Direct thrombin inhibitors: clinical uses, mechanism of action, and laboratory measurement.
Table 1. FDA approved direct thrombin inhibitors. Class Bivalent Indication PCI & PCI in patients with HIT Administration Parenteral Time to peak concentration 0.5 to 1.5 hours Clearance Enzymatic Monitoring ACT; aPTT Class Bivalent Indication Anticoagulation in HIT Administration Parenteral Time to peak concentration 2.0 to 3.0 hours Clearance Kidneys Monitoring aPTT; ECT; ACT in CPB Class Univalent Indication Anticoagulation in HIT Administration Parenteral Time to peak concentration 2.0 to 4.0 hours Clearance Liver Monitoring aPTT;ACT in CPB Class Univalent Indication Stroke Prevention Administration Oral Time to peak concentration 1.5 to 2.0 hours Clearance Kidneys Monitoring None*; ECT if necessary *Except in certain clinical conditions, see text. ACT: activated clotting time; aPTT: activated partial thromboplastin time; CPB: cardiopulmonary bypass; ECT: Ecarin clotting time; HIT: Heparin- induced thrombocytopenia; PCI: percutaneous coronary intervention.
Direct thrombin inhibitors
Direct thrombin inhibitors (DTIs) are a class of anticoagulants that act by directly inhibiting thrombin to delay clotting and are typically used during HIT and in acute coronary syndrome (see Table 1). Hirudin, the first parenteral DTI to be used, was isolated in the late 1800s from the medicinal leech.
Hirudo medicinalis. Issues related to purification and availability, however, led to its disuse following the introduction of heparin. (2) DTIs are classified as either univalent or bivalent according to where they bind thrombin (see Figure 1). The bivalent DTIs include bivalirudin and lepirudin, and bind both the active site and exosite 1 of thrombin. Univalent DTIs bind only the active site and include argatroban and dabigatran.
Bivalirudin'. As a bivalent DTI, bivalirudin is a short synthetic peptide that consists of the N-terminal and C-terminal active peptide domains of hirudin joined by a linker. (5) It blocks thrombin activity by binding the active site (N-terminus of bivalirudin) and the fibrinogen-binding exosite (C-terminus of bivalirudin) of circulating and clot-bound thrombin. The majority of bivalirudin is cnzymatically eliminated and considered safest to use in the presence of both hepatic and renal dysfunction.
Lepirudin'. Approved for anticoagulation in patients with HIT by the Food and Drug Administration (FDA) in 1998, lepirudin is a desulfated recombinant hirudin. Not only can it inhibit free and clot-bound thrombin, (6) it is not activated by platelet factor 4, making it more effective in the presence of platelet-rich thrombi. (7) In addition, lepirudin does not interact with HIT antibodies. (7) Because lepirudin is excreted mainly through the kidneys, dose reductions are recommended in patients with renal dysfunction.
Argatroban: Argatroban is an L-arginine derivative that binds to the active site of free, fibrin-bound, and clot-associated thrombin,8 thereby preventing fibrin formation, Factor V, VIII, and XIII activation, protein C activation, and platelet aggregation. (2) This DTI is cleared by the liver, and, therefore, not recommended for patients with hepatic dysfunction. In 2000, the FDA approved argatroban for the treatment of thrombosis in patients with HIT, and in 2002 approved its use during percutaneous coronary interventions (PCI) in patients with or at risk of HIT.
Dabigatran etexilate: As the first oral anticoagulant approved in the U.S. since warfarin in 1951, (9) dabigatran etexilate is classified as a univalent DTI. It is administered as a prodrug and is quickly absorbed and converted to the active metabolite dabigatran by esterases in the liver. (10,11) Dabigatran binds the active site of free and fibrin-bound thrombin to impede the coagulation process by preventing both the conversion of fibrinogen to fibrin and the feedback activation of Factors VII, XI, and V by thrombin and thrombin-related platelet activation. Because the drug is excreted by the kidneys primarily as active drug, it is contraindicated in patients with renal failure. (11,12) In 2010, dabigatran etexilate was approved by the FDA for prevention of stroke in patients with non-valvular atrial fibrillation. (13) Given the predictable pharmacokinetics of dabigatran, routine laboratory monitoring is not recommended by the FDA, except in certain clinical conditions (see Table 2). (10)
[FIGURE 1 OMITTED]
Laboratory monitoring of DTIs
Because there are no reversal agents for DTIs, elevated levels of these drugs carry the risk of life-threatening bleeding complications. (14) For example, underestimation of argatroban has been shown to exacerbate certain clinical situations and lead to extended coagulopathy. (15) To prevent overdosing and ensure adequate dosing, therapeutic drug monitoring is required for the majority of DTIs. (16) Currently, the majority of the assays that measure DTIs in clinical laboratories are activity based and provide indirect measurements of levels making them vulnerable to interferences from a variety of sources. (16)
Activated partial thromboplastin time (aPTT): If monitoring is indicated, all manufacturers of DTIs in the U.S. recommend using aPTT as the method of choice. The aPTT test measures the efficacy of both the intrinsic and the common coagulation pathways, and is used to detect clotting abnormalities and monitor patient response to anticoagulant therapy. aPTT assays require the presence of fibrinogen, Factors II, V, VIII, IX, X, XI, and XIII.
