Tapping Into the Natural PZ-Independent Anticoagulant Function of ZPI to Inhibit Thrombosis With Minimal Effect on Hemostasis.

Abstract

Background: The protein ZPI (Z-dependent protease inhibitor) binds to PZ (protein Z), which enables ZPI to inhibit membrane-bound FXa (activated factor X). ZPI also inhibits FXIa (activated factor XI) independently of PZ.

Methods: To study the PZ-independent ZPI function, we tested the in vitro and in vivo effect of disrupting the ZPI-PZ interaction by mutating ZPI Asp 293 to Ala (D293A).

Results: D293A mutation reduced PZ-dependent FXa inhibition without affecting FXIa inhibition. D293A also diminished FXIIa (activated FXII)-induced thrombin generation but reduced TF (tissue factor)-induced thrombin generation only at low TF concentrations. This suggests that D293A selectively inhibits the intrinsic pathway and the thrombin-FXI (factor XI) feedback loop that enhances low-dose TF-initiated coagulation. Wild-type and D293A ZPI both showed selectivity in inhibiting activated partial thromboplastin time but not prothrombin time. Increasing PZ in plasma enhances activated partial thromboplastin time inhibition and enables prothrombin time inhibition by wild-type but not D293A ZPI, further indicating that D293A ZPI selectively inhibits the intrinsic pathway independently of PZ. In mouse models, D293A inhibited FeCl₃-induced occlusive carotid artery thrombosis and venous thrombosis in the inferior vena cava. Thus, PZ-independent ZPI function plays a major role in ZPI inhibition of occlusive thrombosis, and D293A ZPI is an effective antithrombotic. Importantly, administering D293A ZPI did not affect tail bleeding time and showed improved hemostasis in a saphenous vein hemostasis model as compared with wild-type ZPI.

Conclusions: The PZ-binding defective variant of ZPI, D293A, selectively inhibits the intrinsic coagulation pathway and is a new anticoagulant with reduced bleeding risk.