Mechanism of low-molecular-weight heparin reversal by platelet factor 4

Low-molecular-weight heparins (LMWH) exert their anticoagulant effect by accelerating anti-thrombin (AT) inactivation of factor Xa (fXa) and thrombin. To address the hypothesis of a calcium-dependent template mechanism in LMWH activity, we compared the ability of the heparin neutralising agent Platelet Factor 4 (PF4) to inhibit various therapeutic LMWH in a kinetic assay. Neutralization coefficients by PF4 and apparent affinities of PF4 for various LMWH increased in a molecular weight-dependent manner. Protamine sulphate neutralized heparin via a non-specific mechanism. EDTA abolished the calcium-dependent acceleration of the fXa-AT reaction, indicating that the bridging mechanism contributed significantly to LMWH activity. 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 heparin were independent of each other. Instead, increasing enzyme concentration reversed the negative effect of heparin-bound AT on PF4-dependent neutralization. Inhibition of thrombin by heparin was also neutralized by PF4, albeit to a higher extent than the fXa-AT reaction. Altogether, these results suggested that an interaction of PF4 with protease mediated the association of PF4 to the heparin chain. We propose that PF4 participates in the anti-fXa dependence of LMWH due to its major effect on the anti-thrombin activity of LMWH and that inhibition of fXa via the template mechanism plays an essential role in LMWH activity and pharmacokinetics, because PF4 specifically targets this mechanism.