Abstract

Heparins which contain both a specific pentasaccharide sequence and a protease binding site accelerate antithrombin inactivation of both thrombin and factor Xa (fXa). The bridging effect due to binding of AT and protease to heparin-which is dominating for thrombin- also participates in fXa inhibition. To address the hypothesis that the calcium-dependent bridging effect by heparin is an essential contributor of protease inhibition, we compared the ability of the heparin-neutralizing agent Platelet Factor 4 (PF4) to inhibit various therapeutic low-molecular-weight heparins (LMWH) in a kinetic assay. LMWH were in order of increasing chain-length; Bemiparin, Enoxaparin, Dalteparin and Tinzaparin. As a reference, the activity of the pentasaccharide Fondaparinux was included. Upon neutralization by PF4, the second-order rate constant describing AT inactivation of fXa by LMWH was 4-fold higher for the pentasaccharide (k2 = 3.8 x 105 M−1 s−1) vs the longer chain LMWH Tinzaparin (k2 = 1.0 x 105 M−1 s−1) and about 2-fold higher for the short-chain Bemiparin ( k2 = 2.4 x 105 M−1 s−1) vs Tinzaparin, in agreement with reports showing higher anti-fXa peak for administered short-chain LMWH. These results could be explained by neutralization coefficients by PF4 and apparent affinities of PF4 for various LMWH which increased with molecular weight, indicating a relationship between the affinity of PF4 for heparin and its neutralizing capacity. The molecular weight-enhanced neutralization was also reminiscent of earlier observations that PF4 binding to heparin followed the same chain length requirement as binding of protease. In contrast to PF4, protamine sulphate fully neutralized LMWH in a non-specific manner and EDTA, abolished the calcium-dependent acceleration of the fXa-AT reaction, indicating that the bridging mechanism contributed to LMWH activity. In fact, as LMWH (Dalteparin) concentration was increased (0.05-0.8 U/ml), neutralization by PF4 decreased from 100 % to 35 %, a consequence of the template cofactor mechanism by heparin. Within a low range of LMWH concentration (<0.2 U/ml), excess AT over PF4 (4:1) had no effect on PF4 activity, indicating that PF4 and AT binding to LMWH were independent of each other. Instead, increasing enzyme concentration reversed the negative effect of heparin-bound AT on neutralization. Indeed, neutralization by PF4 of the fXa-AT reaction increased from 5 % to 55 % using fXa concentrations varying from 1 to 10 nM while neutralization of the thrombin-AT reaction required thrombin concentrations varying from 0.2 nM to 1.5 nM to obtain a similar level of neutralization than in the fXa-AT reaction. This was corroborated by saturation plots by PF4 which showed an increased apparent affinity of PF4 for LMWH as the enzyme concentration was increased in both fXa-AT and thrombin- AT reactions, although lower concentrations of thrombin were required to enhance PF4 affinity. Altogether the results suggested that heparin mediated an interaction of PF4 with protease which was of higher affinity in the presence of thrombin. Therefore, PF4 contributes to LMWH pharmacokinetics by specifically targeting the bridging function of heparin (via enzyme-PF4 interference of enzyme-AT interaction) and participates in the antifXa dependence of LMWH that remains after a major effect on the antithrombin activity.

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