Thrombin generation assessment in patients with atherothrombosis Proponents:
Grigoris T. Gerotziafas, Elisabeth Verdy, Meyer M Samama, Ismail Elalamy.
Atherothrombosis and hypercoagulable state
Atherothrombosis is characterised by multifocal, disseminated and progressive atheromatic lesions of arterial vessel wall, platelet activation and activation of blood coagulation which lead to thrombus formation. This process results in either extension of the thrombogenic atheromatic plaque or in thrombosis and obstruction of arterial vessel and clinical manifestations of acute arterial thrombosis (sudden cardiovascular death, acute coronary syndromes, stroke, acute thrombosis in the context of peripheral arterial disease). A sustained inflammatory process is also implicated in the pathogenesis of atherothrombosis.
In atherothrombotic patients with acute coronary syndromes, the short term anticoagulant treatment (during the hospitalisation period) with either UFH or enoxaparin or fondaparinux is a necessary therapeutic strategy revealing that these patients are in an important hypercoagulable state where platelet activation and thrombin generation are both preponderant pathogenetic processes (1).
Theoretical and Methodological aspects of Thrombin Generation Assessment
The global clotting times (i.e. prothrombin time and partial thromboplastin time) are routinely used for the evaluation of human coagulation but they ignore the procedure of thrombin generation since at the time of clot formation only 3% of prothrombin is activated [2, 3]. This is a constant observation either in normal plasma or in the presence of antithrombotic agents (i.e. LMWHs or fondaparinux) . The global clotting times are mandatory for the detection of clotting factor deficiencies and the biological monitoring of treatment with coumarins and unfractionated heparin but they have a very limited value in the detection of hypercoagulable states [1,2]. Moreover, they are not significantly influenced by the presence of therapeutic concentrations of LMWHs or the indirect FXa inhibitors such as fondaparinux . Measuring of anti-Xa activity in plasma from patients treated with LMWHs has a limited predictive value for the clinical outcome  and in some patients’ populations (i.e elderly or obese patients) the anti-Xa levels in plasma are poorly correlated with the administered dose of LMWH [6,7].
The study of thrombin generation performed either with clotting based assays or with chromogenic substrates is an old and established tool in blood coagulation research [8,9,10,11,12,13]. It describes all the phases of thrombin generation process : initiation, amplification and inhibition of thrombin generation as well as the integral amount of generated thrombin expressed by the Endogenous Thrombin Potential (ETP). According to the experimental system used, thrombin generation may be influenced by most of the factors playing a role in blood coagulation. However, thrombin generation assessment used to be a laborious and time-consuming method. The most sophisticated version of thrombin generation assay using a chromogenic substrate developed by Hemker’s group was less time-demanding than the previous one, since it was automated accompanied with a software for the calculation of the area under thrombin generation curve (endogenous thrombin potential) (14).
The available assays for thrombin generation assessment
During the last years the research on thrombin generation substantially progressed. Two fully automated methods for the assessment of thrombin generation are actually available.
The first, and original one described by Hemker in 1986 and 1993, is the assessment of the ETP in platelet poor plasma in the presence of an inhibitor of fibrin polymerization (called ETP-DB) using a chomogenic substrate. This method is fully automated and it has been adapted in the BCS instrument of Dade Behring, which is a widely used instrument available in many hematological laboratories.
The second assay (Thrombogram-Thrombinoscope®) also described by Hemker in 2000, is the evolution of the original. It can be done both in non-defibrinated platelet poor and in platelet rich plasma. Thrombin generation is monitored using a fluorogenic substrate. This assay is semi-automated adapted for a fluoroscan instrument which is not part of the standard equipment of blood coagulation laboratories.
Preanalytical conditions of thrombin generation assessement
The preanalytical conditions for the assessment of thrombin generation is subject of running projects which have been discussed during Meeting of the ISTH Standardization Committee (Sydney 2005). There is a wide consensus regarding the initial trigger of coagulation process in thrombin generation assays. The use of low, physiologically relevant tissue factor (TF) concentrations is recommended for the both the amidolytic and fluorogenic assay.
