Antithrombin

Antithrombin (AT) is produced in the liver and is an important endogenous anticoagulant, inhibiting the activity of most activated coagulation factors, although its greatest inhibitory effect is against thrombin and FXa. Its activity is enhanced by heparin (hence its use as an anticoagulant), which is provided in vivo by heparin-like glycosaminoglycans on the surface of endothelial cells. When thrombin is generated in vivo by an activated hemostatic system, AT binds thrombin to form thrombin-antithrombin complexes (TAT), which are cleared by the liver. High concentrations of TAT indicate excessive generation of thrombin or hypercoagulability, but this assay is generally only performed in research settings. AT is a small protein around the size of albumin and can be lost, concurrently with albumin in protein-losing conditions. Since AT is an important anticoagulant, deficiency of AT (generally activity <50%) is associated with an increased risk of thrombosis in dogs. There is some data that AT also inhibits plasmin, however its effect on the latter is minor (Chang et al 2017). Antithrombin binds to heparin-like glycosaminoglycans on endothelial cells and other receptors on inflammatory cells, such as monocytes, inducing cellular signaling and exhibiting anti-inflammatory and anti-angiogenic properties (Rezaie and Giri 2020).

Methods

Antithrombin is measured indirectly by its ability to inhibit thrombin or FXa in chromogenic substrate-based assays. The latter is used more as an inhibitory target and is the procedure used by the Comparative Coagulation Laboratory at Cornell University (outlined below). In addition to the chromogenic assay, AT concentration can be measured using immunologic assays, however this is not routinely performed. The assays can also be adapted to measurement of pharmacologic inhibitor concentrations in plasma, e.g. unfractionated or low-molecular-weight heparin or the FXa inhibitor, rivaroxaban (see drug monitoring).

Reaction type

Chromogenic substrate cleaved by thrombin or FXa.

Procedure

• Inhibition of thrombin:In this assay, patient plasma is added to a reagent containing heparin, excess thrombin and a chromogen-labeled thrombin-specific substrate. The appearance of the chromogen released by thrombin-mediated cleavage of the substrate is measured. The amount of chromogen is proportional to the residual thrombin activity which is inversely proportional to the amount of antithrombin activity. This assay suffers from interference with heparin cofactor II. This molecule, like AT, is activated by heparin and inhibits thrombin, thus overestimating the AT activity in the sample.
• Inhibition of FXa: This works similarly to the above assay, except excess FXa is added to the system (therefore, the test is not reliant on the activity of FX in patient plasma). In the presence of AT, FXa activity is inhibited and the chromogenic substrate will not be cleaved. With low concentrations of AT, there is less enzyme inhibition and more substrate cleavage, so the AT activity is inversely proportional to the amount of substrate generated. The assay includes heparin to promote full AT activity.

Units of measurement

Results are expressed as a percentage of a species-specific standard pool, designated as 100%.

Sample considerations

Sample type

Plasma

Anticoagulant

Citrate

Test interpretation

Measurement of AT is usually performed as part of DIC panels, because AT activity is usually decreased due to formation of TAT. AT can also be measured in animals with unexplained thrombotic episodes. Reference intervals for AT activity have been generated by the Comparative Coagulation Laboratory at Cornell University (dogs: 65-145%, cats: 75-110%, horses: 85-130%).

Decreased AT activity

• Decreased production:
  • Liver dysfunction or failure: A decrease in synthetic functional mass of the liver (>70%) can result in low AT activity as can hepatic atrophy secondary to abnormal portal blood flow. Low AT activity (<75%) was seen in 84% of 20 dogs with hepatic failure, 43-51% of 95 dogs with congenital or acquired portosystemic shunts, 19% of dogs with microvascular dysplasia, and 21% of 20 dogs with chronic hepatitis (Toulza et al 2006).
  • Inflammation: AT is a negative acute phase protein (maybe not in cats) with production being decreased in the presence of inflammatory cytokines. This was shown in in vitro studies with HepG2 cells treated with interleukin (IL)-6 and IL-1β and in vivo studies of IL-6 infusion in baboons (Niessen et al 1997).
  • Drugs: L-asparaginase has been postulated to decrease AT through decreased production, which is supported by in vitro studies (Bushman et al 2000).
• Increased consumption
  • Disseminated intravascular coagulation: Due to excessive thrombin generation and formation of TAT complexes, AT is frequently low in dogs and horses with DIC and is a sensitive test for this purpose (Stokol et al 2000, Stokol et al 2005). In contrast, AT activity is not usually low in cats with laboratory evidence of DIC (unpublished observations, Connor et al 2015).
• Increased loss
  • Protein-losing conditions: Both protein-losing enteropathy and nephropathy are associated with concomitant losses of albumin and AT. Low AT activity is not always correlated with hypercoagulability (albeit as measured by viscoelastic testing, which is a dubious test for hypercoagulability) or clinical evidence of thrombosis in these dogs (Goodwin et al 2011, White et al 2016).
  • Protein-rich secretions: Potentially, AT can be lost with albumin in other conditions, including protein-rich effusions into body cavities (e.g. exudates) and exudative dermatopathies. Studies in horses have shown that AT activity is increased in the peritoneal fluid of horses with colic due to gastrointestinal disease (e.g. strangulating obstruction). This was attributed to inflammation-induced leakage of serum proteins into the fluid (Collatos et al 1995).

Increased AT activity

High AT activity is of unknown diagnostic relevance, but has been reported in cats with feline infectious peritonitis, cardiac disease and hyperthyroidism (Boudreaux et al 1989, Welles et al 1994, Keebaugh et al 2021). The cats with FIP had laboratory evidence of DIC (Boudreaux et al 1989). The authors of two studies postulated that high AT activity was due to AT acting as a positive and not negative acute phase reactant in this species (Boudreaux et al 1989, Welles et al 1994), but this intriguing hypothesis (considering the absence of low AT activity in cats with DIC) remains untested and unproven. With regards to hyperthyroidism, there is evidence that thyroid hormone can mildly increase AT production in cultured HepG2 cells (a cell line derived from a hepatoma, Niessen et al 1994). In the hyperthyroid cats, AT activity decreased post radioactive iodine therapy (Keebaugh et al 2021).