Laboratory tests for antithrombin deficiency

Authors


  • Conflict of interest: Nothing to report.

Abstract

Hereditary antithrombin deficiency is a hypercoagulable state associated with an increased risk for venous thrombosis. The recommended initial test for antithrombin is an activity (functional) assay. The advantages and disadvantages of the various testing options are presented. The causes of acquired antithrombin deficiency are much more common than hereditary deficiency. Therefore, this article describes the appropriate steps to take when antithrombin activity is low, in order to confirm or exclude a hereditary deficiency. The causes of falsely normal results are also described, including direct thrombin inhibitors. Am. J. Hematol. 85:947–950, 2010. © 2010 Wiley-Liss, Inc.

Introduction

Heterozygous antithrombin (formerly called antithrombin III) deficiency occurs in 0.02–0.17% of the general population and in 0.5–4.9% of patients with venous thromboembolism (VTE) [1–5]. It is inherited in an autosomal dominant fashion with variable clinical penetrance. Antithrombin deficiency is associated with a 5- to 50-fold increased risk for venous thrombosis [6–9]. A recent study found that antithrombin deficiency is not associated with an increased risk of arterial thrombosis [10]. As in protein C and protein S deficiency, the first thrombotic event tends to present between the ages of 10 and 50 years (peak 15–35 years). There is also a high risk of developing VTE during the patient's lifetime (50%) or during pregnancy (50%) [11]. In general, the risk of thrombosis appears to be higher for antithrombin deficiency than for protein C or protein S deficiency, activated protein C resistance, or prothrombin G20210A. Homozygous type I antithrombin deficiency has not been described in patients and is thought to be incompatible with life. Consistent with this notion, homozygous knockout of the antithrombin gene is lethal in mice [12]. However, patients homozygous for type II heparin binding site (HBS) mutations have been described, characterized by severe venous thrombosis as well as an increased incidence of arterial thrombosis [7, 12]. As with other hereditary hypercoagulable conditions, the presence of a second risk factor further increases the risk for thrombosis.

Antithrombin is a natural anticoagulant that circulates in the plasma at a concentration of 112–140 mg/L with a half life of two to three days [7]. It is a serine protease inhibitor (serpin) which inhibits not only thrombin and factor Xa, but also factors IXa, XIa, XIIa, kallikrein, and plasmin [7]. Like other serpins, antithrombin acts as a suicide substrate inhibitor, covalently binding to and inactivating thrombin [7]. Antithrombin's activity is greatly accelerated by interaction with the heparan sulfate family of glycosaminoglycans, including heparin [12]. In vivo, heparan sulfate is found on the endothelial cell surface, thus localizing antithrombin activity [12]. The interaction of antithrombin with heparan sulfate on the endothelial cell surface also appears to result in release of prostacyclin, a platelet inhibitor [13].

Significant advances have been made in understanding the molecular mechanism of antithrombin activity. The gene that encodes antithrombin, SERPINC1, comprises 7 exons spanning 13.5 kb on chromosome 1. The 1392 bp mRNA encodes a 432 amino acid, 58 kDa glycoprotein that contains 3 β-sheets and 9 α-helices with an active site region and a heparin binding site (HBS) [3]. Heparin binds to the D-helix of antithrombin, exposing antithrombin's reactive center and accelerating its inhibitory activity ∼1,000-fold. While inhibition of thrombin requires the formation of a trimolecular complex between antithrombin, thrombin, and a heparin longer than 18 saccharides (including a specific pentasaccharide sequence), inhibition of factor Xa by antithrombin can be accelerated by just the pentasaccharide of heparin [7, 12].

There are two main types of antithrombin assays, activity (functional) and antigen (immunoassays). Antigen assays are immunoassays designed to measure the quantity of protein regardless of the protein's ability to function. Because antigen levels are often normal in type II deficiencies, activity assays should be used instead for initial testing. If the result is decreased, an antigen assay can be considered to determine the deficiency subtype. Antithrombin deficiencies can be divided into two types, quantitative (type I) or qualitative (type II). Type I deficiencies are characterized by decreased antithrombin activity and antigen levels; typically both are below 70% [3], although values up to 78–80% have also been observed (unpublished observations). Type II deficiencies are qualitative defects resulting in the production of a variant protein with decreased function.

