Antithrombin Levels Are Unaffected by Warfarin Use Michael J. Sanfelippo, MS, MT; Jessica M. Engel, MSN; Adedayo A. Onitilo, MD, MSCR

 Context.—The results of studies among patients with antithrombin deficiency have suggested that the use of warfarin will increase the level of antithrombin. Objective.—To reevaluate the effect of warfarin on antithrombin levels using an automated amidolytic method in current use. Design.—Antithrombin levels were measured in patients who were receiving warfarin for atrial fibrillation and were compared with antithrombin levels in preoperative patients who had not received warfarin.

A

ntithrombin is an important protein in the regulation of hemostasis. It downregulates coagulation by forming an irreversible inactive complex with thrombin and activated factor X (Xa). Partial deficiency of antithrombin is associated with a high incidence of thrombotic disease. Because of its importance in the regulation of coagulation and the high incidence of thrombotic disease associated with a partial deficiency, antithrombin is frequently measured in patients to assess the risk of thrombotic disease.1 The results of studies2–4 among patients with congenital deficiency of antithrombin have suggested that antithrombin levels were increased when they were treated with warfarin. This has led physicians to avoid the measurement of antithrombin when patients were receiving warfarin for treatment of a thrombotic event.2 Because the methods of assay of antithrombin have changed since early investigations,5 we undertook a study to determine if antithrombin levels are truly increased by warfarin treatment using a current method of assay for antithrombin activity. MATERIALS AND METHODS This study was conducted at the Department of Laboratory Medicine, Marshfield Clinic, in Marshfield, Wisconsin. It was approved by the Institutional Review Board of Marshfield Clinic, with waiver of informed consent.

Accepted for publication August 8, 2013. From the Department of Hematology/Coagulation Services, Marshfield Labs, Marshfield (Mr Sanfelippo), the Marshfield Clinic Cancer Care at St Michaels, Stevens Point (Ms Engel), and the Department of Hematology/Oncology, Marshfield Clinic Weston Center, Weston (Dr Onitilo), Wisconsin. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Michael J. Sanfelippo, MS, MT, Department of Hematology/Coagulation Services, Marshfield Labs, 1000 N Oak Ave, Marshfield, WI 54449 (e-mail: sanfelippo.michael@ marshfieldclinic.org). Arch Pathol Lab Med—Vol 138, July 2014

Results.—Patients receiving warfarin had a mean antithrombin level of 100.40% (range, 81%–153%). Patients not receiving warfarin had a mean antithrombin level of 99.97% (range, 79%–120%). The Student t test was not significant for a difference between the mean antithrombin levels of the 2 populations. Conclusions.—The use of warfarin does not increase the level of antithrombin in patients receiving the drug. (Arch Pathol Lab Med. 2014;138:967–968; doi: 10.5858/ arpa.2013-0065-OA) Study Populations Warfarin.—Plasma from patients receiving warfarin for atrial fibrillation was obtained from the residual plasma after determination of the international normalized ratio performed for monitoring the anticoagulant. All specimens tested had international normalized ratios in the range of 2.0 to 3.0. None of these patients were receiving unfractionated heparin or low-molecularweight heparin. No patient had a history of thrombotic disease. No Warfarin.—Plasma from patients undergoing preoperative evaluation was obtained from the residual plasma after testing for the international normalized ratio. All specimens had international normalized ratios of 0.9 to 1.1. All of these patients were scheduled for elective total hip or knee arthroplasty. No patient had a personal or family history of thrombotic disease, and none were taking an anticoagulant drug.

Assay Technique Antithrombin was assayed on citrated plasma using a commercial automated amidolytic antithrombin assay (Coamatic Antithrombin, diaPharma, West Chester, Ohio). This assay is approved by the Food and Drug Administration for diagnostic testing. The assay is based on inhibition of Xa in the presence of heparin. The Xa remaining after inhibition by antithrombin is measured by reaction with a chromogenic substrate. Antithrombin levels are indirectly related to the residual Xa. The assay was run on an automated analyzer (BCS X2, Siemens Healthcare USA, Inc, Malvern, Pennsylvania). The reference range for this method is 80% to 120%. The significance of the difference between the mean antithrombin levels of the 2 populations was determined by the Student t test.

RESULTS The composition of the 2 study populations with respect to sex and age is given in Table 1. The antithrombin levels in the 2 populations are given in Table 2. COMMENT The assay of antithrombin is an important test in the identification of thrombotic factors because a partial deficiency of this protein is associated with a high risk of thrombotic disease. In a patient seen with a thrombotic Antithrombin and Warfarin—Sanfelippo et al 967