In addition to the presence of anticoagulants, coagulation factor deficiencies and the presence of antiphospholipid antibodies can prolong the aPTT. (17) In order to determine the cause of a prolonged aPTT, mixing studies can be performed in which the patient's plasma is mixed with normal plasma. Following these mixing studies, if the aPTT corrects it typically indicates that a factor deficiency is the cause for the prolonged clot time. If, however, the clotting abnormality does not disappear, the likely reason is due to the presence of an anticoagulant or antiphospholipid antibodies. Because aPTT values are affected by clotting factors not directly influenced by DTIs, caution must be used in interpreting the results. In addition, the dose response is non-linear at increasing concentrations of DTIs. Thus, although the aPTT is sufficient in the low level range, it is not accurate at high, and potentially toxic, doses of DTIs.(2)
Activated clotting time (ACT): This test is similar to the aPTT, and depends on factors that constitute the intrinsic pathway of coagulation. (18) It is approved for monitoring the effectiveness of high-dose heparin therapy. The ACT has also been used in the operating room during PCI to monitor bivalirudin levels in patients with HIT, although it is not currently approved for this use. (19) Fresh blood is added to a tube containing a particulate surface activator of Factors XII and XI. (18) Because neither partial thromboplastin nor a phospholipid substitute is added to the sample, coagulation depends on the presence of adequate endogenous amounts of platelet phospholipids in the sample. (18) ACT has also been used to measure DTIs other than bivalirudin in interventional settings but does not provide linear results at high concentrations. (20)
Table 2. Clinical indications for dabigatran monitoring. (10,11)
* assessment of therapy compliance
* assessment of efficacy or over-treatment in patients with renal or liver failure
* assessment of the anticoagulant effect when drug interactions are suspected
* major or life threatening bleeding complication
* suspected drug failure
* determination of coagulation adequacy in treated patients prior to emergency surgery
Ecarin clotting time (ECT): Ecarin is a metalloproteinase that activates prothrombin independent of calcium, phospholipid, and Factor V. (17) Isolated from the venom of the viper Echis carinatus, ecarin cleaves prothrombin to generate meizothrom-bin. (21) When ecarin is added in a defined amount to whole blood or citrated plasma containing a DTI, it will induce the generation of meizothrombin, which is immediately inhibited by any non-oral anticoagulants. Once the DTIs in the sample have been depleted, any additional meizothrombin that is generated and will induce a time-defined start for coagulation by stimulating the conversion of fibrinogen to fibrin.(21) The Thrombin inhibitor management (TIM)-ECT was developed as a poinl-of-care test in Europe and is based on the same coagulation principles as the ECT, but in a one-step, dry chemistry platform.(22) A chromogenic variant of this assay has also been developed (i.e., the Stago ECA-H for recombinant hirudins and the Stago ECA-Tfor synthetic direct thrombin inhibitors), based on using a chromogenic substrate to measure meizothrombin generation in a plasma sample. (20)
Several studies have suggested that ECT is more sensitive than the aPTT and may be suitable for acute clinical situations as a point-of-care test in the monitoring of DTIs. (21,22) In addition, ecarin is not affected by the presence of heparin or oral anticoagulants making it an attractive assay for use in monitoring patients following a necessary changeover from other anticoagulants such as heparin or warfarin. (21)
