Abstract

Anticoagulation requires a careful balance to achieve antithrombotic efficacy without disrupting normal hemostasis. Although current generation direct oral anticoagulants (DOACs) offer advantages over traditional warfarin or heparin therapy they still carry a bleeding risk, highlighting an unmet need for novel anticoagulants with improved therapeutic indices. DOACs such as apixaban and dabigatran bind to the active sites of their target proteases thereby inhibiting proteolysis of all substrates. In contrast, the recombinant antibody JNJ-64179375 (JNJ-9375) specifically binds to the exosite I region of thrombin without inhibiting active site function. Interestingly, JNJ-9375 has demonstrated an improved therapeutic index compared to apixaban in preclinical models of venous thrombosis (Chintala, M et al. Res Pract Thromb Haemost, 2017:1 (suppl 1): P201). To understand how the mechanism of action of JNJ-9375 contributes to its improved therapeutic index in preclinical models, we evaluated the impact of JNJ-9375 on the activity of thrombin toward physiological substrates in vitro. Fibrinogen is the primary in vivo substrate of thrombin and binds to it via exosite I. In a turbidimetric assay JNJ-9375 dose-dependently inhibited fibrinogen cleavage, reflecting direct competition between fibrinogen and JNJ-9375 for exosite I binding. Factor V (FV), Factor VIII (FVIII), and Factor XI (FXI) are activated by thrombin in positive feedback reactions. Exosites I and II in thrombin have been differentially implicated in its action on Factors V and VIII. Accordingly, JNJ-9375 potently inhibited thrombin-dependent FV activation while exhibiting a more modest inhibition of FVIII activation as determined by quantitative Western blotting. Activation of FXI by thrombin, another exosite I-mediated reaction, was reduced approximately 8-fold in the presence of JNJ-9375. Thrombin also interacts with the anticoagulant regulators Protein C and antithrombin III (ATIII). Thrombin activates Protein C through an exosite I-mediated interaction with thrombomodulin (TM). In chromogenic assays JNJ-9375 reduced the TM-dependent activation of Protein C approximately 7-fold. Plasma supplemented with JNJ-9375 also demonstrated a modest resistance to TM-dependent Protein C activation measured in thrombin generation assays. ATIII inactivates thrombin by binding to the active site of thrombin in a suicide substrate reaction. Despite this reaction being independent of exosite I binding the rate of thrombin inactivation by ATIII was reduced approximately 3-fold in the presence of JNJ-9375, indicating an allosteric effect of exosite I binding on the reactivity of thrombin with ATIII. In contrast to exosite I-dependent reactions, JNJ-9375 had no apparent effect on the exosite II-dependent interaction of thrombin with heparin. Pre-incubation with JNJ-9375 did not alter the elution profile of thrombin in heparin sepharose chromatography. Furthermore, both unfractionated heparin and low molecular weight heparin dramatically accelerated inactivation of thrombin by ATIII in the presence of JNJ-9375, indicating that exosite II function was preserved. In summary these in vitro studies illustrate a global mechanism wherein JNJ-9375 attenuates the activity of thrombin toward both pro- and anti-coagulant components of the coagulation cascade through competitive binding to the exosite I region. This distinct mechanism in which JNJ-9375 modulates thrombin without inhibiting its proteolytic activity or exosite II-mediated activity differentiates JNJ-9375 from active site-specific DOACs. We hypothesize that the residual catalytic activity of JNJ-9375-bound thrombin enables antithrombotic efficacy without the concomitant bleeding risk seen with current generation DOACs, resulting in an improved therapeutic index.

Disclosures

Krishnaswamy:Baxalta: Consultancy; Portola: Research Funding; Janssen Research & Development: Research Funding.

Author notes

*

Asterisk with author names denotes non-ASH members.