Low Molecular Weight Heparin Inhibits Plasma Thrombin Generation Via Direct Targeting of Factor IXa: Contribution of the Serpin-Independent Mechanism

Abstract 2242

The relevance of factor IXa as a therapeutic target for heparin therapy remains incompletely defined. To evaluate the contribution of factor IXa inhibition, particularly serpin-independent inhibition of the intrinsic tenase complex (factor IXa-factor VIIIa), the ability of heparin preparations to inhibit tissue factor (TF)-triggered thrombin generation was examined in human plasma. Thrombin generation was determined by a fluorogenic substrate assay employing Technothrombin TGA evaluation software to determine lag time, time to thrombin peak, peak thrombin concentration, and velocity index (slope). The inhibitory potency (EC₅₀) of each heparin was determined by plotting concentration versus relative velocity index (ratio of the velocity index in presence and absence of heparin). Inhibition of thrombin generation was initially compared under factor IX-dependent (limiting TF) and independent (excess TF) conditions, respectively, by addition of either 0.2 or 4 pM TF to pooled normal human plasma containing increasing concentrations of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), Fondaparinux, or unfractionated heparin (UFH). UFH and Fondaparinux demonstrated complete or near complete inhibition with identical EC₅₀ values at both tissue factor concentrations, suggesting that inhibition of intrinsic tenase activity does not significantly contribute to their mechanism of action in plasma. In contrast, LMWH and ssLMWH demonstrated 2.9- and 5.1-fold lower EC₅₀ values, respectively, in the presence of the limiting TF concentration. These results suggest that inhibition of intrinsic tenase activity contributes to the mechanism of action for LMWH in plasma. The effect of heparins on the time course of plasma thrombin generation by Western blotting under similar conditions correlated well with results of the fluorogenic substrate assay. At their EC₅₀ values, LMWH, ssLMWH and Fondaparinux clearly reduced prothrombin/meizothrombin consumption in plasma, while UFH primarily accelerated formation of the thrombin-antithrombin complex. The contribution of antithrombin to inhibition of thrombin generation was determined by comparison of antithrombin- and mock-depleted human plasmas. As expected, antithrombin depletion markedly increased the EC₅₀ values for UFH (62–fold) and Fondaparinux (42-fold) to levels that are ∼3–8 fold higher than their expected therapeutic ranges, confirming an antithrombin-dependent mechanism of action. In contrast, antithrombin depletion increased the EC₅₀ values more modestly for LMWH (9.4-fold) and ssLMWH (2-fold), with the EC₅₀ for LMWH (∼ 0.7 U/ml) remaining within the therapeutic range. In the absence of antithrombin, LMWH demonstrated partial inhibition of plasma thrombin generation with a plateau representing ∼8–10% of the starting activity, remarkably similar to the inhibition of intrinsic tenase activity with purified components. The molecular target for LMWH and ssLMWH was evaluated in dual factor IX/antithrombin-depleted plasma supplemented with 90 nM recombinant factor IX possessing mutations in the heparin-binding exosite. Based on both relative EC₅₀ values for reduction in the velocity index and the maximal degree of inhibition, plasma supplemented with mutant factor IX demonstrated relative resistance to inhibition of thrombin generation by LMWH as follows: R233A > (K126A/R165A/K132A) > R170A > WT. Factor IX R233A demonstrated an 11.4-fold increase in the EC₅₀ for LMWH relative to the wild type protease. The magnitude of differences between mutant factor IX proteins was reduced for ssLMWH relative to LMWH, but the rank order was similar. The effect of these mutations in human plasma recapitulates their effects on in vitro inhibition of intrinsic tenase activity, confirming the factor IXa heparin-binding exosite as the molecular target for antithrombin-independent inhibition of thrombin generation by LMWH and ssLMWH. The ability of therapeutic concentrations of LMWH to directly target factor IXa in antithrombin-depleted plasma suggests that this mechanism may contribute to antithrombotic effects. Furthermore, chemo-enzymatic synthesis of “non-anticoagulant” LMWH preparations acting via this exosite-mediated antithrombotic mechanism may offer advantages in high-risk populations, particularly patients with underlying malignancy.