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Chromogenix Coamatic® LR Antithrombin is an assay kit for the quantitative determination of the heparin cofactor activity of antithrombin (AT) in human citrated plasma. All components of the antithrombin test kit are in liquid formulation (LR=Liquid Reagents).

Reagents, Packaging, Storage and Stability

  1. Substrate solution S-2772™, 26 mg, 2 vials Contains chromogenic substrate S-2772™ (Ac-D-Arg-Gly-Arg-pNA·2HCl) in tartaric acid buffer pH 4.2. Stability once opened: 6 months at 2-8°C in the original vial.
  2. Factor Xa solution, 90 nkat, 6 vials Contains purified bovine Factor Xa, bovine albumin and heparin, in Tris buffer, pH 8.2. Stability once opened: 1 month at 2-8°C in the original vial.

Unopened reagents and calibrator are stable until the expiration date shown on the vial when stored at 2-8°C. WARNING: Do not use reagents beyond the expiry date printed on the package label. Discard if the substrate solution appears yellow. Avoid contamination by microorganisms.

AT + Heparin————–>[AT · Heparin]
[AT · Heparin] + FXa (excess)————–>[AT · Heparin · FXa] + FXa (residual)
FXa (residual)
S-2772™————–>Peptide + pNA

AT = Antithrombin

Antithrombin is the most important natural inhibitor of the coagulation cascade. By inhibiting the coagulation proteases, especially thrombin, factor Xa and factor IXa, antithrombin prevents uncontrolled coagulation and thrombosis. Plasma is incubated with an excess of Factor Xa (FXa) in the presence of heparin. The residual activity of FXa is determined by the rate of hydrolysis of the chromogenic substrate S-2772™. The pNA release measured at 405 nm is inversely proportional to the AT level in the range 15 -125% of normal plasma. Read more…

What is the Chromogenix Coamatic® LR Antithrombin measurement principle?
chromogenic antithrombin measurement assay test kit

Go to Chromogenix Coamatic® LR Antithrombin

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.
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.

What is the function of antithrombin and what is its interaction with thrombin and heparin?
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 concentrations in plasma
ComponentMolecular
Weight kDa
Plasma
Concentration
mg/l
Plasma
Concentration
μmol/l
Fibrinogen33030009
Prothrombin721502
Factor V330200.05
Factor VII500.50.01
Factor VIII3300.10.0003
Factor IX5650.09
Factor X5980.13
Factor XI16050.03
Factor XII80300.4
Factor XIII320100.03
Protein C6240.06
Protein S7010 (free)0.14
Protein Z6220.03
Prekallikrein86500.6
HMW kininogen120700.6
Fibronectin4503000.7
Plasminogen922002
t-PA600.0050.0001
Urokinase530.0040.0001
Antithrombin581452.5
Heparin Cofactor II66801.2
Plasmin Inhibitor63601
Protein C Inhibitor5740.07
α2-Macroglobulin72520003