Effects of Anti-FVIII Inhibitors On Factor VIIa/Tissue Factor-Catalyzed Activation and Inactivation of Factor VIII.
Authors: Yada Koji , Nogami Keiji , Ogiwara Kenichi , Nishiya Katsumi , Takeyama Masahiro , Shim Midori 
Published: October 2019

Abstract
Abstract 3169
Poster Board III-110
Factor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. We have recently demonstrated that FVIIa/TF rapidly activated FVIII by proteolysis of the heavy chain (HCh), and served physiologically as a potent activator for up-regulation of FVIII activity in very early-timed phase (ASH #1036, 2008). FVIII inhibitors develop as alloantibodies in multi-transfused patients with hemophilia A and also arise as autoantibodies in normal individuals. FVIII inactivation by inhibitors is associated with impairment of FVIII(a) cofactor function through the binding to functional crucial epitopes in FVIII. Anti-C2 inhibitors prevent FVIII binding to phospholipid, von Willebrand factor, and FXa. Anti-A2 inhibitors prevent FVIII binding to FIXa and thrombin. However, effects of these inhibitors on FVIIa action for FVIII have remained to be studied. In this study, we prepared 13 of anti-FVIII inhibitor IgGs (2 of anti-A2, 7 of anti-C2 with type 1 behavior, and 4 of anti-C2 with type 2). We first examined FVIIa/TF-catalyzed FVIII activation in the presence of anti-FVIII inhibitors in one-stage clotting assay. The levels of FVIII activity (10 nM) elevated rapidly by ∼2.0-fold within 30 sec after adding of FVIIa/TF (1 nM), and subsequently decreased to the initial level within 20 min. The presence of anti-FVIII inhibitors did not significantly affect FVIIa/TF-catalyzed FVIII activation (by 1.7∼2.2-fold) compared to normal IgG. This action was independent of the difference of inhibitor epitopes. In addition, FVIIa-catalyzed FVIIIa inactivation with anti-A2 or anti-C2 with type 2 inhibitors was little affected, similar to that with normal IgG. However, of note, all of anti-C2 with type 1 significantly inhibited FVIIa-catalyzed inactivation of FVIIIa. Inactivation rates of FVIIa with anti-C2 with type 1 (k ∼0.15) was ∼40% less than that with control IgG (k ∼0.24), supporting that the presence of anti-C2 with type 1 might persist the activity of FVIIIa generated by FVIIa. To clarify this inhibitory mechanism of anti-C2 with type 1, we performed FVIIa-catalyzed FVIII cleavage in Western blotting. FVIIa/TF (1 nM) proteolyzed the HCh of FVIII (10 nM) rapidly by cleavages at Arg372 (and Arg740), whilst cleavage at Arg336 in the A1 domain was appeared at ∼2.5 min, supporting that cleavages at Arg372 and Arg336 by FVIIa contribute to the up- and down-regulation of FVIII(a) activity, respectively. All inhibitors, independent of recognizing epitopes, did not affect FVIIa-catalyzed cleavage at Arg372. However, the presence of anti-C2 type 1 delayed the cleavage at Arg336 in timed- and dose-dependent manners, whilst either anti-A2 or anti-C2 type 2 did not affect, consistent with the functional inactivation results. FVIIa binds to the A2, A3, and C2 domains in FVIII. Based on our findings, FVIIa-interactive sites on FVIII unlikely overlapped with anti-A2 and -C2 inhibitor epitopes, and inhibition of Arg336 cleavage may be due to conformational change caused by antibody binding. Furthermore, FVIIa indeed activates FVIII even in the presence of anti-FVIII inhibitors, different from thrombin, FXa, etc, and it would be important to predict the effect of FVIIa for FVIII to determine the characteristics of anti-FVIII inhibitors.

Disclosures
No relevant conflicts of interest to declare.