Summary
Low molecular weight (LMW) heparin preparations have unknown distributions of ATIII-binding
material, so mean molecular weights as such might bear little information on their
anti-factor Xa and anti-thrombin activities, and on the neutralization of these activities
by platelet factor 4 (PF4). These properties were investigated in pure systems with
proteins of human origin. Pseudo-first order rate constants of inactivation of factor
Xa and thrombin by antithrombin III were determined as function of heparin concentration,
in the presence of 4.0 mM CaCl2. Despite a large variation in the mean molecular weights, the ratios of the anti-factor
Xa over the anti-thrombin activities were essentially the same for the 4th International
Standard for heparin (0.46), the 1st International Standard for LMW heparin (0.32),
CY216 (0.42) and enoxaparin (0.50). The ultra LMW heparin CY222 had only a 2-times
higher ratio (0.98). Analysis of CY216 subfractions, obtained by gel filtration, showed
that the heparin molecules of the upper region of the molecular weight distribution
are responsible for the anti-thrombin, but also to a large extent for the anti-factor
Xa activities. The results indicate that depolymerization of unfractionated heparin
does not result in an increased anti-factor Xa/anti-thrombin ratio, because in the
presence of Ca2+-ions the rate constants of inactivation of factor Xa are lowered as compared to those
of native heparin. PF4-dependent neutralization of anti-factor Xa and anti-thrombin
activities of fixed concentrations of the LMW heparins was studied by measuring rate
constants as function of PF4 concentration. All anti-thrombin and 50% of the anti-factor
Xa activities were readily neutralized. Excess PF4 was required to neutralize another
35-50% of the anti-factor Xa activities. At PF4 levels obtained at maximal release
of the content of platelet α-granules, all anti-thrombin and most (≥85%) of the anti-factor
Xa activities can be neutralized.
