Elevated function of blood clotting factor VIIa mutants that have enhancedaffinity for membranes - Behavior in a diffusion-limited reaction

Blood clotting factor VIIa is involved in the first step of the blood coagu lation cascade, as a membrane-associated enzyme in complex with tissue fact or (TF). Factor VIIa is also an important therapeutic agent for hemophilia where its function may include TF-independent as well as TF-dependent mecha nisms. This study compared the activity of wild type factor VIIa (WT-VIIa) with that of a mutant with elevated affinity for membrane (P10Q/Q32E, QE-VI Ia). Phospholipid and cell-based assays showed the mutant to have up to 40- fold higher function than WT-VIIa in both TF-dependent and TF-independent r eactions. Tissue factor-dependent reactions displayed the maximum enhanceme nt when binding had reached equilibrium in competition with another TF-bind ing protein. In liposome-based assays, the association rate of WT-VIIa with TF occurred at a physical maximum and could not be improved by site-direct ed mutagenesis. A practical consequence was identical function of WT-VIIa a nd QE-VIIa in assays that depended entirely on assembly kinetics. Thus, fac tor VIIa mutants provided unique reagents for probing the mechanism of fact or VIIa action. They may also offer superior agents for therapy.