Emicizumab Augments Thrombus Formation in Whole Blood from Patients with Hemophilia A under High Shear Flow Conditions

Abstract

Background Emicizumab is a bispecific antibody to factor (F) IXa and FX that mimics the FVIIIa cofactor function. Emicizumab prophylaxis markedly decreases bleeding episodes in patients with hemophilia A (PwHAs), irrespective of the presence of FVIII inhibitors. However, thrombotic microangiopathy (TMA) was reported when repeated high doses of activated prothrombin complex concentrates (aPCC) were concomitantly used with emicizumab. Although bypassing agents (BPAs) are vital in the hemostatic treatment for PwHAs with inhibitors, the mechanism of emicizumab-related TMA remains unclear. Aim To assess the risk of excessive thrombus formation associated with BPAs and emicizumab under high shear conditions. Methods Perfusion flow-chamber experiments under high shear conditions were performed using whole blood from PwHAs in the presence of emicizumab without or together with FVIII or BPAs ex vivo. Results Emicizumab (100 μg/mL) added ex vivo to whole blood from PwHAs improved defective thrombus formation in a similar manner to that observed with the addition of recombinant FVIII at the early phase, while FVIII continued to be important at the later stages. aPCC (1.2 U/mL equivalent to 100 U/kg) or recombinant FVIIa (1.1 µg/mL; equivalent to 90 µg/kg) together with emicizumab further promoted platelet interactions and fibrin formation ex vivo but did not induce excessive thrombus formation. Conclusion Emicizumab enhanced thrombin generation at local sites and improved defective hemostasis in whole blood from PwHAs under high shear conditions. Simple concomitant use of BPAs with emicizumab did not mediate excessive thrombus formation and remains an option for hemostatic management of emicizumab-treated PwHAs with inhibitors.

Emicizumab improves thrombus formation of type 2A von willebrand disease under high shear condition

“…In assessment of thrombogenesis, surface coverage (SC) would be more influenced by the VWF function whilst thrombus height (TH) is by fibrin formation regarded to be associated with enhanced stability of thrombus. 18,24 An earlier report demonstrated normal SC but defective TH in a patient with a fibrinogenemia. 29 By contrast, both parameters were defective in haemophilia A patients and the role of FVIII was essential even in the early phase of thrombus formation, which was different from fibrinogen.…”

“…Perfusion experiments were performed as previously described. 18,24 Briefly, a six-channel slide (µ-Slide VI 0.1, ibidi) was coated with collagen I/III (300 μg/ml) at room temperature (…”

“…Following perfusion, thrombi were fixed with Cytoperm/Cytofix ® and immunofluorescent staining was performed to visualize platelets and FVIII. 18,24 The fixed thrombus was permeabilized with PBS with 1% Triton X-100 for 10 min at RT and blocked with serum free protein block for 20 min, followed by incubation with phalloidin-Alexa488 (6 µg/ml), anti-VWF polyAb (10 µg/ml) labelled with Alexa568, and anti-FVIII polyAb (10 µg/ml) overnight at 4°C.…”

“…In addition, the action of emicizumab appeared to be more rapid, whilst that of FVIII was more robust, supporting the findings in our earlier study using whole blood samples from PwHA. 18…”

“…More recently, we have demonstrated that emicizumab enhanced thrombin generation at local sites and improved defective haemostasis in whole blood from PwHA under high shear condition using an ex vivo perfusion chamber experiment. 18 However, it remains unknown whether emicizumab may provide somewhat influence on thrombus formation in patients with 2AVWD characterized by the low levels of FVIII as well as the lack or impaired platelet adhesion and aggregation. In the present study, the similar ex vivo perfusion chamber experiments were devised to compare the effects of FVIII, FVIII/VWF products and emicizumab using whole blood from patients with 2AVWD.…”