DIFFERENTIAL NEUTRALIZATION OF UNFRACTIONATED HEPARIN (UH) AND LOW MOLECULAR WEIGHT HEPARINS (LMWHS) BY HUMAN PLATELET FACTOR FOUR (PF-4)
Platelet factor 4 neutralization profiles of four newly developed LMWHs, PK 10169 (Pharmuka, Gennevilliers, France) CY 216 (Choay, Paris, France), KABI 2165 (KabiVitrum, Stockholm, Sweden), OP 2123 (Opocrin, Corlo Italy) and an unfractionated porcine mucosal heparin were studied utilizing amidolytic (anti-factor Xa, anti-factor Ila) and clot based (APTT, Heptest ) assays. The neutralization studies were carried out in normal human pooled plasma in two experimental protocolsIn one set of
... one set of experiments, PF-4 (10μg/ml) was added to varying amounts of UH or LMWH (10-2.5 μg/ml). In the second set of experiments, the concentration of heparins was fixed at 5 μg/ml and varying amounts of PF-4(10-2.5 μg/ml) were added. The relative neutralization profiles of these heparins were assay dependant. The anticoagulant effects of UH and LMWHs as measured by APTT were equally neutralizable. Similarly the anti-factor Ila actions of both UH and LMWH were effectively neutralized, whereas the anti-factor Xa activity of the LMWHs was less neutralizable than the UH. Different levels of residual anti-Xa activity of the LMWHs were seen whereas the UH was effectively neutralized. Interestingly, although the anti Xa activity of the UH and Kabi 2165 as measured by amidolytic and clot based methods were similiar and more potent than the other LMWHs, however there were differences between their PF-4 neutralization profile. At a gravimetric ratio of 2:1 , the anti Xa activity of the UH was completely neutralized whereas the Kabi 2165 was least neutralized of all of the LMWHs. The slope of the PF-4 titration curves in the anti-factor Xa assays for all of the LMWHs were similar and varied significantly from the UH. PF-4 neutralization of UH and LMWHs was not dependant on the molecular weight or pharmacopoeial potency (USP or antifactor Xa). These results show that UH and LMWHs exhibit a dissociation between the anti-factor Xa and anti-factor Ila neutralization which may contribute to their sustained antithrombotic effects.