title: Mechanism of low-molecular-weight heparin reversal by platelet factor 4.
abstract: 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.
authors.affiliation: Haemostasis Research Unit, Department of Hematology, University College London, United Kingdom. m.fiore@ucl.ac.uk
authors.forename: Martine M
authors.initials: MM
authors.lastname: Fiore
authors.forename: Ian M
authors.initials: IM
authors.lastname: Mackie
mesh_terms: D000925:Anticoagulants / Q000737:chemistry*; D000991:Antithrombins / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003029:Coagulants / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D065427:Factor Xa Inhibitors; D006495:Heparin, Low-Molecular-Weight / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010978:Platelet Factor 4 / Q000378:metabolism / Q000494:pharmacology*; D011479:Protamines / Q000494:pharmacology; D011485:Protein Binding; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D002614:Chelating Agents; D003029:Coagulants; D065427:Factor Xa Inhibitors; D006495:Heparin, Low-Molecular-Weight; D011479:Protamines; D010978:Platelet Factor 4; D004492:Edetic Acid; D013917:Thrombin