Table 1. Composition of the 2 Study Populations Group No warfarin Warfarin

Male, No. Female, No. Age, Mean (Range), y 57 72

43 59

61.3 (39–92) 75.7 (46–93)

event, the measurement of this protein can be problematic. Its measurement on admission is not recommended because the antithrombin level may be reduced because of the generation of the thrombin that caused the thrombotic event. Furthermore, treatment of the patient with unfractionated or low-molecular-weight heparin will reduce the half-life of the antithrombin by accelerating the binding of antithrombin to thrombin and Xa. In addition, the findings of a 1974 study2 suggested that the use of warfarin caused an increase in the level of antithrombin, which could prevent detection of an underlining deficiency. Therefore, testing for a congenital antithrombin deficiency would have to be delayed until the patient had discontinued warfarin. The increase in antithrombin concentration during warfarin therapy in that study2 was based on assay methods using serum and demonstrating inhibition of human thrombin. The current assay methods use citrated plasma and are based on inhibition of bovine thrombin or Xa. Both techniques involve amidolytic methods that use chromogenic substrates to demonstrate inhibition of thrombin or Xa. The old methods used the clotting time of a fibrinogen solution to demonstrate inhibition of thrombin. We undertook this study to determine if an increase in antithrombin in patients receiving warfarin could be demonstrated using a method that is in current use. We measured antithrombin levels in 2 populations, one receiving warfarin for prophylaxis and another not receiving warfarin. The 2 populations had approximately equal numbers of men and women. In addition, the age range was approximately equal, with the exception that the mean age of the warfarin group was older by about 14 years. A difference in antithrombin levels between men and women has been reported using an amidolytic assay but was only significant in the age range of 25 to 35 years.6 In addition, the same study was unable to demonstrate significant agerelated differences in antithrombin levels in individuals older than 35 years.6 None of the patients in either of our populations were younger than 35 years. If warfarin increases antithrombin levels by some unknown mechanism, we would expect to see significantly higher antithrombin levels in the warfarin population than in the nonwarfarin population. The reported increase in antithrombin during warfarin therapy was based on assay procedures using a serum specimen.5 The use of serum as a test specimen produced misleading results because of variable production of thrombin and Xa, with subsequent variable consumption of antithrombin. Warfarin use reduces vitamin K–dependent coagulation factor activities due to reduced numbers of gamma-carboxyglutamic acid residues per molecule. The reduction in coagulation factor activities results in decreased generation of thrombin and Xa. The reduction in these 2 targets of antithrombin will bind to and consume less antithrombin than would be consumed in the clotting of plasma with normal levels of vitamin K-dependent factor

968 Arch Pathol Lab Med—Vol 138, July 2014

Table 2. Antithrombin Levels in the 2 Study Populations Group

No.

Antithrombin Level, Mean (Range), %

t Statistic

No warfarin Warfarin

100 131

99.97 (79–120) 100.40 (81–153)

... 0.496

activities. The result will be an increased level of antithrombin in the serum of warfarin-treated patients because of decreased consumption. Our data demonstrated no significant difference in antithrombin levels between a population of patients receiving warfarin and another population not receiving warfarin. Because the observations of increased antithrombin levels during warfarin therapy were made in patients with congenital partial deficiencies of antithrombin, we would have liked to test this patient population with an amidolytic assay using a citrated plasma specimen. Unfortunately, no patients with partial congenital antithrombin deficiency were available for study. However, in a 2008 study by Kearon et al,7 there were 23 patients identified with partial deficiency of antithrombin among a population of 645 patients receiving warfarin for treatment of thrombotic events. The antithrombin deficiency was identified in these patients using a citrated specimen and an amidolytic assay identical to the one we used.8 Variables such as techniques of defibrination, detection of end points, interference from heparin cofactor II, and different levels of the target enzyme thrombin contributed to inaccuracies of the preamidolytic methods. These variables have all been eliminated in the automated amidolytic assays of antithrombin in current use. We conclude that the use of warfarin does not increase the level of antithrombin in patients receiving the drug. Furthermore, the increases reported in earlier studies were artifactual, related to the specimen and the methods used. We thank Marie Fleisner of the Marshfield Clinic Research Foundation’s Office of Scientific Writing and Publication for editorial assistance in the preparation of the manuscript. This study was supported by Marshfield Clinic laboratory general funds. References 1. Bick RL. Clinical relevance of antithrombin III. Semin Thromb Hemost. 1982;8(4):276–287. 2. Marciniak E, Farley CH, DeSimone PA. Familial thrombosis due to antithrombin 3 deficiency. Blood. 1974;43(2):219–231. 3. O’Brien JR, Etherington MD. Effect of heparin and warfarin on antithrombin III. Lancet. 1977;2(8050):1231–1232. 4. Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 3rd ed. Philadelphia, PA: JB Lippincott; 1994:1553. 5. von Kaulla E, von Kaulla KN. Antithrombin 3 and diseases. Am J Clin Pathol. 1967;48(1):69–80. 6. Dolan G, Neal K, Cooper P, Brown P, Prestton FE. Protein C, antithrombin III and plasminogen effect of age, sex, and blood group. Br J Haematol. 1994;86(4): 798–803. 7. Kearon C, Julian JA, Kovacs MJ, et al. Influence of thrombophilia on the risk of recurrent venous thromboembolism while on warfarin: results from a randomized trial. Blood. 2008;112(12):4432–4436. 8. Anderson NE, Menschik M. New chromogenic ATIII activity kit which is insensitive to heparin cofactor II and designed for use on automated instruments [abstract]. Thromb Haemost. 1991;65(6):912.

Antithrombin and Warfarin—Sanfelippo et al

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Antithrombin levels are unaffected by warfarin use.

The results of studies among patients with antithrombin deficiency have suggested that the use of warfarin will increase the level of antithrombin...
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