An in Vitro Pilot Study of the Role of Factor IX As an Alternative to Bypassing Agents in the Treatment of Breakthrough Bleeds in Patients with Hemophilia a and Inhibitors Treated with Emicizumab

Abstract

Introduction: Hemophilia A(HA) is an X linked disorder characterized by a deficiency in Factor VIII. 30% of patients with severe HA develop alloantibodies that inhibit the coagulant activity of FVIII. Emicizumab, a substitution therapy that bridges factor IX and X, is approved in patients with severe HA with and without inhibitors. Indeed, it is now the standard of care prophylactic therapy for patients with inhibitors. Breakthrough bleeds and perioperative management require the use of bypassing agents in addition to emicizumab, with recombinant VIIa deemed the safest of these options. Nevertheless there are still concerns about thrombotic risks and inability to monitor bypassing agents given to patients on emicizumab. Since the rate limiting step for FXa generation in the plasma of patients receiving emicizumab is the concentration of generated FIXa, we conducted an in vitro pilot study to evaluate the hemostatic effect of adding FIX to the plasma of patients on emicizumab prophylaxis. Methods: After informed consent was obtained, blood was collected in citrated tubes prefilled with 1.45mM of corn trypsin inhibitor(CTI) from 25 patients with HA receiving emicizumab prophylaxis. Aligning with previously published data, the added hemostatic effect of rFVIIa was studied in platelet rich plasma (PRP), while all other products were studied in platelet poor plasma (PPP). PRP was spiked with 0, 45, 90 and 270mg/kg of rFVIIa, and PPP was spiked with 0, 10, 20, 30 and 50 IU/kg of activated prothrombin complex concentrate (aPCC); 0, 25, 50 and 100 IU/kg of recombinant FIX(rFIX) and 0, 25, 50 and 100 IU/kg of plasma derived FIX(pdFIX). Thrombin generation was run on these spiked samples using the CAT method. Coagulation was triggered in PRP samples with tissue factor 1pM. PPP samples were activated using 1pM of TF and 1.5pM FIXa. Mean endogenous thrombin potential (ETP, area under the thrombin generation curve) values were compared between the different experiments and with samples obtained from controls. Baseline FIX concentrations were determined in these patients using bovine chromogenic assay. The mean plasma emicizumab concentration (+Standard Deviation) was 44.45 µg/mL (+15.83). Results: As shown in fig 1, ETP was significantly improved with increasing concentrations of aPCC, rFIX and pdFIX in PPP(p<0.005). Fig 2 shows that ETP normalized with the addition of rFVIIa 45mg/kg (p<0.0001) in most of the patients tested. Moreover, we did not see ETP above the upper limit of the normal range when FIX was added, while 50 IU/kg of aPCC and 270 mg/kg of rFVIIa clearly surpassed the upper limit of the normal range, suggesting a risk of hypercoagulability and thrombosis. FIX levels varied between 63.9% and 248.9% with a mean of 134.27%. ETP obtained after adding rFIX 100 IU/kg was significantly greater than that attained with pdFIX (p=0.027), that might be explained by the presence of the activated form of FIX in rFIX concentrates. No statistical significant difference between the doses of 50 IU/Kg of FIX and 100 IU/KG was observed (p>0.05), with a noted tendency to increased ETP with 100IU/KG dose. Conclusion: Our in vitro study demonstrates excess thrombin generation (and potential thrombosis risk) with higher concentrations of rFVIIa and aPCC that are used in the clinical care of patients. While addition of FIX at a range of doses appears to improve thrombin generation without exceeding the upper normal limits. These data suggest that FIX therapy may safely improve hemostasis in patients with HA and inhibitors receiving emicizumab prophylaxis. A clinical study looking at a starting dose of rFIX at 50 IU/KG for mild to moderate bleeds and 100 IU/Kg for more severe bleeds is warranted. In addition to the advantage of monitoring levels with FIX therapy, the longer half-life of FIX offers the potential advantage of controlling hemostasis with single dose administration. Future prospective clinical trials are needed to investigate the efficacy and safety of this therapy in this patient population.