The ability to anticoagulate patients at elevated risk for thrombosis without inducing a bleeding diathesis remains an unmet need of patient care both in a hospital setting and at home. Traditional anticoagulants including warfarin and heparin effectively inhibit clotting, but incur a bleeding risk and require continued monitoring after a stable profile has been established. Current generation direct oral anticoagulants (DOACs) including dabigatran, rivaroxaban, and apixaban overcome many of the liabilities of warfarin and heparin (e.g. lack of target specificity and need for monitoring), but are still susceptible to bleeding diathesis. Thus there remains a need for novel anticoagulants that offer an improved therapeutic index. The recombinant human antibody JNJ-64179375 (JNJ-9375) is a novel thrombin inhibitor that binds to the exosite I region with high affinity and selectivity. In contrast to DOACs that inhibit the protease active site, JNJ-9375 inhibits the interaction of thrombin with its substrates including fibrinogen and PAR-1 while retaining active site and exosite II mediated interactions. This targeted and distinct mechanism of action which modulates the actions of thrombin offers the potential for efficacious anticoagulation with the potential for reduced risk of bleeding compared to active site-specific DOACs.

To assess the ability to measure exosite I-specific thrombin inhibitors effects in blood, we compared JNJ- 9375 to the active site ligand dabigatran in clotting assays, thromboelastography (TEG), and calibrated automated thrombography (CAT) assays. We focused on clinically relevant concentrations of these inhibitors based on the efficacious doses reported in the RE-LY study for dabigatran (NCT00262600) and a Phase 1 study for JNJ-9375 (NCT02949206). In conventional clotting assays (prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT)) dabigatran and JNJ-9375 each prolonged clotting times in a dose-dependent manner. TT assays were the most sensitive to dabigatran and JNJ-9375, with 80 nM dabigatran prolonging the clotting time by 5-fold and 100 nM JNJ-9375 prolonging the clot time 4-fold over vehicle control. PT and aPTT assays were less sensitive than the TT assay, requiring ≥ 300 nM dabigatran and ≥ 1 µM JNJ- 9375 to double the PT or aPTT clotting times. In TEG assays both JNJ-9375 and dabigatran prolonged the reaction time (R), with 200 nM JNJ-9375 prolonging R by 1.3- to 1.5-fold and 100 nM dabigatran prolonging R from 1.6- to 1.8-fold over buffer control. In CAT assays initiated with 5 pM tissue factor (PPP Reagent, Stago), dabigatran inhibited peak thrombin and Endogenous thrombin potential (ETP) with an approximate IC50 of 1 µM. In contrast, titration of JNJ-9375 up to 1 µM increased both the Peak thrombin and ETP ≥ 20% above buffer control. This increase in peak thrombin and ETP with JNJ-9375 was also observed with other exosite I-specific ligands such as hirugen, suggesting a general effect of exosite I binding on the primary endpoints of the CAT assay. This insensitivity of the CAT assay to exosite I ligands likely reflects the inability of these molecules to inhibit proteolysis of the peptidyl substrate z-Gly-Gly-Arg-AMC, which directly binds to the active site and is used as the readout of thrombin activity.

In summary, of the assays tested the TT clotting assay offers the greatest sensitivity to detect multiple classes of direct thrombin inhibitors in plasma, while TEG can detect direct thrombin inhibitors in whole blood. In contrast, the CAT assay is insensitive to exosite I ligands such as JNJ-9375. These results highlight the importance of the relationship between the endpoint of a given coagulation assay and the mechanism of action of the anticoagulant being tested.


Bunce: Janssen Research & Development: Employment. Huang Devine: Janssen Research & Development: Employment. Chintala: Janssen Research & Development: Employment.

Author notes


Asterisk with author names denotes non-ASH members.