Thrombolysis by chemically modified coagulation factor Xa

Essentials Factor Xa (FXa) acquires cleavage‐mediated tissue plasminogen activator (tPA) cofactor activity. Recombinant (r) tPA is the predominant thrombolytic drug, but it may cause systemic side effects. Chemically modified, non‐enzymatic FXa was produced (Xai‐K), which rapidly lysed thrombi in mice. Unlike rtPA, Xai‐K had no systemic fibrinolysis activation markers, indicating improved safety. Summary Background Enzymatic thrombolysis carries the risk of hemorrhage and re‐occlusion must be evaded by co‐administration with an anticoagulant. Toward further improving these shortcomings, we report a novel dual‐functioning molecule, Xai‐K, which is both a non‐enzymatic thrombolytic agent and an anticoagulant. Xai‐K is based on clotting factor Xa, whose sequential plasmin‐mediated fragments, FXaβ and Xa33/13, accelerate the principal thrombolytic agent, tissue plasminogen activator (tPA), but only when localized to anionic phospholipid. Methods The effect of Xai‐K on fibrinolysis was measured in vitro by turbidity, thromboelastography and chromogenic assays, and measured in a murine model of occlusive carotid thrombosis by Doppler ultrasound. The anticoagulant properties of Xai‐K were evaluated by normal plasma clotting assays, and in murine liver laceration and tail amputation hemostatic models. Results Xa33/13, which participates in fibrinolysis of purified fibrin, was rapidly inhibited in plasma. Cleavage was blocked at FXaβ by modifying residues at the active site. The resultant Xai‐K (1 nm) enhanced plasma clot dissolution by ~7‐fold in vitro and was dependent on tPA. Xai‐K alone (2.0 μg g−1 body weight) achieved therapeutic patency in mice. The minimum primary dose of the tPA variant, Tenecteplase (TNK; 17 μg g−1), could be reduced by > 30‐fold to restore blood flow with adjunctive Xai‐K (0.5 μg g−1). TNK‐induced systemic markers of fibrinolysis were not detected with Xai‐K (2.0 μg g−1). Xai‐K had anticoagulant activity that was somewhat attenuated compared with a previously reported analogue. Conclusion These results suggest that Xai‐K may ameliorate the safety profile of therapeutic thrombolysis, either as a primary or tPA/TNK‐adjunctive agent.