Mechanisms of factor VIIa-catalyzed activation of factor VIII.
BACKGROUND
Factor (F)VIIa, complexed with tissue factor (TF), is a primary trigger of blood coagulation, and has extremely restricted substrate specificity. The complex catalyzes limited proteolysis of FVIII, but these mechanisms are poorly understood.


OBJECTIVE
In the present study, we investigated the precise mechanisms of FVIIa/TF-catalyzed FVIII activation.


RESULTS
FVIII activity increased ~4-fold within 30 s in the presence of FVIIa/TF, and then decreased to initial levels within 20 min. FVIIa (0.1 nM), at concentrations present physiologically in plasma, activated FVIII in the presence of TF, and this activation was more rapid than that induced by thrombin. The heavy chain (HCh) of FVIII was proteolyzed at Arg(740) and Arg(372) more rapidly by FVIIa/TF than by thrombin, consistent with the enhanced activation of FVIII. Cleavage at Arg(336) was evident at ~1 min, whilst little cleavage of the light chain (LCh) was observed. Cleavage of the HCh by FVIIa/TF was governed by the presence of the LCh. FVIII bound to Glu-Gly-Arg-active-site-modified FVIIa (K(d), ~0.8 nM) with a higher affinity for the HCh than for the LCh (K(d), 5.9 and 18.9 nm). Binding to the A2 domain was particularly evident. Von Willebrand factor (VWF) modestly inhibited FVIIa/TF-catalyzed FVIII activation, in keeping with the concept that VWF could moderate FVIIa/TF-mediated reactions.


CONCLUSIONS
The results demonstrated that this activation mechanism was distinct from those mediated by thrombin, and indicated that FVIIa/TF functions through a 'priming' mechanism for the activation of FVIII in the initiation phase of coagulation.
Soeda
T
T
Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
Nogami
K
K
Matsumoto
T
T
Ogiwara
K
K
Shima
M
M
D001777:Blood Coagulation; D002384:Catalysis; D020134:Catalytic Domain; D002624:Chemistry, Clinical / Q000379:methods; D005169:Factor VIII / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000737:chemistry; D013925:Thromboplastin / Q000378:metabolism; D013997:Time Factors
D011994:Recombinant Proteins; D005169:Factor VIII; D013925:Thromboplastin; D015942:Factor VIIa; D013917:Thrombin