Antithrombin deficiency: no sugar, no diagnosis!

In this issue of Blood, de la Morena-Barrio et al¹ describe 2 novel variants of antithrombin (AT) with altered glycosylation profiles. This study found that although these mutations are undoubtedly associated with thrombophilia, carriers are not identified using routine testing for AT deficiency.

Several studies have reported that, among genetic risk factors for thrombophilia, inherited AT deficiency is underdiagnosed, emphasizing the need for the development of new genetic and functional assays.² In their study, de la Morena-Barrio et al add further evidence of this need by identifying 2 novel mutations in the SERPINC1 gene (encoding for AT) in 4 unrelated thrombophilic families whose proband had normal (or near-normal) AT activity in functional assays routinely used to diagnose AT deficiency. By a combination of molecular and biochemical analyses, they established that both were missense mutations close to or in an N-glycosylation sequon, resulting in an impaired or null N-glycosylation of Asn224.

AT is a major endogenous anticoagulant, whose deficiency causes the most severe form of thrombophilia. It is a serine protease inhibitor (serpin) that primarily targets thrombin (factor IIa [FIIa]) and activated factor X (FXa), but also targets other procoagulant enzymes such as FVIIa, FIXa, and FXIa. AT is a glycoprotein with 4 sites of N-glycosylation (Asn128, Asn167, Asn187, and Asn224). Most of the AT in the plasma is fully glycosylated (α-AT), although a minor form, lacking a glycosyl sidechain on Asn167 (β-AT), is also present.³ 

This study presents the case of a patient who had an unprovoked portal vein thrombosis at the age of 44 years. His mother and his 2 daughters also had venous thrombosis at the ages of 70, 19, and 18 years, respectively. The screening for inherited risk factors for thrombophilia, including for AT deficiency, led to negative or inconclusive results. Although the patient had slightly decreased AT activity during the acute episode, a second assay a few months later was normal. Based on the severe clinical manifestations in this family, genetic analysis of the proband was undertaken by whole-genome sequencing. It revealed a heterozygous mutation in the SERPINC1 gene resulting in the Glu227Lys substitution, near the N-glycosylation site 224. This mutation, never described before, has also been found in another patient with recurrent thrombotic events starting at an early age.

The biochemical characterization of the variant AT-Glu227Lys, in a recombinant system, showed both reduced secretion and heterogeneous glycosylation, presumably from partial glycosylation in position 224. In addition, the β glycoform of AT-Glu227Lys exhibited impaired anti-FXa and anti-FIIa activities compared with β-AT.

Two other unrelated patients are also described in this study. Both were carriers of another mutation in the same glycosylation sequon, resulting in the Asn224His substitution. These patients, and some of their relatives, also presented with recurrent thrombosis at a young age, with normal or near-normal AT activity. As expected, biochemical analysis of the variant AT-Asn224His, in a recombinant system, confirmed the loss of a glycan chain. Interestingly, although this mutation had no effect on the AT anticoagulant activity in its α-glycoform, it abolished both anti-FXa and anti-FIIa activities of the β glycoform.

This study highlights the difficulty of diagnosing congenital AT deficiency. Routine laboratory tests for AT are primarily functional assays based on the ability of plasma AT to inhibit exogenous FIIa- or FXa-chromogenic activity. Even if these tests are able to detect functional defects, they sometimes lack the sensitivity to detect milder forms. The biochemical analysis of AT variants revealed that mutations had no effect on its anticoagulant activity, at least of its α glycoform, which is predominant in plasma. However, hypoglycosylation of Asn224 affected the anticoagulant activity of the β-AT, which accounts for ∼10% of plasma AT. This may explain why the carriers of this mutation were not diagnosed as having a congenital AT deficiency. The discrepancy between the results of functional assays and the severity of prothrombotic phenotype suggests that β-AT could play a preponderant role in the anticoagulant activity of AT, as previously reported in animal models.⁴ 

AT anticoagulant activity is closely related to its metastable structure that undergoes a profound conformational change on interaction with its target proteases to form an irreversible covalent complex through a mousetrap mechanism (common to serpins). Thus, a glycosylation defect could alter AT folding and stability, making it more sensitive to external conditions that alter its conformation and thereby its anticoagulant activity.⁵ This hypothesis is supported by the higher sensitivity of plasma from patients with Glu227Lys or Asn224His mutation to heat stress. Patient plasma had lower AT activity when heated to 41°C than normal control plasma. It is conceivable that mutations impairing glycosylation could be associated with transient AT deficiency with thrombotic consequences brought on by external factors. This transient AT deficiency state would also make it difficult to diagnose.⁶ 

The development of more sensitive functional assays could help in the diagnosis of AT deficiency. Indeed, plasma from carriers of the Glu227Lys mutation exhibited a hypercoagulable state in a thrombin generation assay, evidenced by a faster and greater thrombin generation compared with control plasma. In addition, when assessed for anti-FVIIa activity, carriers of the Glu227Lys or Asn224His mutation appeared to have reduced levels of FVIIa-AT complexes compared with healthy controls. Because anti-FXa and anti-FIIa activity of carriers were nearly normal, this result implies that hypoglycosylation of Asn224 could have distinct effects depending on the AT target, and that, in this case, an anti-FVIIa assay could be more sensitive/specific than anti-FIIa or anti-FXa assays. This result is very interesting and requires further investigations to determine the molecular mechanism responsible for this observation.

Finally, even if these results encourage further development of screening methods for inherited AT deficiency; overall, the usefulness of testing for hereditary thrombophilia remains debated.⁷ In this context, the development of too sensitive or overly broad-spectrum assays has to be considered carefully as, although it may be useful for the management of thrombophilia, it may also generate many off-target results that could be difficult to interpret.