrombomodulin is essential, but probably not sufficient to mimic in vivo conditions. Platelets and leucocytes are presumably needed, but the assay design (though possible) would be difficult in practice. These limitations notwithstanding our results in a cohort of individuals selected on the basis of their VTE risk support the concept that the more thrombin is generated the higher is the risk of VTE. Accordingly, individuals with high ETP levels measured in the presence of thrombomodulin may have nearly 5-fold increased risk of VTE compared to those with lower levels. Some limitations of this study must be addressed. First, the definition of risk categories is heterogeneous with respect to the reasons for referral, type of thrombophilia, circumstantial risk factors and previous VTE. However, the aim of this study was to evaluate the levels of a global test as ETP in individuals with different risk of VTE and not to assess the risk of first or recurrent VTE associated with ETP. Second, our study population is formed by highly selected individuals referred to the Thrombosis Center for thrombophilia screening, and therefore the results do not necessarily apply to the general population. Third, the selection criteria of our patient population do not allow any conclusion on the levels of ETP as predictor of the risk of VTE.
358 In conclusion our study shows that ETP measured in the presence of thrombomodulin may help to distinguish patients with different risk of VTE, although it does not clearly distinguish intermediate- from high-risk categories as shown by the similar estimates (4.03 versus 4.96) and by the overlap of the confidence intervals. This likely reflects the limitation concerning the definition of risk categories due to the limited data available in the literature. The advantages of measuring thrombin generation in plasma using the global test ETP instead of testing for single anti- or procoagulant proteins are obvious, but its utility in clinical practice remains to be evaluated in prospective studies.
A. Tripodi et al.
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