Mechanism of Tissue Factor (TF) Enhancing Factor (F)VIII Activity on FXa Generation in Initial Phase of Coagulation and Interaction Between TF and FVIII C2 Domain
Authors: Shoko Furukawa , Nogami Keiji , Ogiwara Kenichi , Shima Midori 
Published: November 2019

Abstract
In the cell-based coagulation model, factor (F)VIIa complex with tissue factor (TF) initiates the blood coagulation by generating FXa as the extrinsic tenase complex and activates FIX which composes the intrinsic tenase complex. We demonstrated that FVIIa/TF directly activated FVIII in an early coagulation phase (Soeda, JTH, 2010), and TF enhanced the intrinsic tenase activity via possible interaction with FVIIIa (Ogiwara, ASH, 2010). In this study, we clarified the enhancing mechanism of intrinsic tenase activity in the TF-related up-regulation of FVIII, and identified the TF-interactive region on FVIII.
To explore the enhancing mechanism of TF for FVIII regulation, we performed the FXa generation assay with various amounts of FVIII or thrombin-mediated FVIIIa, constant FIXa (1 nM), FX (300 nM) and phospholipid vesicles (PL; 20 µM) in the presence of recombinant lipidated TF (rTF, Innovin®). The Km value for FVIII in the presence of rTF was ~2.4-fold lower than that its absence (Km; 5.6±0.5, 13.3±3.7 nM, respectively, p<0.05). Similarly, the Km for FVIIIa in the presence of TF was ~1.5-fold lower than that in its absence (Km; 6.3±0.6 nM, 9.7±1.6 nM, respectively, p<0.05), supporting that the presence of TF could promote the FXa-catalyzed activation of FVIII and FVIIIa-dependent generation of intrinsic FXa. To further evaluate the effect of TF on FVIII-dependent FXa generation, the FXa generation assay with FVIIa/TF-activated FVIIIa (FVIIIa-VIIa/TF) was also performed. The initial velocity on FXa generation with FVIIIa-VIIa/TF was 22.6 nM/min. However, the initial velocity on FXa generation with FVIIIa-VIIa/TF by addition of FVIIa-inhibitor (E-76), not to generate FVIIa/TF-dependent FXa, was 3.4 nM/min, and that with FVIII alone was 0.05 nM/min. In addition, the initial velocity with FVIIa/TF alone was 10.4 nM/min. These findings supported that the TF increased FXa generation greater than the additive effect of FVIII-dependent and FVIIa/TF-dependent FXa generation in early initiation phase of coagulation prior to thrombin generation. Since tissue factor pathway inhibitor (TFPI) is present in physiological circulating whole blood, a similar experiment on FXa generation assay was repeated under the presence of TFPI, estimated to be present at 0.5 nM in the pre-coagulant state or at 15 nM in the coagulant state in circulating blood. The initial velocity on FXa generation with FVIIIa-VIIa/TF was reduced by the presence of 0.5 or 15 nM TFPI (17.9 and 12.6 nM/min, respectively). By contrast, the initial velocity on FVIIIa-VIIa/TF-dependent FXa generation with addition of FVIIa inhibitor was little reduced by 0.5 nM TFPI, whilst was reduced by 15 nM TFPI (3.5 and 2.5 nM/min, respectively). These findings supported that the TFPI possibly didn't inhibit TF on the enhanced intrinsic tenase on association with FVIII in the pre-coagulant state.
We further reported that TF enabled FXa to activate FVIII, irrespective of von Willebrand factor (VWF), and the direct association of rTF and non-lipidated TF with FVIII (Furukawa, ISTH, 2015). Since TF is transmembrane protein, however, we performed a surface plasmon resonance (SPR)-based assay (BIAcore®) and solid phase-based ELISA to identify the interactive region(s) on FVIII to recombinant soluble TF (sTF; Altor BioScience), a portion of TF outside of the PL membrane. An SPR-based assay revealed the direct binding of intact FVIII, LCh (a3-A3C1C2, A3C1C2) subunit, C2 domain to immobilized sTF (Kd; 2.3±0.6, 5.8±1.0, 10.5±3.5, 11.8±0.5 nM, respectively). The intact HCh, A1 or A2 domain to sTF failed to bind, however. A non-equilibrium ELISA also revealed that sTF bound to immobilized C2 domain with moderate affinity (Kdapp; 16.9±2.2 nM), and the interaction was dependent on ionic strength and Ca2+. In addition, the presence of VWF significantly competitively inhibited the C2 and sTF binding by ~90% (IC50; 5.7 µg/ml) at the maximal concentration employed, suggesting that the C2 domain-TF interaction could activate FVIII by FXa even in the presence of VWF.
We concluded that it might be possible that TF enhanced the FVIII-mediated FXa generation by not only FVIIa but also FXa, additionally this enhancing mechanism might not be suppressed by TFPI in the initiation phase of coagulation. Furthermore, TF might function to FVIII activation, irrespective of presence of VWF, by the binding to C2 domain through the competition with VWF.

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