Our group recently published data regarding the standardization of the Thrombogram-Thrombinoscope assay and an extensive analysis on the precision of the assay and the optimal experimental conditions (15).
Aim of the project Thrombin generation and atherothrombosis
The analysis of thrombin generation process in atherothrombotic patients could allow to evaluate the “hypercoagulable state” and to understand the pathogenesis of thrombosis in such patients. In addition it will propose new biological markers of hypercoagulability suitable for patients with atherothrombosis.
Patients with atherothrombosis. Inclusion criteria :all PAD patients (after PAD questionnaire) and exclusion criteria : concomitant thrombin affecting drugs.
- Gerotziafas GT, Zafiropoulos A, Van Dreden P, Karavaggeli E, Goutzoumas N, Nikolaidis P, Combot C, Lagoudaki P, Zervas K, Arzoglou P, Samama MM. Inhibition of factor IIa generation and prothrombin activation by treatment with enoxaparin in patients with unstable angina. Br J Haematol 2003;120:611-17.
- Hemker HC, Beguin S. Phenotyping the clotting system. Thromb Haemost 2000; 84:741–751.
- Mann KG, Butenas S, Brummel K. The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol 2003; 23:17–25.
- Gerotziafas GT, Depasse F, Chakroun T, Van Dreden P, Samama MM, Elalamy I. Comparison of the effect of fondaparinux and enoxaparin on thrombin generation during in vitro clotting of whole blood and platelet rich plasma. Blood Coag Fibrinol 2004;15:149-156.
- Bara L, Planes A, Samama MM. Occurrence of thrombosis and haemorrhage, relationship with anti-Xa, anti-IIa activities, and D-dimer plasma levels in patients receiving a low molecular weight heparin, enoxaparin or tinzaparin, to prevent deep vein thrombosis after hip surgery. Br J Haematol. 1999;104:230-40.
- Mahe I, Drouet L, Chassany O, Grenard AS, Caulin C, Bergmann JF. Low molecular weight heparin for the prevention of deep venous thrombosis: a suitable monitoring in elderly patients? Pathophysiol Haemost Thromb. 2002;32:134-6.
- Heizmann M, Baerlocher GM, Steinmann F, Horber FF, Wuillemin W. Anti-Xa activity in obese patients after double standard dose of nadroparin for prophylaxis. Thromb Res. 2002;106:179-81.
- 8 Provenzale L, Casolo P, Bifani I. Value of an original thrombogram in the early diagnosis of thrombosis and the recognition of potential thrombosis Policlinico Chir 1952;59:257-79.
- Macfarlane RG, Biggs R: A thrombin generation test. J Clin Pathol 1953;6:3–7.
- Pitney WR, Dacie J: A simple method of studying the generation of thrombin in recalcified plasma. J Clin Pathol 1953;6:9–13.
- Seegers WH: A personal perspective on hemostasis and thrombosis (1937–1981). Semin Thromb Hemost 1981;7:177–307.
- Houbouyan L, Padilla A, Gray E, Longstaff C, Barrowcliffe TW. Inhibition of thrombin generation by heparin and LMW heparins: a comparison of chromogenic and clotting methods. Blood Coagul Fibrinolysis 1996;7:24-30
- Hemker HC, Willems GM, Beguinb S. A computer assisted method to obtain the propthrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb Haemost 1986;56:9-17.
- Hemker HC, Wielders S, Kessels H, Beguin S. Continuous registration of thrombin generation in plasma, its use for the determination of the thrombin potential. Thromb Haemost 1993; 70:617–624.
- Gerotziafas GT, Depasse F, Busson J, Leflem L, Elalamy I, Samama MM. Towards a standardization of thrombin generation assessment: The influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay. Thrombosis Journal 2005; 3:16