Thrombolysis by chemically modified coagulation factor Xa

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 nonenzymatic thrombolytic agent and an anticoagulant. Xai-K is based on clotting factor Xa, whose sequential plasmin-mediated fragments, FXa beta 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 FXab by modifying residues at the active site. The resultant Xai-K (1 nM) enhanced plasma clot dissolution by similar to 7-fold in vitro and was dependent on tPA. Xai-K alone (2.0 mu g g(-1) body weight) achieved therapeutic patency in mice. The minimum primary dose of the tPA variant, Tenecteplase (TNK; 17 mu g g(-1)), could be reduced by > 30-fold to restore blood flow with adjunctive Xai-K (0.5 mu g g(-1)). TNK-induced systemic markers of fibrinolysis were not detected with Xai-K (2.0 mu g 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.