PB1072 - A Novel Factor VIII Specific APC Resistance Assay

Background: (F)actor VIII is inactivated by spontaneous A2-domain dissociation and activated protein C (APC) cleavage. We described a FVIII variant (FVIIIR336Q,R562Q; FVIII-QQ) resistant to APC cleavage with ~4-fold enhanced hemostatic function in vivo (Wilhelm et al. Blood 2021). Our ongoing work is investigating the use of FVIII-QQ for hemophilia A gene therapy; however, one important translational consideration is that plasma-based FVIII activity assays do not capture APC inactivation of FVIIIa. The generation of APC is largely dependent on binding the endothelial protein C receptor, which is absent in plasma. Additionally, FVIIIa bound to FIXa inhibits FVIIIa inactivation and licensed chromogenic FVIII:C assays use saturating FIXa concentrations. Lastly, APC also inactivates FVa; a reliable means of distinguishing APC inactivation/resistance of FVIIIa versus FVa is not clinically available.

Aims: We developed a plasma-based FVIII-specific APC resistance (APCR) assay.

Methods: An existing licensed chromogenic FVIII activity assay was modified by diluting the FIXa concentration to permit < 50% of generated FVIIIa (0.1-2nM) to be saturated by FIXa.

Results: Factor VIII:C of equimolar amounts of recombinant FVIII-WT versus FVIII-QQ were analyzed in the presence or absence of 1nM APC. FVIII-WT was sensitive to APC inactivation while FVIII-QQ activity ±APC was unchanged (Figure 1A). FVIII-specific plasma APCR of FVIII-QQ was determined relative to FVIII-WT+APC as a standard and found to be 3.9 to 6 within [FVIII] 0.1-1nM (normal plasma [FVIII] is ~1nM); the FVIII-QQ determined APCR relative to FVIII-WT was analogous to the observed in vivo enhanced hemostatic effect (Figure 1B).

Conclusion(s): We developed a modified chromogenic FVIII:C assay to incorporate APC inactivation of FVIIIa and determine APCR. This may inform potential translational efforts with FVIII-QQ for HA gene therapy. More broadly, assessment of FVIII-specific APCR in the setting of supraphysiologic FVIII-WT FVIII:C is a previously established prothrombotic risk factor; assessment of FVIII-specific APCR may inform clinical prothrombotic risk assessment.