Background: Tissue factor pathway inhibitor (TFPI) plays a major role in regulation of tissue factor (TF)-initiated coagulation in a FXa-dependent manner. Andexanet alfa (AnXa) is a modified, recombinant, catalytically inactive form of human FXa being developed to reverse the anticoagulant activity of FXa inhibitors in patients during episodes of major bleeding. As a modified FXa, AnXa retained high binding affinity to fXa inhibitors, including TFPI. Our previous in vitro studies demonstrated that AnXa reverses rivaroxaban-induced inhibition of thrombin generation (TG) initiated by either the extrinsic or intrinsic pathways. However, the thromboelastography (TEG) results showed that AnXa-TFPI interaction enhanced thrombin formation at low TF conditions, which suggested potential increase in the fibrin degradation product in the presence of recombinant tissue plasminogen activator (rtPA) (Lu, G., et al, ASH2016). In the current study, we further characterized the differential effect of AnXa on thrombin formation and clot lysis via the two different pathways, and correlated the TG and TEG parameters with the coagulation markers in the TF-initiated reaction in plasma, including prothrombin fragment (F1+2), thrombin-antithrombin complex (TAT), and fibrin degradation product (D-dimer).
Methods: TF-initiated TG in human plasma was measured using a calibrated automated thrombogram (TF-CAT) and the PPP-reagent (5 pM TF, Diagnostica Stago). Non-TF-initiated TG was measured using CAT and an aPTT reagent, Actin FS (Siemens Healthcare) as the activator (Actin FS-CAT). Clot formation was measured in plasma using a TEG 5000 analyzer (Haemonetics). TF (1 pM) and Actin FS (1:240 final dilution) were used as the activators with or without rtPA (150 ng/mL). Time-dependent thrombin formation and clot lysis were monitored by quenching samples at various times (0, 2.5, 5, 10, 15, 30, 60 min) following initiation of the reaction by TF (1 pM) in plasma with or without rtPA (150 ng/mL) and AnXa (4.0 µM). Corn trypsin inhibitor (CTI, 50 µg/mL) was added to block contact activation. F1+2, TAT and D-dimer were quantified according to manufacturers' recommendations.
Results: The effect of AnXa on TF-CAT and Actin FS-CAT were compared side-by-side with AnXa at low (0 - 20 nM) and high (0 - 4.0 µM) concentration ranges. AnXa (5 nM) increased TF-CAT parameters sensitive to TFPI activity (i.e., peak thrombin), with less effect on endogenous thrombin potential (ETP). No further increase in these parameters was observed at AnXa therapeutic concentrations (e.g., 2.0 and 4.0 µM), consistent with the expected effect of AnXa on the activity of TFPI, which is present at low plasma concentration (~2.4 nM). In contrast, AnXa did not significantly affect the Actin FS-CAT parameters.
The effect of AnXa-TFPI interaction on coagulation and fibrinolytic pathways in human plasma were compared using TEG, with or without rtPA. AnXa (4.0 µM) reduced the TF-TEG-R parameter (lag time, equivalent to clotting time) and resulted in an increased area under the fibrinolytic profile with rtPA as previously shown. In contrast, AnXa and the buffer controls had similar profiles in the Actin FS-TEG assay under similar conditions, with or without rtPA (150 ng/mL) in the current studies, suggesting lack of an AnXa-TFPI effect.
We further investigated the effect of AnXa-TFPI interaction on coagulation markers following TF (1pM)-initiated thrombin formation in plasma. AnXa (4 µM) increased F1+2 and TAT levels at early time points following initiation (2.5, 5, 10, 15 min), but had no difference at or after 30 min, compared to buffer control. A similar pattern was observed for D-dimer level when exogenous rtPA (150 ng/mL) was added. Compared to buffer control, AnXa caused elevation of D-dimer level at early time points but had no difference at or after 30 min following initiation of the reaction.
Conclusions: AnXa-TFPI interaction can modulate the TF/TFPI activity in the initiation phase of the extrinsic coagulation pathway and increase parameters sensitive to TFPI activity in the TF-CAT and TF-TEG assays, but has minimal effect on the later phase of the reactions and the overall thrombin formation potential. Consistent with clinical observations, AnXa-TFPI interaction increased the F1+2 and TAT levels, as well as D-dimer level in the presence of rtPA in the early phase of the reaction following initiation of the reaction by TF.
Lu: Portola Pharmaceuticals, Inc.: Employment. Lin: Portola Pharmaceuticals, Inc.: Employment. Curnutte: 3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties. Conley: Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties.
Asterisk with author names denotes non-ASH members.