Prothrombinase-induced clotting time (PiCT): Currently under consideration for FDA approval, (24) studies have demonstrated the ability of the PiCT assay to determine activities of both direct and indirect thrombin inhibitors in a linear manner over a wide concentration range. (25) PiCT is a plasma-based anticoagulant assay that is sensitive to Factor Xa and Factor IIa (thrombin). Patient samples are mixed with activated Factor Xa, phospholipids, and an enzyme from the venom of the Russell viper (RVV-V). RVV-V is a specific activator of Factor V and will stimulate the formation of the prothrombina.se complex upon recalcification. Once the prothrombinase complex has formed, Factor II is activated, fibrinogen is converted to fibrin, and clotting time of the sample can be measured. (17,26) Because the assay uses RVV-V to activate Factor V, results of the PiCT are not dependant on coagulation factors normally required for Factor V activation. Deficiencies in Factor II, Factor V, fibrinogen, and lupus anticoagulants, however, can prolong clot time and lead to an overestimation of the amount of DTI present.(17)
Chromogenic anti-IIa: This method is used to determine the concentration of DTIs based on their anti-IIa activity in the presence of a constant and in excess amount of thrombin (Flla). (27) Thrombin is added in excess to diluted patient plasma, and its activity is neutralized in proportion to the amount of DTI in the sample. The remaining active thrombin then hydrolyzes a chromogenic substrate to stimulate the release of a chromogen that can be measured photometrically. Thus, the remaining active thrombin is inversely proportional to the amount of DTI in the sample. Because thrombin is added in excess to the patient sample, the chromogenic anti-IIa is not sensitive to lupus anticoagulants or coagulation factor deficiencies which can interfere with thrombin generation. While FDA-approved assay are available, they have not yet been fully validated and approved for monitoring DTI levels. (17)
Diluted thrombin time (dTT): The thrombin time test (TT) measures the time to clot formation in a patient sample following addition of excess thrombin. It is prolonged in the presence of low fibrinogen levels, elevated fibrin degradation products, heparin, and DTIs. (20)Because TT is over-sensitive to low levels of heparin, hirudin, and argatroban, the alternative dTT assay was developed. These tests, such as the Hemoclot assay, are based on the same principle as TT, except that the thrombin is added to dilute patient plasma. (10,28)
DTIs are used as alternative therapy in patients diagnosed with HIT who require anticoagulation. As relatively new therapeutic agents, there is currently a lack of standardized laboratory tests for these anticoagulants. While the global clotting assays are widely available to monitor DTI therapy, they are not particularly specific, and the results have been shown to vary with reagents from different manufacturers. (28) As an alternative to clotting based tests, the chromogenic assays are specific, but currently lack widespread availability. (28) An increased focus has been placed on developing new laboratory tests suitable for the measurement of DTIs that are reproducible, accurate, and sensitive, such as the ability to directly measure drug concentrations by mass spectrometry. (29-31)
Jeanne M. Rhea, PhD, is a post-doctoral Fellow working with Ross J. Molinaro, MT(ASCP), PhD, D(ABCC), F(ACB), within the Department of Pathology and Laboratory Medicine at Emory University School of Medicine in Atlanta, GA.
(1.) Sikka P, Bindra VK. Newer antithrombotic drugs. Indian J Crit Care Med. 2010; 14(4): 188-195.
(2.) Frenkel EP, Shen YM, Haley BB. The direct thrombin inhibitors: their role and use lor rational anticoagulation. Hematol Oncol Clin North Am. 2005;19(11:119-145, vi-vii.
(3.) Bauer KA. New anticoagulants. Hematology Am Soc Hematol Educ Program. 2006:450-456.
(4.) Ahmed I, Majeed A, Powell R. Heparin induced thrombocytopenia: diagnosis and management update. Postgrad Med J. 2007;83(983):575-582.
(5.) Hirsh J. New anticoagulants. Am Heart J. 2001;142<2 Suppl):S3-8.
(6.) Weitz Jl, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin Ill-independent inhibitors. J Clin Invest. 1990;86(2):385-391.
(7.) Greinacher A, Janssens U, Berg G, et al. Lepirudin (recombinant hirudin) for parenteral anticoagulation in patients with heparin-induced thrombocytopenia. Heparin-Associated Thrombocytopenia Study (HAT) investigators. Circulation. 1999;100(61:587-593.
(8.) Koster A, Fischer KG, Harder S, Mertzlufft F. The direct thrombin inhibitor argatroban: a review of its use in patients with and without HIT. Biologies. 2007;M2V105-112.
(9.) Tran A,Cheng-Lai A. Dabigatran etexilate: the first oral anticoagulant available in the United States since warfarin. Cardiol Rev. 2011;19(3):154-161.
