Summary
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.
Keywords
Factor VIIIa C2 domain - Factor IXa Gla domain - peptide 2228–2240 - anticoagulant
effect - propagation of clot formation