Presentation Details
| Procoagulant Rescue of Hemophilia B Causing Factor IX Variants by Factor FVIII Mimetics Kyumin Lee1, 2, Yani Suber1, 2, Julia Q.Chau1, 2, Sebastian E.Leyes Porello1, 2, Bhavya S.Doshi1, 2, Ben J.Samelson-Jones1, 2. 1The Children’s Hospital of Philadelphia, Division of Hematology, Philadelphia, PA, USA.2University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA |
Abstract
Background: The intrinsic Xase enzyme complex, composed of activated coagulation factor (F) IX (FIXa) and its cofactor FVIIIa, activates coagulation FX through proteolytic cleavage. Within the intrinsic Xase, FVIIIa binding allosterically activates FIXa and enhances FX substrate binding, though the details of the FVIIIa/FIXa interaction remain poorly understood. Deficiencies in the assembly of the intrinsic Xase complex result in bleeding disorders hemophilia B (HB) and hemophilia A (HA). The FVIII-mimetic emicizumab, a bi-specific antibody for FIXa and FX, has been widely adopted for prophylaxis for HA patients. However, treatment for HB patients still requires frequent intravenous FIX administration. Because FVIIIa and FVIII-mimetics are biochemically distinct, we hypothesized that the hemostatic activity of some HB-causing FIX variants with dysfunctional Xase assembly may be rescuable by FVIII-mimetics such as emicizumab. Method: Initial screening of HB-causing FIX variants rescued by emicizumab was completed with conditioned media of transiently transfected HEK293 cells using one-stage FIX assays (OSA) and thrombin generation assay (TGA). To account for the OSA activity of emicizumab, the FIX activity of the HB-causing variants was compared against the activity of wild-type (WT) FIX in the presence of an equal concentration of emicizumab. Additional biochemical characterization of variants identified in the initial screen was done with FIX protein purified via affinity chromatography from stable FIX-expressing HEK293 cell lines. Enzyme kinetic parameters in the absence or presence of emicizumab were determined using a purified-protein intrinsic Xase assay. Result: To date, we have identified >40 HB-causing factor IX variants that exhibit at least a twofold increase in OSA activity with a therapeutic concentration of emicizumab (300 nM), including the highly prevalent FIX-R333Q variant. Most of these rescuable variants also display a similar enhancement with the addition of emicizumab in a TGA. Consistent with the screening results, purified FIX-R333Q TGA and OSA activity was 40- to 100- fold lower than purified FIX-WT, but increased 3- to 20-fold with the addition of 300 nM emicizumab. The specific activity of recombinant FIX-R333Q increase twentyfold in the presence of 300 nM emicizumab (Figure 1). Interestingly, the specific activity of FIX-R333Q with emicizumab doubled with the addition of monoclonal FVIII inhibitors, suggesting that FIXa-R333Q is likely able to bind FVIIIa, but this complex has minimal enzymatic activity. Indeed, in the purified-protein intrinsic Xase assay, FIXa-R333Q failed to generate detectable FXa even at high concentrations 90 nM of FVIIIa. However, FIXa-R333Q with emicizumab in the intrinsic Xase assay produced FXa. Combined, these results suggest that the loss-of-function of the HB-causing high prevalence FIX-R333Q variant is due to an impaired interaction between FIXa/FVIIIa and that this impairment can be bypassed with emicizumab. Conclusion: The procoagulant activity of select HB-causing FIX variants with dysfunctional Xase complex assembly can be rescued with therapeutic amounts of emicizumab. Specifically, activated FIX-R333Q is likely unable to functionally interact with FVIIIa, but can be rescued with emicizumab. FVIII-mimetics may be an alternative prophylactic hemostatic therapy for HB patients with FIX variants with similar loss-of-function mechanisms.
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