Type II deficiencies can be further subdivided into three subtypes: reactive site (RS), heparin binding site (HBS), and pleiotropic defects (mutations clustered in a region called s1C-s4B) [3, 14]. Both type II HBS and RS mutations are associated with decreased activity and normal antigen levels; however, only type II HBS mutations are associated with progressive activity (see below) [3, 15]. Type II pleiotropic defects are associated with a moderate decrease in both antithrombin activity and antigen levels (typically antithrombin activity is lower than antigen levels) with a genetic mutation in the s1C-s4B region [3, 16]. The decreased antigen level may be due to a combination of factors, including reduced synthesis and secretion as well as increased catabolism [7]. Clinically, type II HBS mutations occur in the general population at about 0.03–0.04% and are associated with a low risk of thrombosis in heterozygous carriers [15, 17, 18]. This raises the possibility that it may be useful to distinguish HBS defects from other type II defects.

Commercially available antithrombin activity assays predominantly use chromogenic amidolytic methods. These assays can be factor IIa (thrombin)- or factor Xa-based. With thrombin-based assays, heparin and excess thrombin are added to the patient's plasma and the patient's endogenous antithrombin inactivates the thrombin. The amount of thrombin remaining is spectrophotometrically measured by its cleavage of a chromogenic peptide substrate, and is inversely proportional to the patient's antithrombin level. The factor Xa-based assay is similar except factor Xa is used in place of thrombin, since antithrombin also inhibits factor Xa. Heparin cofactor II, a natural inhibitor of thrombin, in theory can cause overestimation of antithrombin levels by thrombin-based assays but not by factor Xa-based assays [19]. However, one study suggested that the factor Xa-based assay may be less sensitive to type II deficiencies than the thrombin-based assay [20]. Some assays use bovine thrombin, which is resistant to heparin cofactor II, or protease inhibitors such as aprotinin, to reduce nonspecific cleavage of the substrate by other natural proteases [7].

Because antithrombin activity assays involve heparin, the result depends on both the HBS and the RS of antithrombin, and thus identifies all types of antithrombin deficiency and is not able to distinguish type II HBS from other type II defects [15]. A variant of this assay, performed in the absence of heparin with a prolonged incubation time (300 sec), is much slower (progressive activity) and measures activity independent of the HBS. Thus, a type II HBS deficiency would exhibit progressive activity (i.e. increased activity with prolonged incubation time), unlike a type II RS defect [15, 21, 22]. Because the progressive activity assay can be affected by other inhibitors such as trypsin inhibitor and α2-macroglobulin, it is not commonly used as a screening assay and is not routinely available at present [23].

Overall, with antithrombin III activity assays the ECAT study shows an interlaboratory and intralaboratory variability of 7.4% and 5.8–10.3%, respectively, lower than of assays for protein C and protein S activity [24]. This could reflect the higher complexity in the performance of clotting-based assays compared to chromogenic assays.

Antigen levels were first tested by radial immunodiffusion and Laurell rocket electrophoresis. Newer methods include ELISAs and automated immuno-turbidimetric methods. The 2008 ECAT proficiency test data shows a similar CV of 6.0% and 9.1% for measurements of antithrombin activity and antigen respectively (ECAT 2008 data) [7].

Over 127 distinct mutations are known to confer antithrombin deficiency [25–27]. Therefore, DNA testing is generally not available outside of specialized research laboratories. Although most of these mutations are private mutations dispersed throughout the gene, a recent study identified one mutation (antithrombin Cambridge II, A384S) that is relatively common in British and Spanish populations and seems to confer a 9-fold increased risk of venous thrombosis [26]. Notably, this mutation is not associated with decreased activity or antigen levels and could be under-diagnosed. However, a review of the Paris PATHROS cohort showed a much lower prevalence of this mutation, and its significance remains unclear [3].

