Antithrombin Testing

Physiologic Role and Mechanism of Action

AT is the most important and potent physiologic inhibitor of thrombin. AT is essential for postembryonic life, with complete knockout in mice resulting in embryonic lethality due to thrombosis. The protein belongs to the class of serine protease inhibitors (serpins). Serpins are suicide substrates that mimic the natural substrates of the target protease. On partial digestion, these inhibitors form covalent bonds with the target, irreversibly blocking further protease activity by steric hindrance. In addition to thrombin, AT inhibits numerous other proteases involved in the clotting cascade, including factors IXa, Xa, XIa, and tissue factor bound factor VIIa. AT activity is enhanced approximately 1000-fold by heparin-like glucosaminoglycans (GAG). This potentiation requirement provides a mechanism for localizing AT activity to the endothelial surface where heparan sulfate is expressed. A single pentasaccharide moiety of heparin-like GAG is sufficient for potentiation of AT activity toward most proteases other than thrombin. Inactivation of thrombin requires additional interaction of the GAG with thrombin. For this to occur, at least 18 pentasaccharide moieties are necessary.

Congenital Antithrombin Deficiency

Congenital AT deficiency occurs in 0.2%–0.02% of the general population. The deficiency is overrepresented in subjects who have suffered a VTE. The prevalence of AT deficiency in younger patients with unprovoked first VTE is approximately 1%–3%. Heterozygous AT deficiency is a moderately strong risk factor for venous thrombosis and pregnancy complications, with deficient subjects experiencing a 5- to 50-fold increased risk over their relatives without AT deficiency. The peak incidence of thrombotic events in AT deficiency may be earlier than for other congenital risk factors. VTE incidence peaks between ages of 15 and 35 years. Role of AT deficiency in arterial thrombosis is controversial and may be specific mutation-dependent.

Most AT deficiencies are a result of an inheritance of a single defective allele of the SERPIN1 gene located on chromosome 1. Thus, an autosomal dominant inheritance pattern is the norm. Complete AT deficiency, resulting from inheritance of two defective AT alleles with no protein activity, appears to be incompatible with postembryonic life in humans. Cases of double heterozygosity for defective alleles with a milder phenotype for each have been reported. Deficiencies resulting from such mutations have variable presentation, with a subset of patients having severe thrombotic complications in the neonatal period. Most AT deficiencies (about 80%) are classified as type I (quantitative) defects with declines in AT protein levels and corresponding declines in AT inhibitory activity. The remaining deficiencies are classified as type II (qualitative) defects where critical aspects of the protein function are compromised. In type II deficiencies, a decline of protein activity is out of proportion to the protein levels.

Among type II deficiencies there are three recognized subtypes. Type II RS occurs due to mutations that directly affect AT interaction with the target proteases. Mutations of this type are expected to affect AT inhibitory activity toward its targets regardless of the presence of heparin. Type II heparin-binding site (HBS) mutations affect potentiation of the thrombin activity by heparin. These types of defective AT show normal inhibitory activity when heparin is not present but will show substantial defect when heparin is added to potentiate the reaction. Pleiotropic (PE) subtypes are due to mutations that affect the structure of the protein in the way that compromises both functions. Overall, some HBS mutations show milder clinical phenotype than the RS mutations.

Testing Indications

AT deficiency is quite rare in the general population. Assuming that low end of the reference range excludes ∼2.5% of the population and the incidence of AT deficiency is at most 0.2%, false-positive results would far exceed true positives in a general population screening. AT deficiency testing should be reserved for patients with a high pretest probability of a true-positive result. Confirmation of AT deficiency in a first-degree relative of a suspected case greatly improves the confidence in diagnosis. Outside of a diagnosis of congenital AT deficiency, AT measurements may be warranted in patients undergoing very high-dose heparin therapy for extended periods, such as neonates receiving extracorporeal membrane oxygenation. Under those circumstances, the testing is performed to assess the need for AT replacement using AT concentrates.

Preanalytical Considerations

Preanalytical variables must be taken into consideration when deciding on the timing of AT testing, as both false-positive and false-negative results may occur if testing is mistimed. Like other factors involved in coagulation, AT is consumed in thrombosis, burns, trauma, disseminated intravascular coagulation, and postoperatively. AT is reduced by long-term full-dose unfractionated heparin therapy. It is advisable to delay testing after a recent thrombotic event or long-term heparin administration. Liver insufficiency and treatment with l -asparaginase can lead to substantial decreases in AT levels due to decreased liver synthetic function. In addition, renal loss in nephrotic syndrome can lower AT levels. Mild declines have been reported in diabetics and in women taking oral contraceptives or hormone replacement therapy. AT levels may be mildly increased by oral anticoagulant therapy with warfarin.