title: Interactions between residues 2228-2240 within factor VIIIa C2 domain and factor IXa Gla domain contribute to propagation of clot formation.
abstract: Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 domain (residues 2228-2240) interacts with the FIXa Gla domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228-2240. The peptide 2228-2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 μM). This effect was significantly greater than that obtained by peptide 1804-1818 (IC50; ~180 μM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228-2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228-2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 domain (residues 2228-2240) with the FIXa Gla domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.
authors.affiliation: Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Soeda
authors.forename: K
authors.initials: K
authors.lastname: Nogami
authors.forename: K
authors.initials: K
authors.lastname: Ogiwara
authors.forename: M
authors.initials: M
authors.lastname: Shima
mesh_terms: D001777:Blood Coagulation; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015949:Factor IXa / Q000737:chemistry / Q000378:metabolism*; D015944:Factor VIIIa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009363:Neoplasm Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011517:Prothrombin Time; D011994:Recombinant Proteins / Q000378:metabolism; D013916:Thrombelastography
chemical_list: D009363:Neoplasm Proteins; D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins; D015944:Factor VIIIa; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant