Identification of a Factor IXa-Interactive Site within the C2 Domain of Factor VIIIa and a Crucial Role of These Associations for the Process of Clot Formation (2008)

Abstract Factor VIIIa functions as a cofactor for the factor IXa in the membrane-dependent conversion of factor X to factor Xa. We recently have reported the C2 fragment (residues 2182–2259) of factor VIIIa directly interacts with the Gla domain of factor IXa via both the electrostatic- and calcium-dependent interactions and this association plays a significant role in the factor Xase complex (Blood2007; 110, 2687). In this study, we further localized a factor IXa-interactive site within the 2182–2259 region of C2 domain. The competitive binding assays in ELISA using several overlapping synthetic peptides encompassing residues 2182–2259 demonstrated that one peptide 2228–2240 (EWLQVDFQKTMKV; C22228–2240), supposed to be exposed on the molecular surface according to the crystal structure of FVIII, significantly inhibited the binding of active-site modified EGR-factor IXa to the recombinant C2 domain (residues 2169-2332) by ~80% (IC50; ~400 μM), whilst a control peptide, comprising the 2228-2240 residues in a random sequence, failed to inhibit. This peptide did not affect both bindings of factor VIII and factor IXa to phospholipid. The addition of C22228–2240 inhibited the factor VIIIa/factor IXa-mediated factor X activation in the presence of phospholipid dose-dependently (IC50; ~10 μM), suggesting that residues 2228–2240 of the C2 domain significantly contribute to the interaction with the Gla domain of factor IXa. The amino acid sequences were well-conserved, independently of species. Of note, this inhibitory effect was much greater than those obtained by the 484–509 peptide and 1804–1818 peptide, corresponding to other factor IXa-interactive sites (IC50; ~60 and ~180 μM, respectively). Furthermore, we studied the inhibitory effect of C22228–2240 on the blood coagulation quantitatively using both the clot-waveform analyses by a photo-optical automated coagulation analyzer and thromboelastography. This peptide (500 μM) slightly prolonged the activated partial thromboplastin time (APTT) by ~1.3-fold. The maximum coagulation velocity (min1) and maximum coagulation acceleration (min2) were significantly reduced (by ~95%) by the addition of peptide, whilst the prothrombin time (PT) was little affected. A thromboelastography revealed that the C22228–2240 prolonged the value of clot formation time and decreased α angel and maximal clot firmness in dose-dependent manners. These inhibitory effects obtained by both assays were equivalent to those of representative anticoagulants, low weight molecular heparin (at ~0.3 IU/ml) and fondaparinux (at ~0.5 IU/ml). Of interest, this peptide showed little effect on the precoagulation phase (clotting time) in both assays. These data indicated that C22228–2240 possessed an anticoagulant effect for the process of clot formation. In conclusion, we indentified a factor IXa-interactive site within a 2228–2240 region of the C2 domain of factor VIIIa. Furthermore, these interactions on the factor Xase assembly likely play an essential role for propagation of intrinsic coagulation.