(11.) Weitz Jl, Hirsh J, Samama MM. New antithrombotic drugs: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines, 8th ed.). Chest. 2008;133I6 Suppl):234S-256S.
(12.) Harbrecht U. Old and new anticoagulants. Hamostaseologie. 2011;31(1):21-27.
(13.) Wann LS, Curtis AB, Ellenbogen KA, et al. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (update on Dabigatran): a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation. 1144;123(10):1144-1150.
(14.) Follis F, Filippone G, Montalbano G, et al. Argatroban as a substitute of heparin during cardiopulmonary bypass: a safe alternative? Interact Cardiovasc Thorac Surg. 2010;10(4):592-596.
(15.) Genzen JR, Fareed J, Hoppensteadt D, et al. Prolonged elevation of plasma argatroban in a cardiac transplant patient with a suspected history of heparin-induced thrombocytopenia with thrombosis. Transfusion. 2010;50(4):801-807.
(16.) Love JE, Ferrell C, Chandler WL Monitoring directthrombin inhibitors with a plasma diluted thrombin time. Thromb Haemost. 2007;98(1):234-242.
(17.) Castellone DD.Van CottEM. Laboratory monitoring of new anticoagulants. Am J Hematol. 2010;85(3):185-187.
(18.) Barash PG, Cullen BF, Stoelting RK, Cahalan MK, Stock MC. Clinical Anesthesia. 6th ed. (Lippincott Williams & Wilkins, Philadelphia, PA, 2009).
(19.) Casserly IP, Kereiakes DJ, Gray WA et al. Point-of-care ecarin clotting time versus activated clotting time in correlation with bivalirudin concentration. Thromb Res. 2004; 113(21:115-121.
(20.) Bates SM,Weitz Jl. Coagulation assays. Circulation. 2005;112(4):e53-60.
(21.) Nowak G. Clinical monitoring of hirudin and direct thrombin inhibitors. Semin Thromb Hemost. 2011;27(5);537-541.
(22.) Cho L, Kottke-Marchant K, Lincoff AM et al. Correlation of point-of-care ecarin clotting time versus activated clotting time with bivalirudin concentrations. Am J Cardiol. 2003;91(9):1110-1113.
(23.) Nowak G.Bucha E. Quantitative determination of hirudin in blood and body fluids. Semin Thromb Hemost. 1996;22(2):197-202.
(24.) JeskeWP.Fareed J,HoppensteadtDA, Lewis B.Walenga JM. Pharmacologyo!argatroban. Expert Rev HematoL 2010;3(5):527-539.
(25.) Fenyvesi T, Jorg I, Harenberg J. Effect of phenprocoumon on monitoring ot lepirudin, argatroban, melagatran and unfractionated heparin with the PiCT method. Palbophysiol Haemost Thromb. 20D2;32 (4):174-179.
(26.) CalatzisA, PeetzD, Haas S etal. Prothrombinase-irtduced clotting time assay for determination of the anticoagulant effects of unfractionated and low-molecular-weight heparins, fondaparinux, and thrombin inhibitors. Am J Clin Pathol. 2008;30(3): 44B-454;
(27.) Biophen DTI Package Insert. Anaria Hyphen Biomed. 2008.
(28.) SamamaMM.GiiinetC. Laboratory assessment of new anticoagulants. Clinical Chemistry and Laboratory Meo7cHie.2011;49(5):761-772.
(29.) Mplinaro RJ. Quantitation of argatroban in plasma using liquid chromatography electrospray tandem mass spectrometry (UPLC-ESl-MS/MS). Methods Mol Biol. 201Q;6Q357-60363.
(30.) Swan SK,Hursting MJ. The pharmacokinetics and pharmacodynamics of argatroban: effects of age, gender, and hepatic or renal dysfunction. Pharmacotherapy. 2000;20(3):318-329.
(31.) Tran JQ, Di Cicco RA, Sheth SB, et al. Assessment of the potential pharmacokinetic and pharmacodynamic interactions between erythromycin and argatroban. J Clin Pharmacol 1999;39(5I:513-519.
By Jeanne M. Rhea, PhD, and Ross J. Molinaro, MT(ASCP), PhD, D(ABCC), F(ACB)
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||Clinical issues|
|Author:||Rhea, Jeanne M.|
|Publication:||Medical Laboratory Observer|
|Date:||Aug 1, 2011|
|Previous Article:||Acetaminophen: hidden complexities of a simple overdose.|
|Next Article:||Where does the LIS go from here?|