Renaparin test

RENAPARIN-test (pharmaceutical preparations control)

A set of reagents for determining the activity of low-molecular-weight heparin.

A set of reagents for determining the anti-Xa and anti-IIa activity of low-molecular-weight heparin (Renaparin-test) is designed for the quantitative determination of anti-Xa and anti-IIa activity in pharmaceutical preparations and substances of low-molecular-weight heparin (LMWH) using chromogenic substrates.

Method principle: The method for determining LMWH activity is based on the ability of the antithrombin III-heparin complex (AT III-LMWH) to neutralize activated factors Xa and IIa.

LMWH activity in preparations and substances is determined by adding an excess of antithrombin III and factor Xa or IIa to them. This results in the inhibition of factors Xa or IIa by the AT III-LMWH complex in proportion to the amount of LMWH in the preparation or substance. The remaining amount of factor Xa or IIa catalyzes the cleavage of paranitroaniline (pNA) from synthetic chromogenic substrates. The absorption of free pNA, determined at a wavelength of 405 nm, is inversely proportional to the anti-Xa or anti-IIa activities of LMWH.

The process follows these schemes:

1. When determining anti-Xa activity of LMWH: AT III (excess) + LMWH ⇒ AT III-LMWH. AT III-LMWH + factor Xa (excess) ⇒ AT III-LMWH-Xa + factor Xa (remainder). Factor Xa (remainder) + factor Xa-Substrate-pNA ⇒ Peptide + pNA.

2. When determining anti-IIa activity of LMWH: AT III (excess) + LMWH ⇒ AT III-LMWH. AT III-LMWH + factor IIa (excess) ⇒ AT III-LMWH-IIa + factor IIa (remainder). Factor IIa (remainder) + factor IIa-Substrate-pNA ⇒ Peptide + pNA.