Emizicumab Promotes Factor Xa Generation on Activated Endothelium in a Blood Cell-Independent Manner

Abstract

IntroductionHemophilia A (HA) is an inherited bleeding disorder caused by the deficiency of coagulation factor VIII (FVIII) resulting in severe hemorrhage if untreated. Recombinant and plasma derived FVIII products have long been the standard of care in hemophilia. However, approximately 25-30% of patients with severe HA develop inhibitors, neutralizing alloantibodies to FVIII, a significant complication in the treatment of patients with HA that leads to bleeding despite factor therapy.First approved for bleed prophylaxis in HA with inhibitors in the US by the FDA in 2018, emicizumab (Genentech, USA) has initiated a new era of HA treatment. This drug is a bispecific, monoclonal antibody that binds to activated Factor IX (FIXa) and Factor X (FX), mimicking activated FVIII (FVIIIa) by bringing FIXa and FX into proximity to enable FX activation, even in the presence of inhibitors.Emicizumab prophylaxis drastically reduces bleed episodes. However, thromboses and thrombotic microangiopathy (TMA) were observed in trials, all associated with concomitant use of activated prothrombin complex concentrates (aPCC) (Callaghan et al., 2021). The mechanism of this devastating condition is uncertain, as emicizumab is not known to bind to phospholipid or vascular surfaces. We report that FX is more readily activated by FIXa and emicizumab on endothelium that has been activated by tumor necrosis factor alpha (TNF). This finding may partially explain the development of TMA in these patients.MethodsWe utilized novel, microfluidic devices that are inexpensive to manufacture and were modified from a technique previously described (Alapan et al. 2016). These devices are fabricated using a laser cut double-sided adhesive film sandwiched between a clear, gas-permeable polymer (Ibidi, Germany) and an acrylic top that is laser cut (Universal Laser Systems Inc., USA) (Figure 1).Human umbilical endothelial cells (HUVEC, Lonza, Switzerland) were harvested at passage 3 to 4 and seeded into the devices. These were then cultured under flow conditions using a non-peristaltic, air-driven pump (Ibidi GmbH, Germany) to achieve a confluent and quiescent endothelial surface. HUVEC are then activated by incubating with 5 nM TNF in serum-free growth medium for 4 hours. This treatment induced markers of endothelial activation without gross apoptosis. Non-activated HUVEC were incubated with endothelial cell growth medium (2% serum) until time of experiments.Factors IXa, X (Haemtech, USA), and/or emicizumab (discarded clinical material) were mixed in HEPES-buffered saline with 5 mM calcium chloride for all experimental conditions. Concentrations used of FIXa (30 nM), FX (170 nM), and emicizumab (55 ug/mL) were constant for all conditions. Combinations of factors and emicizumab were then incubated in the endothelialized device for 30 minutes at 37° C. The entire volume of the mixture was then aspirated (20 uL) and stored at -80° C.FXa activity was assayed on the effluent for 60 minutes using a chromogenic FXa substrate (Pefachrome, Pentapharm, Switzerland).ResultsNo significant generation of Xa was noted in the presence of healthy or activated endothelium with emicuzumab alone, emicizumab and FIXa, emicizumab and FX, or factors IXa and X. Median Xa generation observed with the combination of emicizumab, FIXa, and FX on healthy endothelium was 2 nM. Median Xa generation with the same combination on activated endothelium was 8.1 nM, a four-fold increase (P = 0.028, Mann-Whitney test) (Figure 2).DiscussionEmicizumab represents an evolving standard of care for hemophilia A. Considering data showing diminishing FVIII expression in the months to years after AAV gene therapy, (Pasi et al., 2020) it may well be the dominant treatment paradigm in HA for some time.However, much remains to be answered in the use of emicizumab, and the mechanism of thrombosis and TMA with concomitant aPCC use has resulted in the avoidance of aPCC use for breakthrough bleeding in patients on emicizumab therapy, even up to 6 months after cessation.Our data demonstrate that activated endothelial cells promote FX activation more readily than quiescent endothelial cells in the presence of FIXa and emicizumab. These findings demonstrate the potential of thrombotic angiopathy in an in vitro system. Further investigation of the interaction of endothelium with FIXa, FX, and FVIIIa-mimetic antibodies is warranted. [Display omitted] DisclosuresEllsworth: Takeda: Other: Salary supported as part of NHF-Takeda Clinical Fellowship Award. Monroe: Medexus Pharmaceuticals: Consultancy; Takeda: Consultancy; Otello Medical: Current equity holder in publicly-traded company. Hoffman: Takeda: Research Funding; CSL Behring: Consultancy; Sanofi: Consultancy; BPL (Bio Products Laboratory): Consultancy. Key: BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator; Sanofi: Consultancy.