Measurement of Maximum Thrombin Generation Capacity in Blood and Plasma Using the Thrombin Generation Assay (THROGA) Q1 Götz Nowak, M.D., 1 Ute Lange, Ph.D., 2 Annett Wiesenburg, 1 and Elke Bucha, M.D. Q11 ABSTRACT Q2 Q3 Diagnostics of a hyper- or hypocoagulable state has been very difficult Q2. The first attempt to solve this problem was the method of endogenous thrombin potential (ETP) by Hemker Q3. In ETP, activators and a chromogenic substrate are added to diluted plasma samples and the thrombin generation is measured. By analysis of acquired data, three characteristics of ETP are seen: lag phase, peak thrombin, and velocity index. ETP is not suited for exact determination of maximum activated thrombin. Therefore, a new method was developed: the thrombin generation assay (THROGA). With the use of THROGA, the maximum generated thrombin in a blood or plasma sample can be measured easily. The background of the method is the addition of a certain amount of recombinant hirudin (r-hirudin) to the blood or plasma sample. After activation, the generated thrombin is bound quantitatively and neutralized by r-hirudin so that at the end of the activation phase the amount of generated thrombin can be determined easily and exactly by measurement of residual r-hirudin in the sample. KEYWORDS: Thrombin generation assay (THROGA), thrombin generation capacity, drug monitoring, r-hirudin, global coagulation state Blood coagulation represents one of the fundamental vital functions of a living organism. By a variety of control and repair mechanisms, blood coagulation guarantees the lifelong circulation of blood through the whole organism. Blood coagulation is a complex process triggered by cellular elements (platelets, white blood cells, endothelial cells) and plasmatic coagulation. The physiological function of blood coagulation is to induce an immediate arrest of bleeding at the site of a lesion or discontinuity of the endothelium (i.e., to cover endothelial defects to prevent the leakage of blood from the vessels). Active components of this process are the platelets and plasmatic coagulation factors that convert soluble fibrinogen to fibrin at the end of a complex procedure. The multifactor process is controlled by several serine proteases that are activated from inactive precursors. These inactive precursors of the most important coagulation enzymes (factor [F] XI, FIX, FX, FVII, and FII) are fixed on negatively charged phospholipid surfaces, mediated by calcium ions and 508 1 Friedrich Schiller University Jena, Medical Faculty, Research Group Pharmacological Haemostaseology, Jena, Germany; 2 HaemoSys GmbH, Jena, Germany. Address for correspondence and reprint requests: Prof. Dr. Götz Nowak, Friedrich Schiller University Jena, Medical Faculty, Research Group Pharmacological Haemostaseology, Drackendorfer Str. 1, D-07747 Jena, Germany. E-mail: AGPHH@med.uni-jena.de. New Anticoagulants; Guest Editor, Job Harenberg, M.D. Semin Thromb Hemost 2007;33:508 514. Copyright # 2007 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584 4662. DOI 10.1055/s-2007-982082. ISSN 0094-6176.
MAXIMUM THROMBIN GENERATION CAPACITY IN BLOOD AND PLASMA/NOWAK ET AL 509 Q4 Q5 Q6 thrombin-activated cofactors (i.e., they are activated to active serine proteases Q4 ). The involved serine proteases are fixed to the surfaces by g-carboxy anchors. The serine protease thrombin is the most important enzyme in activation of plasmatic coagulation. Thrombin is the last activation product and leaves its activation complex, the so-called prothrombinase, in which the activated FXa induces the limited proteolysis of prothrombin Q5. During activation of coagulation, thrombin Q6 is the only enzyme that is freely available in blood circulation. Here, it encounters different substrates. Its most relevant substrate is fibrinogen, which is converted to fibrin by thrombin. Furthermore, thrombin finds several cellular receptors at platelets, leukocytes, endothelial cells, and many other cells of the body that are known as protease-activated receptors. Thereby, thrombin has a central role and a bridging function between plasmatic coagulation and the cells of the organism. Conversely, on the phospholipid surfaces of platelets that are aggregated with leukocytes (especially monocytes), coagulation activation takes place by which great amounts of thrombin are released (so-called thrombin burst). In blood circulation, thrombin can induce physiological but often also pathological processes, depending on the amount of available thrombin. Therefore, from the maximum activated thrombin amount in blood or plasma, the global coagulation state of a patient can be determined. From this actual thrombin generation capacity, information can be obtained about both hypercoagulability (thrombophilia) and hypofunction of thrombin generation (hemophilia or bleeding tendency). METHOD The thrombin generation assay (THROGA) is based on the binding of recombinant hirudin (r-hirudin) to thrombin that is formed following activation of both endogenous and exogenous pathway of blood coagulation. The r-hirudin binds to the generated thrombin in a slow, tight-binding manner in a stoichiometric 1:1 enzyme/inhibitor complex. In THROGA, thrombin generation is induced by addition of a patient s plasma or blood sample to a vial with lyophilized activator reagent (activator vial), which contains a defined Q7 Q9 Figure 1 Q7 Optimum activation of coagulation in the thrombin generation assay (THROGA). Phospholipids contained in the activator reagent start the intrinsic pathway of coagulation by activation of factor (F) XII and formation of a high molecular complex in which FXI is activated. This factor and further coagulation factors (FII, FVII, FIX, and FX) are permanently fixed on negatively charged phospholipid surfaces (PL ) using Ca 2 þ -bridging by g-carboxy anchors. Together with FVIIa, the tissue factor (TF) contained in the activator reagent forms an activation complex that primarily activates FIX to FIXa. FIXa is the central serine protease of tenase complex. FXa that was activated in the tenase complex, together with FVa in the prothrombinase complex, is involved in the generation of thrombin from prothrombin. Thrombin is freely available in blood (thrombinemia Q8 ). Thrombin reaches a variety of substrates, mostly fibrinogen that is converted to fibrin. V XIII, coagulation factors V to XIII; Va XIIIa, activated coagulation factors V to XIII; HMWK, high molecular weight Q9 kininogen; PK, prekallikrein. Q8
510 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 33, NUMBER 5 2007 Figure 2 assay. Schematic overview of thrombin generation assay procedure. r-hirudin, recombinant hirudin; ECA-H, ecarin chromogenic amount of r-hirudin as well as tissue factor, phospholipids, and calcium. As reference, blood or plasma is added to a vial with lyophilized buffer and r-hirudin (reference vial). Both vials are shaken in a defined manner (550 rpm, 30 minutes) at room temperature. The proceeding optimum activation of plasmatic coagulation is finished by addition of an ethylenediaminetetraacetic acid containing stop reagent. In the next step, the amount of r-hirudin in both vials is measured using a quantitative r-hirudin determination method (ecarin chromogenic assay [ECA-H]; HaemoSys GmbH, Jena, Germany). In ECA-H, from a linear calibration line the measured antithrombin units (ATU) in blood or plasma are read. The maximum thrombin generation (MTG) in the patient s sample is calculated as the difference between the r-hirudin amount measured in the reference (R) and in the activator vial (A): MTG ¼ R A. Considering the equivalence of a thrombin unit (TU) and an antithrombin unit of r-hirudin (ATU), the thrombin amount canbeprovidedeasilyintu/ml.aschematicoverview of the THROGA procedure is shown in Fig. 2. An example for an ECA-H calibration line for r-hirudin is shown in Fig. 3, as is an example for calculation of maximum thrombin generation. THROGA is product of HaemoSys. Whereas THROGA is designed for an activation time of 30 minutes, in our investigations we measured the thrombin generation also at 10 and 20 minutes of activation time to evaluate the time-dependent dynamics of thrombin activation. RESULTS Determination of Thrombin Generation Capacity in Blood and Plasma of Healthy Volunteers The thrombin generation has been determined in 50 male and female healthy volunteers (age 19 to 65 years). In plasma, 126.4 23.7 TU/mL have been measured; in blood, 84.2 18.2 TU/mL have been measured. Regarding the measured mean hematocrit of 36, a calculative thrombin generation in blood of 81 TU/mL was observed Q10. In Fig. 4 the measured thrombin activation is depicted. It is seen that after 10 minutes of activation, 40% of maximum thrombin already has been generated in blood and plasma. After 20 minutes of activation, this value has increased to 85% in plasma and 90% in blood, respectively. Determination of Thrombin Generation Capacity in Blood and Plasma of Patients THROGA was used in blood and plasma of patients suffering from various diseases, and it was seen that a hypercoagulable state can be diagnosed using THROGA. In Fig. 5 the course of thrombin generation is depicted in a patient who had to undergo platelet function analysis due to multiple transitory ischemic attacks. When the platelet adhesion assay (PADA) was used, 2 an increased adhesion index of 68 was measured. In Fig. 5 it is seen that this patient has a significantly Q10
MAXIMUM THROMBIN GENERATION CAPACITY IN BLOOD AND PLASMA/NOWAK ET AL 511 Figure 3 Example of ecarin chromogenic assay (ECA-H) calibration line and calculation of maximum thrombin generation. ATU, antithrombin unit. Figure 4 Determination of normal values of thrombin generation in healthy volunteers (n ¼ 50; age 19 to 65 years). In plasma, 126.4 23.7 thrombin units (TU)/mL have been measured; in blood, 84.2 18.2 TU/mL has been measured.