Acquired causes of low antithrombin are much more common than hereditary antithrombin deficiency. Therefore, it is essential to exclude potential acquired etiologies of low antithrombin before making a diagnosis of hereditary antithrombin deficiency. Several conditions can lead to an acquired antithrombin deficiency. Liver disease can decrease antithrombin levels because of decreased hepatic synthesis. As protein C has a shorter half-life than antithrombin and is also synthesized in the liver, protein C is usually also decreased if the cause of low antithrombin is liver dysfunction. Protein S can be low or normal in liver disease, depending on the severity of the liver dysfunction, and this is speculated to be attributable to synthesis of protein S in endothelial cells as well as in the liver. Recent or active thrombosis, surgical procedures, or disseminated intravascular coagulation (DIC) consume antithrombin and thus lower antithrombin levels (and usually also decrease protein C and protein S). L-asparaginase therapy decreases antithrombin (and protein C and protein S) probably by decreasing hepatic synthesis [28]. In addition, low antithrombin levels can be seen with active Crohn's disease or ulcerative colitis, [29] poor nutrition, and significant proteinuria such as in nephrotic syndrome. Full-dose heparin administration can cause up to a 30% reduction in antithrombin levels within several days; antithrombin levels return to normal when heparin is discontinued [30]. Antithrombin levels may increase during oral anticoagulation, in contrast with protein C and protein S. Argatroban is a direct thrombin inhibitor, and therefore argatroban can falsely elevate the apparent antithrombin level in factor IIa (thrombin)-based activity assays (but not factor Xa activity or antigen assays). Antithrombin levels can be decreased by oral contraceptives and pregnancy, and are lower in premenopausal women but higher in postmenopausal women, compared with men [31]. At birth, antithrombin levels are on average decreased to 63% (range 39–87%) of adult normal values. Antithrombin rises to adult normal values within six months [32]. Alpha-2-macroglobulin, a natural thrombin inhibitor, is elevated in newborns and children relative to adult values. It has been proposed that the elevated α-2-macroglobulin helps compensate for the low antithrombin observed at birth [33]. Between the ages of 1–16 years, antithrombin levels tend to be higher than adult levels [34, 35].

Interestingly, a growing body of work describes other possible roles for antithrombin testing. Antithrombin levels appear to decrease significantly in septic patients (as expected if DIC is present), and a study of patients with systemic inflammatory response syndrome (SIRS) found antithrombin activity to be the most useful predictor of organ dysfunction [36, 37]. Moreover, antithrombin appears to have thrombin-independent effects on the function of endothelial cells and leukocytes [38, 39]. Recent work suggests this may be in part due to antithrombin interacting with cell-surface glycosaminoglycans, leading to blocking NF-κB activation and modulation of gene expression [40]. These studies raise the possibility that testing for distinct functions of antithrombin in various clinical contexts may become important in the future.

Summary

An activity (functional) assay should be used for screening. If the result is decreased, an antigenic assay may be considered to distinguish type I and type II deficiencies, and repeat testing at a later date is recommended to determine if the result is reproducible. Concomitant testing of protein S and protein C activity can help with the interpretation of the results, as shown in Figure 1. Concomitant testing of prothrombin time (PT) and activated partial thromboplastin time (PTT) can also help with the interpretation by (1) verifying the integrity of the specimen if the PT and PTT are normal (or appropriate for the patient's known medical condition), and (2) revealing possible alternative explanations for abnormal antithrombin results, if the PT and/or PTT are prolonged. Acquired causes of antithrombin deficiency must be excluded prior to making a diagnosis of hereditary antithrombin deficiency, and testing should be repeated after any such conditions have resolved. Laboratory tests that can identify some acquired causes of decreased antithrombin include liver function tests, urinalysis to detect proteinuria, and a DIC screen (D-dimer, PT, PTT, fibrinogen, platelet count, and if available, PTT waveform) [41]. A biphasic transmittance waveform in the PTT is due to the formation of a Ca(++)-dependent complex of C-reactive protein with very-low-density lipoprotein and is a novel marker of impending DIC [41]. Since no confirmatory DNA test is readily available, definitive confirmation of a hereditary deficiency often requires additional approaches such as demonstrating antithrombin deficiency in a relative.

Figure 1.

A diagnostic algorithm for hereditary antithrombin deficiency, using functional assays and, if needed, antigen assays. *If protein C and/or protein S are decreased to a similar extent, then an acquired etiology is likely to account at least partially for the decreases, such as liver dysfunction, thrombosis, disseminated intravascular coagulation (DIC), or surgery. Repeat testing at a later date (after resolution of potential acquired etiologies) would be recommended [42]. **An antithrombin antigen measurement may also be considered, particularly if protein C and protein S are normal. DTI, direct thrombin inhibitor, such as argatroban or hirudin.

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