Interactions of Factor VIII with Tissue Factor Contributes to the Acceleration of Factor Xa Generation in the Initiation Phase of Blood Coagulation.
Authors: Ogiwara Kenichi , Nogami Keiji , Okuda Masahiro , Nishiya Katsumi , Takeyama Masahiro , Shima Midori 
Published: October 2019

Abstract
Abstract 3177
Poster Board III-116
Activated factor (F)VII complex with tissue factor (FVIIa/TF) initiates the blood coagulation by generating FXa as extrinsic Xase complex (ex-Xase). Although FVIIa/TF also activates FIX, FIXa little functions without its cofactor, FVIIIa. A tiny amount of thrombin generated by FXa activates FV and FVIII, followed by forming of intrinsic Xase complex (in-Xase) and prothrombinase complex, respectively. These formations result in ‘thrombin burst’ and successful hemostasis. Although thrombin is thought to be a unique potent activator of FVIII in vivo, FXa and FVIIa/TF also activate FVIII in vitro. We have recently reported the detailed mechanism by which FVIIa/TF activated FVIII more rapidly in early timed-phase than thrombin (Blood Abst.1036, 2008). In this study, we further developed to examine whether TF affected FVIII(a) function. (1) FVIIa/TF rapidly increased FVIII activity by 4.7-fold of initial in the presence of Ca2+ and phospholipid (PL), following by inactivation, in one-stage clotting assay. However, since even in the presence of TF alone, FVIII activity elevated by 1.8-fold of initial, actual increase of FVIII activity by FVIIa/TF was 2.6-fold. A possibility that TF might bind to FVIIa contained in FVIII-deficient plasmas used, was negligible, since FVIIa-inhibitor used blocked an ex-Xase effect >95%. In the presence of FVIIa-inhibitor, residue FVIII activity with TF was ∼50%, thus TF alone affected FVIII cofactor activity independently of FVIIa. (2) Using SDS-PAGE, the addition of TF accelerated FVIII cleavage by FVIIa, whilst decelerated that by thrombin and FXa. (3) Surface plasmon resonance-based assays showed that FVIII(a) directly bound to TF with high affinity (Kd; ∼3 nM). (4) The effect of FVIIa/TF on in-Xase was evaluated in FXa generation assay. 0.1 nM FVIIa/TF, 1 nM FVIII, 90 nM FIX and 20 μM PL were reacted with 150 nM FX at various combinations. FVIIa/TF and FVIIa/TF/FVIII/FIX generated FXa with 3.9 and 10.4 nM/min, respectively. When FVIIa-inhibitor was added prior to addition of FX, FXa generated by FVIIa/TF and FVIIa/TF/FVIII/FIX were 5% and 46% (0.2 and 4.8 nM/min) of those without FVIIa-inhibitor, respectively. The latter was considered as FXa generated by in-Xase. Therefore, FXa derived from in-Xase was ∼40% of total FXa in this condition. (5) FVIIIa/FIXa (1 nM/2 nM)-dependent FXa generation in the presence of TF was evaluated. FXa generation in the presence of TF (0.02 and 0.3 nM) increased by ∼2 and ∼6-folds, respectively, of that in its absence. Furthermore, the functional affinity of FVIIIa for in-Xase complex in the presence of TF (0.1 nM), showed an ∼1.5-fold greater than that in its absence (Km; 4.9 ± 0.4 and 7.1 ± 0.9 nM, respectively). In conclusion, FVIIa/TF can generate FVIIIa in early timed-phase in vitro as well as FXa and FIXa, and possess potential of forming in-Xase. In addition, TF directly binds to FVIII(a), and functions in-Xase complex more efficiently by enhancing the affinity of FVIIIa for in-Xase. Although TF-dependent these reactions may be terminated rapidly via anticoagulant systems such as tissue factor pathway inhibitor, our data suggest that interactions of FVIII with TF might contribute to the acceleration of FXa generation in the initiation phase of blood coagulation.

Disclosures
Okuda: Sysmex Corporation: Employment.