An Engineered Factor Fva Prevents Bleeding Induced By Anticoagulant Wild Type Activated Protein C
Authors: Von Drygalski Annette , Gale Andrew J. , Burnier Laurent , Cramer Thomas J. , Griffin John H. , Mosnier Laurent O. 
Published: October 2019

Abstract
Increased risk of bleeding is observed in patients receiving therapy with a variety of anticoagulant drugs, and there is an unmet need for prohemostatic agents that reduce bleeding risk. In some clinical trials, wild type (wt) APC therapy was associated with an increased risk of serious bleeding. In most animal models of inflammatory injury and disease where APC was beneficial, APC’s cytoprotective effects were responsible for the protective effects of APC therapy whereas its anticoagulant effects were neither required nor contributing. Thus, wt APC’s anticoagulant activities and associated risk of bleeding may be a limiting factor for potential novel wt APC therapies. The availability of a wt APC-anticoagulant specific antidote or reversal agent that does not affect wt APC’s cytoprotective activities would be highly desirable.
We hypothesized that superFVa, an engineered FVa-variant that potently normalizes hemostasis in hemophilia, fits the criteria for a prohemostatic biologic that could reduce wt APC-anticoagulant-induced bleeding, and here we test this hypothesis. SuperFVa has enhanced specific activity compared to wt FVa due to an engineered disulfide bond (Cys609-Cys1691) between the A2 and A3 domains (FV(A2-SS-A3)) and, its biological activity is augmented by mutations of the APC cleavage sites (Arg506/306/679Gln). As a result of these modifications, superFVa was found to be resistant to wt APC and normalized hemostatic properties and prevented bleeding in a hemophilic mouse model much more efficiently than wt FVa.
In aPTT clotting assays both superFVa and wt FVa dose-dependently normalized clotting times when wt-APC was used to prolong clotting (aPTT > 100 sec). However, superFVa normalized the aPTT at 100-fold lower concentrations compared to wt FVa. In thrombin generation assays using either human or murine plasma, superFVa fully restored thrombin generation at concentrations where wt FVa did not show effects.
In an ex vivo whole blood aPTT assay, intravenous (iv) injection of murine recombinant wt APC (0.5 mg/kg) in Balb/c mice doubled clotting times from 30 sec to ∼ 60 sec (n=40). Addition of superFVa (1 nM) to whole blood significantly normalized aPTT clotting times whereas wt FVa failed to show a significant effect.
In a tail clip-bleeding model in Balb/c mice, injection (iv) of human recombinant or plasma-derived wt APC induced significant bleeding at 1.25 mg/kg and mean blood loss increased from 3.4 µL/g with saline to 27 µL/g with wt APC treatment. SuperFVa injected (iv 3.5 mg/kg) 2 min prior to wt APC administration reduced bleeding significantly to 9.2 µL/g (n∼10 per group).
In another bleeding model, liver laceration was used because it provides important information concerning microvessel-mediated bleeding after acute organ injury. After adaptation and technical modifications of the model used in rats and rabbits for mouse anatomy and validation in hemophilia versus control mice, this model provided a reliable assessment of bleeding with a ∼ 4-fold reduced inter-individual range of blood loss compared to the tail clip-bleeding model. Injection (iv) of human wt APC increased blood loss from 29 µL/g to 49 µL/g, and the excessive bleeding was associated with a ∼40% mortality rate. SuperFVa reduced wt APC-induced bleeding after 20 min significantly to 29 µL/g and abolished bleeding-induced mortality.
Remarkably, bleeding patterns in the tail clip and liver laceration models were different when blood loss was determined separately for the 1st and 2nd 10 min after injury. In the tail clip-model wt-APC-induced bleeding during both 1st and 2nd 10 min after tail clip and superFVa decreased blood loss during both phases. In the liver laceration model, most blood loss occurred immediately after injury and bleeding during the 2nd 10 min was less pronounced. In this model, however, superFVa corrected blood loss entirely during the 1st 10 min phase and fully prevented bleeding during the 2nd 10 min phase.
Our results provide proof of principle that superFVa is effective in the prevention and reversal of wt APC-induced bleeding. Thus, in addition to improving hemostasis in hemophilia, superFVa protects against bleeding in 2 different mouse models where bleeding was induced by wt APC. Hence, superFVa deserves future consideration for development as a prohemostatic agent in situations where bleeding is a serious risk.

Disclosures:
No relevant conflicts of interest to declare.