PB0553 - The antithrombotic protein C activator thrombin mutant W215A/E217A (AB002, E-WE) is slowly inhibited by the endogenous thrombin inhibitors antithrombin, protein C inhibitor, and heparin cofactor II

Background: AB002 is a recombinant thrombin analog (W215A/E217A, E-WE thrombin) in phase 2 clinical development. Compared to thrombin, AB002 exhibits diminished activity toward prothrombotic substrates but can activate protein C when bound to thrombomodulin. These properties, in addition to a reduced rate of inhibition by antithrombin, presumably combine to produce a potent antithrombotic effect. However, the relative contributions of plasma serpins to AB002 inhibition is unknown.

Aims: To investigate the ability of antithrombin, protein C inhibitor (PCI), and heparin cofactor II (HCII) to inhibit AB002 in the absence or presence of cofactors, and to evaluate circulating serpin-enzyme complexes following intravenous AB002 administration.

Methods: The time course of inhibitor complex formation following AB002 addition to prothrombin-depleted plasma or buffer containing purified antithrombin, PCI, or HCII +/- heparin or thrombomodulin was evaluated by Western blot and SDS-PAGE, respectively. Human plasma samples (n=3) from an AB002 phase 1 clinical trial (NCT03453060) were evaluated for serpin-enzyme complexes by ELISAs.

Results: AB002-inhibitor complexes were not detected within 90 min of AB002 addition to plasma without addition of heparin or thrombomodulin (Figure 1). In buffer, the addition of either antithrombin or HCII to AB002 did not produce observable complex formation within 20 min, compared to PCI, which formed complexes within minutes. Thrombin rapidly complexed with all inhibitors. Heparin and thrombomodulin had the greatest effect on AB002-HCII and AB002-PCI, respectively. In AB002-dosed subjects (4 mcg/kg), there was a temporal increase in PCI, HCII, and antithrombin complexes (Figure 2). HCII complexes were the most prevalent and peaked at 15 min post-dose, returning to baseline by ~2 hr post-dose.

Conclusion(s): Our in vitro data suggest that, compared to thrombin, AB002 is relatively slowly inhibited by serpins including antithrombin, PCI, and HCII. All three inhibitors may play a role in regulating AB002’s effects in vivo, with relative contributions likely dependent on local availability of cofactors.