Interactions between residues 2228–2240 within factor VIIIa C2 domain and factor IXa Gla domain contribute to propagation of clot formation^[*]

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

* An account of this work was presented at the 50th annual meeting of the American Society of Hematology, 2008, San Francisco, CA, USA. This work was supported by grants for MEXT KAKENHI 21591370.

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