Even though there is documentation that the anti-FXa method is better, how could a researcher determine antithrombin in plasma based on a thrombin method?
A thrombin-based chromogenic heparin cofactor assay for the determination of antithrombin activity can be performed using Chromogenix S-2238™ as described on the Antithrombin Method tab of the Chromogenix S-2238™ product page.
Studies have shown that thrombin-based AT assays, such as Coatest® AT, show an overestimation of AT activity in patients on heparin therapy due to the influence of heparin cofactor II. The FXa-based method provides more valid results in patients on heparin therapy. There is no influence from heparin cofactor II, a2-macroglobulin, or a2-antitrypsin. The AT FXa assay is a better discriminant between AT deficient and non-AT deficient individuals than the thrombin based assay.
In plasma where contact activation has occurred, a contribution to the substrate activity might be produced. An underestimation of AT level may follow. A blank can therefore be performed, and the value obtained in the absence of FXa can be subtracted from the sample value.
How should I make my standard pre-dilutions? It is not very clear from the Chromogenix Coamatic® Antithrombin package insert.
A suggested method for the predilution, although it cab be up to the analyst what volumes to use:
100%: 400 ul normal plasma, 0 ul Saline
75%: 300 ul normal plasma, 100 ul Saline
50%: 200 ul normal plasma, 200 ul Saline
25%: 100 ul normal plasma, 300 ul Saline
From here, follow the package insert.
How does heparin interact with antithrombin? Why is the anti-FXa method a better way to measure heparin activity?
Slow protease-antithrombin interactions are enhanced dramatically in the presence of certain sulfated polysaccharides like heparan sulfate. Heparin is a commercial preparation of heparan sulfate, and binds antithrombin, the major inhibitor of coagulation in plasma and thrombin, thereby catalyzing the thrombin-AT reaction. Binding to antithrombin induces a conformational change in AT that facilitates its reaction with thrombin. Thrombin binds to heparin in a non-specific manner and slides along the chain until it encounters the bound AT. The affinity of heparin to the thrombin-AT (TAT) complex is much lower than to free AT. Heparin will therefore dissociate from the TAT complex, which is rapidly removed from the blood circulation by the liver and the result is a stable protease inhibitor complex, which is rapidly removed and catabolized. The anti-FXa assays are more specific since they measure the ability of heparin-accelerated antithrombin to inhibit a single enzyme. Either plasma or purified AT can be used. More precise determination of unfractionated heparin and low molecular weight heparin are possible.
What are the types of antithrombin deficiency, and the clinical manifestations. What treatment options are available?
A normal AT range is assumed to be 80-120%. Individuals with low antithrombin levels have an increased thrombosis risk. The most common presentation of antithrombin deficiency is venous thrombosis of the lower limbs. A history of recurrent thrombosis occurs in about 60% of patients and is the clinical feature that usually prompts a search for AT deficiency.
AT deficiency is usually transmitted as an autosomal dominant trait in may in some countries affect up to 0.3% of the general population. In patients with a history of venous thrombosis presented before the age of 40-45, the incidence is estimated to be 3-5%. Levels of functionally active protein are usually around 40-70% of normal. There are two types of AT deficiency. Type I is the “classic” form of disorder and is characterized by a 50% reduction in both antigen and functional activity levels. Type II deficiency covers cases in which approximately half the plasma antithrombin is a variant protein with reduced activity. In other words, the antigen level is normal, but there is a mutation of the molecule that leads to decreased activity.
Acquired AT deficiency is also possible, and can be caused by such things as liver disease, DIC, and drugs. In some cases the risk of thrombosis is similar to that in hereditary AT deficiency. Acquired AT deficiency is usually accompanied by a decrease in other coagulation proteins, however, and is therefore difficult to determine an independent risk factor.
Management of AT deficiency includes the administration of heparin, warfarin, or antithrombin concentrates.
Antithrombin is the most important natural inhibitor of the coagulation cascade, accounting for approximately 80% of the thrombin inhibitory activity in plasma. By inhibiting the coagulation proteases, especially thrombin, FXa, and FIXa, AT prevents uncontrolled coagulation and thrombosis. Inhibition of antithrombin involves the formation of a stable 1:1 complex between the active domain of the serine protease such as thrombin, and the reactive site of antithrombin, which proteases initially recognize as a substrate. During the cleavage of the reactive site bond in antithrombin, a conformational change occurs in the inhibitor that traps the protease.
Slow protease-antithrombin interactions are enhanced dramatically in the presence of certain sulfated polysaccharides like heparan sulfate. Heparin is a commercial preparation of heparan sulfate, and binds antithrombin and thrombin, thereby catalyzing the thrombin-AT reaction. Binding to antithrombin induces a conformational change in AT that facilitates its reaction with thrombin. Thrombin binds to heparin in a non-specific manner and slides along the chain until it encounters the bound AT. The affinity of heparin to the thrombin-AT (TAT) complex is much lower than to free AT. Heparin will therefore dissociate from the TAT complex, which is rapidly removed from the blood circulation by the liver.
|Protein S||70||10 (free)||0.14|
|Heparin Cofactor II||66||80||1.2|
|Protein C Inhibitor||57||4||0.07|
5 IU corresponds to 1 mg. Therefore a 25 IU vial corresponds to 5 mg AT. The molecular weight of AT is 58 kDa.