512 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 33, NUMBER 5 2007 Q11 Figure 5 Thrombin generation assay (THROGA) in a patient with transient ischemic attacks (US Q11, female, age 56 years). Analysis of coagulation state: pathological thrombin generation in blood and plasma in THROGA. Subsequent diagnostics: activated protein C APC resistance (factor V Leiden) and thrombophilia. TU, thrombin unit. increased thrombin generation after 10, 20, and 30 minutes, both in blood and plasma. When considering her hematocrit of 39, a very high thrombin generation capacity in plasmatic coagulation becomes obvious in this patient. Similar pathologic courses of thrombin generation have been detected in other patients. In the investigated population (220 patients), such prothrombotic or hypercoagulable states have been detected in 8 to 10% of patients. According to preliminary consecutive analyses, mainly hereditary thrombophilic disorders have been diagnosed in these patients, mostly FV Leiden. Within the investigation of patients, those who showed a very high thrombin generation in blood have been identified, with values much higher than the hematocrit-corrected calculative thrombin generation. In Fig. 6 the thrombin generation in a patient after infarct is presented as an example. In addition to a Q13 Figure 6 Thrombin generation assay (THROGA) in a postinfarct patient (HQ Q12, male, age 65 years). Cardiac infarction December 2005, and cardiopulmonary bypass; February 2006, analysis of coagulation state: ASA Q13 resistance, pathologic thrombin generation in blood in THROGA ( þ 18%). TU, thrombin unit. Q12
MAXIMUM THROMBIN GENERATION CAPACITY IN BLOOD AND PLASMA/NOWAK ET AL 513 pathologically high adhesion index in PADA of 87, this patient showed in blood nearly the same amount of generated thrombin as in plasma (measured hematocrit, 41). Such a certainly pathologic thrombophilic state has been detected in only 2 to 3% of investigated patients. Less severe forms, where in blood only 10 to 15 TU/mL more is generated than the calculative value would predict, have been found more frequently, mostly in patients who suffered from a platelet-induced hypercoagulable state. Influence of Anticoagulants on THROGA Quantification of FX inhibitors or FIX inhibitors in blood has been difficult. Global clotting assays such as activated partial thromboplastin time or prothrombin time are only partly suited or not suited at all. Chromogenic assays make use of activators that release serine proteases in plasma and react with FIX and FX inhibitors. The efficacy of these inhibitors on the complex coagulation process cannot be measured. THROGA can solve these problems because the assay imitates the physiologic blood coagulation process. It has been tested whether fondaparinux can be measured by THROGA. Fondaparinux acts as highly effective FX inhibitor in the fondaparinux antithrombin III complex. In Fig. 7 the influence of fondaparinux on maximum thrombin generation in pooled plasma is depicted. A dose-dependent inhibition of thrombin generation is seen. At 1 mg/ml fondaparinux, the thrombin generation is reduced to 67%; at 2.5 mg/ml fondaparinux, the thrombin generation is reduced to 39%. Furthermore, the lag phase (10 minutes in control plasma) is prolonged to 15 and 20 minutes, respectively. According to these data, THROGA provides a new assay for quantification of fondaparinux. In an additional subset of studies, the maximum thrombin generation in heparin-treated patients was measured. Fig. 8 shows the thrombin generation in a patient who had been anticoagulated with 27,000 units of heparin during hemodialysis (HD). In Fig. 8A it is seen that due to this high heparin dose, the thrombin generation in blood and plasma is inhibited completely. Given that the patient had tested positive for heparininduced thrombocytopenia Q14 II antibodies both before and at the end of HD, he was switched to r-hirudin anticoagulation. In Fig. 8B his thrombin generation after the third r-hirudin anticoagulated HD is depicted (dose, 0.1mg/kg lepirudin Q15 ). The heparin effect was washed out and a nearly normal thrombin generation capacity was regained. Given that in THROGA, r-hirudin is used as thrombin trap, the direct determination of anti-iia inhibitors is not possible. However, in patients receiving long-term treatment with r-hirudin or other direct thrombin inhibitors, their influence on blood coagulation can be assessed. In patients who showed an increased thrombin generation in blood, a normalization of the coagulation state can be demonstrated following long-term application of r-hirudin. THROGA is also suited for verification of efficacy of oral anticoagulants. In patients receiving oral anticoagulation, quantitative drug monitoring can be performed using THROGA. Especially for patients receiving low-dose oral anticoagulation, drug monitoring using THROGA is optimal because with this assay, the patient s level can be set up to a 50% inhibition of blood coagulation, for example. According to our experiences Q14 Q15 Figure 7 Influence of fondaparinux on thrombin generation in plasma. After addition of 1 or 2.5 mg/ml fondaparinux, the thrombin generation is significantly reduced to 66% and 39%, respectively. Furthermore, the lag phase is prolonged from 10 minutes in control to 15 and 20 minutes, respectively, and the slope is decreased. TU, thrombin unit.
