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In this work, we aimed to demonstrate that different methods can give different results when the same pair of heparin samples are compared, even when specific antiprotease assays are performed. For this purpose, the effect of heparin on factor Xa (Xa) or thrombin (IIa) inhibition by antithrombin III (AT III) was examined in the presence of varying amounts of AT III, during different incubation times.  The molecular weight of the two heparins were 5,200 (LMW) and 42,000 (HMW). Solutions containing 2 y.g per ml of heparin and a 1.4 to 15 μM freshly purified human AT III were incubated with either bovine Xa or human Ila. After a 20, 30, 60 or 90 secondes incubation at 30° C, the remaining protease activity was measured by the initial velocity of a chromogenic substrate. The method was entirely automated using a reaction rate analyser and an adapted program.  The antiprotease activity of the LMW heparin (related to the HMW heparin activity) varied from 0.23 to 0.89 in the anti Xa system, from 0.30 to 0.77 in the anti Ila system. The ratio of LMW to HMW activity was a parabolic function of either AT III concentration or incubation time. No meaning differences were observed between anti Xa and anti Ila activity when the inhibiting capacity was assayed in the same experimental conditions. These results suggest that the relative activities of HMW and LMW fractions depend upon the assay procedure. AT III concentration as well as incubation time are of particular importance.

thrombosis and haemostasis

Effect Of Some Experimental Conditions On The Activity Of A Low Molecular Weight (LMW) Heparin Fraction Compared To A Hight Molecular Weight (HMW) Fraction