Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.

Discrepancies exist for some of the modified coagulation factors when assayed with different one-stage clotting and chromogenic substrate assay reagents. The aim of this study was to evaluate the performance of a recombinant factor VIII Fc fusion protein (rFVIIIFc), currently in clinical development for the treatment of severe haemophilia A, in a variety of one-stage clotting and chromogenic substrate assays in clinical haemostasis laboratories. Haemophilic plasma samples spiked with rFVIIIFc or Advate(®) at 0.05, 0.20 or 0.80 IU mL(-1) were tested by 30 laboratories using their routine procedures and plasma standards. Data were evaluated for intra- and inter-laboratory variation, accuracy and possible rFVIIIFc-specific assay discrepancies. For the one-stage assay, mean recovery was 95% to 100% of expected for both Advate(®) and rFVIIIFc at 0.8 IU mL(-1). Intra-laboratory percent coefficient of variance (CV) ranged from 6.3% to 7.8% for Advate(®), and 6.0% to 10.3% for rFVIIIFc. Inter-laboratory CV ranged from 10% for Advate(®) and 16% for rFVIIIFc at 0.8 IU mL(-1), to over 30% at 0.05 IU mL(-1) for both products. For the chromogenic substrate assay, the average FVIII recovery was 107% ± 5% and 124% ± 8% of label potency across the three concentrations of Advate(®) and rFVIIIFc, respectively. Plasma rFVIIIFc levels can be monitored by either the one-stage or the chromogenic substrate assay routinely performed in clinical laboratories without the need for a product-specific rFVIIIFc laboratory standard. Accuracy by the one-stage assay was comparable to that of Advate(®), while marginally higher results may be observed for rFVIIIFc when using the chromogenic assay.

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. 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 (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.