Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin-independent mechanism.
BACKGROUND
Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined.


OBJECTIVE
To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin-independent inhibition of intrinsic tenase (FIXa-FVIIIa) activity.


METHODS
Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC(50) s) of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), fondaparinux and unfractionated heparin (UFH) were determined by plotting concentration vs. relative velocity index (ratio ± heparin). Inhibition was compared under FIX-dependent and FIX-independent conditions (0.2 or 4 pm tissue factor [TF], respectively) in normal plasma, and in mock-depleted or antithrombin/FIX-depleted plasma supplemented with recombinant FIX.


RESULTS
UFH and fondaparinux demonstrated similar potency under FIX-dependent and FIX-independent conditions, whereas LMWH (2.9-fold) and ssLMWH (5.1-fold) demonstrated increased potency with limiting TF. UFH (62-fold) and fondaparinux (42-fold) demonstrated markedly increased EC(50) values in antithrombin-depleted plasma, whereas LMWH (9.4-fold) and ssLMWH (two-fold) were less affected, with an EC(50) within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing FIX/antithrombin-depleted plasma with 90 nm recombinant FIX possessing mutations in the heparin-binding exosite. Mutated FIX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition.


CONCLUSIONS
Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin-independent interaction with the FIXa heparin-binding exosite.
Department of Pathology, University of Wisconsin-Madison, Madison, WI, USA.
Y
Y
Buyue
T M
TM
Misenheimer
J P
JP
Sheehan
D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology*; D000990:Antithrombin III / Q000378:metabolism; D058833:Antithrombin Proteins / Q000378:metabolism*; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects*; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D015949:Factor IXa / Q000009:adverse effects* / Q000235:genetics / Q000378:metabolism; D000077425:Fondaparinux; D006495:Heparin, Low-Molecular-Weight / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism
D000925:Anticoagulants; D058833:Antithrombin Proteins; D006495:Heparin, Low-Molecular-Weight; D011134:Polysaccharides; D011994:Recombinant Proteins; C046193:antithrombin III-protease complex; D000990:Antithrombin III; D013925:Thromboplastin; D010447:Peptide Hydrolases; D015949:Factor IXa; D013917:Thrombin; D000077425:Fondaparinux