Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin-independent mechanism
Version of Record online: 1 OCT 2012
© 2012 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 10, Issue 10, pages 2086–2098, October 2012
How to Cite
BUYUE, Y., MISENHEIMER, T. M. and SHEEHAN, J. P. (2012), Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin-independent mechanism. Journal of Thrombosis and Haemostasis, 10: 2086–2098. doi: 10.1111/j.1538-7836.2012.04892.x
- Issue online: 1 OCT 2012
- Version of Record online: 1 OCT 2012
- Accepted manuscript online: 20 AUG 2012 10:25AM EST
- Received 20 December 2011, accepted 10 August 2012
- factor IXa;
- low molecular weight heparin;
- thrombin generation
Summary. 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.
Objectives: To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin-independent inhibition of intrinsic tenase (FIXa–FVIIIa) activity.
Patients/Methods: Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC50s) 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 EC50 values in antithrombin-depleted plasma, whereas LMWH (9.4-fold) and ssLMWH (two-fold) were less affected, with an EC50 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.