Low Molecular Weight Heparin - Anti-factor Xa Activity

Anti-factor Xa Activity

Coagulation cascade is a normal physiological process which aims at preventing significant blood loss or hemorrhage following vascular injury. Unfortunately, there are times when a blood clot (thrombus) will form when it is not needed. For instance, some high risk conditions such as acute medical illness prolonged immobilization, surgery, or cancer can increase the risk of developing a blood clot which can potentially lead to significant consequences.

The coagulation cascade consists of a series of steps in which a protease cleaves and subsequently activates the next protease in the sequence. Since each protease can activate several molecules of the next protease in the series, this biological cascade is amplified. The final result of these reactions is to convert fibrinogen, a soluble protein, to insoluble threads of fibrin. Together with platelets, the fibrin threads form a stable blood clot.

Antithrombin (AT), a serine protease inhibitor, is the major plasma inhibitor of coagulation proteases. LMWHs inhibit the coagulation process through binding to AT via a pentasaccharide sequence (see also heparin: mechanism of action). This binding leads to a conformational change of AT which accelerates its inhibition of thrombin (factor IIa) and activated factor X (factor Xa). Once dissociated, the LMWH is free to bind to another antithrombin molecule and subsequently inhibit more thrombin.

The effects of LMWHs cannot be acceptably measured using the partial thromboplastin time (PTT) or activated clotting time (ACT) tests. Rather, LMWH therapy is monitored by the anti-factor Xa assay, measuring anti-factor Xa activity. The methodology of an anti-factor Xa assay is that patient plasma is added to a known amount of excess factor Xa and excess antithrombin. If heparin or LMWH is present in the patient plasma, it will bind to antithrombin and form a complex with factor Xa, inhibiting it. The amount of residual factor Xa is inversely proportional to the amount of heparin/LMWH in the plasma. The amount of residual factor Xa is detected by adding a chromogenic substrate that mimics the natural substrate of factor Xa, making residual factor Xa cleave it, releasing a colored compound that can be detected by a spectrophotometer. Antithrombin deficiencies in the patient do not affect the assay, because excess amounts of antithrombin is provided in the reaction. Results are given in anticoagulant concentration in units/mL of antifactor Xa, such that high values indicate high levels of anticoagulation and low values indicate low levels of anticoagulation.

LMWHs have a potency of greater than 70 units/mg of anti-factor Xa activity and a ratio of anti-factor Xa activity to anti-thrombin activity of >1.5. (see table 1)

Table 1 Molecular weight (MW) data and anticoagulant activities of currently available LMWH products. Adapted from Gray E et al. 2008.