Blood coagulation involves a highly regulated balance between pro-coagulant and anticoagulant activities, allowing for the abatement of bleeding while limiting clot formation, which, if unchecked, could result in thrombosis. Hemostasis is triggered upon vascular injury when extravascular tissue factor (TF) binds plasma coagulation factor VIIa (FVIIa) forming a FVIIa/TF complex that generates limited amounts of activated factors IX (FIXa) and X (FXa). In normal hemostasis, FIXa with FVIIIa dramatically amplifies the production of FXa; FXa with FVa cleaves prothrombin to thrombin, and thrombin cleaves fibrinogen to form a fibrin clot. FXa generated by FVIIa/TF alone is insufficient for normal coagulation as FVIIa/TF activity is limited due to tissue factor pathway inhibitor (TFPI) and TFPI and other plasma protease inhibitors suppress the action of the initially generated FXa (and thrombin). Lacking FVIII or FIX, hemophiliacs fail to generate the additional FXa that is critical for normal hemostasis. Several methods designed to reduce the activity of endogenous inhibitors of coagulation are currently being investigated as a means to ameliorate the bleeding in hemophilia. These include antibody-mediated reduction in the activities of TFPI and protein S, and siRNA-mediated reduction in antithrombin (AT).

Protein Z (PZ) is a vitamin K-dependent coagulation factor that serves as the cofactor for FXa inhibition by the serpin, PZ-dependent protease inhibitor (ZPI), at phospholipid surfaces. If inhibition of the action of PZ/ZPI significantly enhanced coagulation in hemophilia, it might be associated with a reduced risk of thrombosis than other approaches. In the latter regard it is of interest that TFPI, PS and AT deficiency are associated with clinical thrombotic disease and mice lacking these proteins die in utero or perinatally of thromboembolic disease. In contrast, PZ and ZPI KO mice appear healthy.

Re-bleeding is a hallmark of clinical hemophilia. A tail vein re-bleeding (TVRB) assay, adapted from the saphenous vein re-bleeding assay of Whinna, was developed to assess the effects of PZ and ZPI deficiency on re-bleeding in the hemophilia A mouse model. In this assay, the lateral vein of the tail (diameter 2.25 mm) of an anesthetized mouse is lacerated to a depth of 1 mm with scalpel and the tail placed in warm saline. When bleeding stops, the wound is wiped with a balled Kimwipe to re-induce bleeding and the tail replaced in warm saline. This process is repeated and the number of wound disruptions occurring within 15 minutes is recorded. In normal mice, the period of bleeding following each wound disruption remains relatively constant, whereas in hemophilia mice the bleeding periods tend to progressively prolong with each subsequent disruption leading to a reduced number of total disruptions over 15 minutes.

Disruptions in the mouse TVRB assay were: for C57Bl/6 25.8+1.7 (mean+SEM); for FVIII KO 4.2+1.3; for PZ/FVIII double KOs 15.2+3.2; for ZPI/FVIII doubles KOs 14.6+3.5; for WT/KO littermates 4.0+2.6; and for FVIII KO with 100% human FVIII replacement 25.4+3.7. Based on FVIII dose-response experiments, the results in PZ/FVIII and ZPI/FVIII double KOs were equivalent to ~15% FVIII replacement. This outcome of the TVRB assays was confirmed in tests of the survival of sibling WT/FVIII KO (0%, n=5) and ZPI/FVIII double KO (75%, n=12) mice following tail transection 10 mm from the tip.

To assess the translation of the PZ/ZPI results in mice to humans, the effect of an anti-PZ monoclonal antibody (Mab) was compared to FVIII replacement on TF (1 pM) induced thrombin generation in human hemophilia A plasma. The Mab enhanced thrombin generation in hemophilia plasma to a similar degree as ~15% FVIII.

These studies provide proof of concept, both in vivo in mice and in vitro in human plasma, that blockade of the PZ/ZPI system may be sufficient to alter the phenotype of severe hemophilia to that of mild hemophilia, perhaps with limited thrombotic risk.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.