Activated FVIII Released from FVIII/VWF Complex Facilitates Thrombus Development Under High Shear Flow Condition
Authors: Yaoi Hiroaki , Shida Yasuaki , Kitazawa Takehisa , Hattori Kunihiro , Shima Midori , Nogami Keiji 
Published: October 2019

Abstract
Background : Factor VIII (FVIII) is protected by binding to its carrier protein, von Willebrand factor (VWF) in the circulation. VWF contributes to hemostasis particularly under high shear flow condition by extending its multimeric configuration. By contrast, coagulation factors including FVIII are thought to play a dominant role under low shear. Since FVIII binds to VWF unlike other coagulation factors, FVIII may contribute to thrombus formation even under high shear conditions. FVIII has to be released from VWF and activated to exert its coagulation function. However, the role of interaction between FVIII and VWF on hemostasis under flow conditions needs to be explored in addition to the protective function of VWF. Aims: To analyze the mechanism and role of FVIII and VWF interaction on hemostasis under flow conditions. Methods: Whole blood samples were obtained from patients with type 2A von Willebrand disease (VWD). FVIII, VWF, FVIII/VWF (final concentration; f.c. 1U/mL), FVIII plus ESH8 (f.c. 1 µM), and emicizumab (ACE910; f.c. 100 µg/mL) were added to the blood followed by perfusion into the collagen-coated flow chamber under controlled high (2,500s-1) and low (50s-1) shear conditions. ESH8 is an anti-FVIII antibody that interrupts the release of FVIII from VWF, and emicizumab is a bispecific antibody mimicking activated FVIII (FVIIIa). The concentration of emicizumab was chosen, as it was the highest dose in clinical study. After the perfusion, formed thrombus was fixed and immunostaining was performed to visualize platelets, VWF, thrombin, and FVIII. Thrombi were observed by using confocal laser scanning microscopy and the obtained images were analyzed by Image Pro Premier 3D. Initial thrombus formation was measured as surface coverage (SC) and thrombus development was measured as thrombus volume (TV). Results: SC and TV were impaired in VWD particularly under high shear (high: SC 8.3%, TV 3.9x103μm3, low: SC 2.7%, TV 2.9x103μm3). Addition of FVIII/VWF improved thrombus formation to normal level under both shear conditions (high: SC 37.3%, TV 2.4x104μm3, low SC 4.3%, TV 4.1x103μm3). Addition of VWF improved SC and TV under both shear conditions (high: 40.9%, 1.7x104μm3, low: SC 3.0%, TV 3.3x103μm3), suggesting that the VWF function was crucial under both shear. By contrast, addition of FVIII alone did not improve SC and TV under both shears (high: SC 9.9%, TV 5.0x103μm3, low: SC 2.4%, TV 3.1x103μm3). Since FVIII/VWF had a greater effect on TV than VWF alone, FVIII enhanced thrombus development under high shears, however, this effect required the presence of VWF. FVIII immunostaining demonstrated the binding of FVIII on platelet surface in the FVIII-added experiment and, therefore, FVIII binding alone was not sufficient to initiate coagulation. Since FVIII may need to be activated on the platelet surface in a timely fashion, it was speculated that the optimal delivery from VWF and activation of FVIII on platelets was required for hemostasis. To analyze the role of FVIII and VWF interaction on hemostasis under flow condition, ESH8 was used to modify FVIII/VWF binding by blocking FVIII release. Treatment of FVIII/VWF with ESH8 did not change SC but slightly impaired TV under high shear (high: SC 41.8%, TV 1.8 x104μm3). Thus, the presence of FVIII at thrombus was not enough and the release of FVIII from VWF and presumably activation of FVIII on the platelet surface might be essential for thrombus formation under high shear. Under low shear, both SC and TV were interrupted by ESH8 (SC 2.6%, TV 3.1x103μm3), suggesting the important role of FVIII and coagulation under low shear. Lastly, to analyze the impact of activated FVIII on thrombus formation under flow conditions, emicizumab was evaluated in this system. In contrast to FVIII, emicizumab alone improved thrombus formation (high: SC 26.0%, TV 1.1 x104μm3, low: SC 3.5%, TV 4.2 x103μm3). The rate of improvement in SC and TV was better under low shear than high shear, which implicated the coagulative role of emicizumab. Even under high shear, however, emicizumab enhanced thrombus formation. Therefore, the role of emicizumab in substituting for the activated form of FVIII might be able to rapidly initiate coagulation even under high shear. Conclusion: VWF delivers and releases FVIII in a timely fashion on platelet surface. FVIII has to become active on platelets for hemostasis and FVIIIa can accelerate thrombus formation even under high shear condition.

Disclosures
Yaoi: Chugai Pharmaceutical Co., Ltd.: Research Funding. Shida:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kitazawa:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties; Sysmex Corporation: Patents & Royalties. Hattori:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties. Shima:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Nogami:F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.