514 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 33, NUMBER 5 2007 Q16 Q17 Figure 8 Thrombin generation assay (THROGA) in a patient with renal insufficiency and heparin-induced thrombocytopenia II on chronic hemodialysis (JB Q16, male, age 86 years) at the end of hemodialysis. (A) Under heparin anticoagulation, complete inhibition of coagulation occurred, with no generation of thrombin detectable. (B) Nine days later after the switch to recombinant hirudin anticoagulation, the patient partly recovered thrombin generation, showed nearly normal THROGA in plasma, and had improved THROGA in blood, but still low Q17. TU, thrombin unit. with THROGA, the range between 25% and 50% inhibition of coagulation is regarded as the therapeutic range, depending on severity of underlying thrombophilic disorder. THROGA can also be used to detect a bleeding tendency in patients receiving oral anticoagulants. CONCLUSION THROGA represents a new innovative assay for quantification of maximum thrombin generation capacity in blood and plasma. THROGA is suited for diagnostics of coagulation disorders as well as a monitoring method for indirect anticoagulants, FIX inhibitors, and FX inhibitors. REFERENCES 1. Hemker HC, Beguin S. Thrombin generation in plasma: its assessment via the endogenous thrombin potential. Thromb Haemost 1995;74:134 138 Q18 2. Nowak G, Wiesenburg A, Schumann A, Bucha E. Platelet adhesion assay a new quantitative whole blood test to measure platelet function. Semin Thromb Hemost 2005;31: 470 475 Q18
Author Query Form (STH/01311) Special Instructions: Author please write responses to queries directly on proofs and then return back. Q1: AU: Please provide a link to an affiliation for the last author and add professional degrees for the last two authors, if applicable. Q2: AU: Please clarify. Do you mean "Diagnosis of a hyper- or hypocoagulable state..." or "Development of diagnostic test or procedures for a hyper- or hypocoagulable state..."? Q3: AU: References cannot be cited within the Abstract. Please cite reference 1 within text in numerical order or delete. Q4: AU: Please clarify phrase. Do you mean "...they are activated by active serine proteases"? Q5: AU: Sentence OK as edited for clarity? Q6: AU: Correct as stated that thrombin is an enzyme? Q7: Figure 1 is not cited in the text. Please add an in-text citation or delete the figure. Q8: AU: Please verify spelling. Q9: AU: Correct with "weight" as added for clarity? Q10: AU: OK as changed for clarity from "would arise"? Q11: AU: To what does "US" refer? Are you indicating the patient s country of origin (United States), initials, or something else? Q12: AU: Please define HQ. Are you indicating the patient s initials? Q13: AU; Please spell out ASA. Do you mean acetylsalicylic acid? Q14: AU: HIT correct as defined throughout? Q15: AU: Okay as changed from trade name of drug to generic name? Q16: AU: Please define JB. Are these the patient s initials? Q17: AU: Were all three parameters low, or only THROGA in blood? Q18: The reference 1 "Hemker, Beguin, 1995" is not cited in the text. Please add an in-text citation or delete the reference.
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