PB0562 - Modifications in the Thrombin Active Site S4 Subpocket Induce Resistance to Direct Thrombin Inhibitors

Background: Direct anticoagulants inhibit coagulation serine proteases by reversibly engaging their active site with high affinity. To expand the repertoire of specific reversal agents we have previously successfully modified the S4 active site subpocket to prevent specific ligand binding and preserve catalytic activity in factor Xa [Verhoef 2017 Nature Commun.]. Whether an analogous strategy can be applied to introduce resistance to direct inhibitors in thrombin is unclear.

Aims: Assess if specific substitutions or sequences in or around the S4 subsite derived from naturally occurring serine proteases introduce inhibitor resistance in (pro)thrombin.

Methods: Recombinant prothrombin variants were generated with substitution of the S4 subsite residue I174 or exchange of the 91-99 sequence, directly adjacent to the S4 subsite, with that of human kallikrein-3. Following purification using the CaptureSelect matrix selecting for fully gamma-carboxylated prothrombin, the variants were biochemically assessed for their sensitivity towards direct thrombin inhibitors.

Results: The specific prothrombin clotting activity of the variants was 6-fold (intrinsic clotting) to 10-fold (extrinsic clotting) reduced relative to wild-type prothrombin. Turbidity analyses of purified fibrinogen revealed that modification of the S4 subsite hampers the conversion of fibrinogen by thrombin. Consistent with this, the thrombin variants displayed a reduced catalytic efficiency towards the peptidyl substrate used in thrombin generation assessments. In case of the latter, substantial thrombin generation was attained when supplementing prothrombin-deficient plasma with 180 µg/ml prothrombin variant. In this set-up, the variants displayed a ~2-fold reduced sensitivity for dabigatran relative to wild-type prothrombin, while argatroban inhibition was unaffected. Analyses using a purified component system revealed an up to 24-fold and 4-fold reduced IC50 for inhibition of thrombin by dabigatran and argatroban, respectively (Figure 1).

Conclusion(s): Collectively, prothrombin variants with S4 subpocket modifications are able to generate thrombin in plasma inhibited by dabigatran and as such could have the potential to bypass anticoagulant therapy.