Summary
Three fractions of the low molecular weight heparin CY216 (fraxiparin, mean molecular
weight [MMW] 5,090), with MMWs of respectively, 3,090, 4,400 and 7,910 were prepared
by gel permeation chromatography. From CY222 (MMW 3,770) as well as from CY216 and
its three fractions the material with high affinity to antithrombin III (AT III) was
obtained by chromatography on immobilised AT III. The molecular weight distribution
of each of the ten preparations thus obtained was determined by high performance liquid
chromatography, while the content of AT III binding material was determined by stoichiometric
titration of AT III, monitored by intrinsic fluorescence enhancement.
We measured the effect of all heparins on the decay of endogenous thrombin in plasma
and on the overall generation of thrombin in plasma, triggered via the extrinsic or
via the intrinsic pathway. From these data we calculated the time course of prothrombin
conversion, i. e. the course of factor Xa activity as expressed by prothrombinase
activity.
It was found that in platelet-poor plasma the anticoagulant properties of the heparins
are largely dependent on their antithrombin action, which is determined by their content
of high affinity material with a MW of 5,400 or higher. The specific antithrombin
activity of all heparins, when expressed in terms of material with high affinity to
antithrombin III (HAM) with a MW >5,400 is 13.0 min-1/(μg/ml) (range 10.5-15.9). The anticoagulant potency is not influenced by the presence
of low-affinity material and hardly by material with MW <5,400.
In platelet-rich plasma, however, the presence of non-AT III binding material enhances
the inhibition, presumably by neutralising heparin binding material originating from
activated platelets. The ultra low MW fractions (<3,400) show a similar activity in
PPP and in PRP.
