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Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor
B B Sorensen1, E Persson, P O Freskgârd
1Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Niels Steensens Vej 1, DK-2820 Gentofte, Denmark. bbsn@novo.dk
Abstract:
Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo and in vivo model systems. These studies suggest that inactivated factor VIIa competes efficiently with plasma factor VII(a) for a limited number of tissue factor sites. In the present study, we compared the interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor. Competition studies of factor VIIa and active site-inhibited factor VIIa in a factor X activation assay showed that the affinity of the latter for relipidated tissue factor was 5-fold higher than that of factor VIIa. Radioligand binding studies with a human bladder carcinoma cell line (J82) and surface plasmon resonance studies using soluble tissue factor demonstrated a faster association and a slower dissociation for the active site-inhibited factor VIIa. Studies of equilibrium binding to cell surface tissue factor showed that the affinity of active site-inhibited VIIa was 5-fold higher than that of factor VIIa to non-functional tissue factor sites, whereas both inactivated factor VIIa and factor VIIa bound to functional tissue factor sites with the same high affinity. Comparison of the CD spectra of factor VIIa and active site-inactivated factor VIIa revealed structural differences in the protease domain. The potential physiological implications of these findings are discussed.
Insights
Active site-inhibited factor VIIa shows higher affinity for tissue factor than factor VIIa, impacting thrombus formation. This suggests a potential therapeutic role for inactivated factor VIIa in controlling coagulation.
Area of Science:
- Biochemistry
- Hematology
- Pharmacology
Background:
- Factor VIIa plays a crucial role in the coagulation cascade.
- Active site-inhibited factor VIIa has shown potential in blocking thrombus formation.
- Understanding the interaction between factor VIIa, its inhibitors, and tissue factor is vital for hemostasis research.
Purpose of the Study:
- To compare the binding affinities and interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor.
- To investigate the structural differences between factor VIIa and active site-inhibited factor VIIa.
- To explore the physiological implications of these interactions in coagulation.
Main Methods:
- Factor X activation assays to assess catalytic activity and binding affinity.
- Radioligand binding studies using a human bladder carcinoma cell line (J82).
- Surface plasmon resonance (SPR) using soluble tissue factor.
- Equilibrium binding studies to cell surface tissue factor.
- Circular Dichroism (CD) spectroscopy to analyze structural differences.
Main Results:
- Active site-inhibited factor VIIa exhibited a 5-fold higher affinity for relipidated tissue factor compared to factor VIIa.
- Active site-inhibited factor VIIa demonstrated faster association and slower dissociation rates with tissue factor.
- While both forms bound functional tissue factor sites with high affinity, active site-inhibited factor VIIa showed significantly higher affinity for non-functional tissue factor sites.
- CD spectra revealed structural differences in the protease domain between factor VIIa and active site-inhibited factor VIIa.
Conclusions:
- Active site-inhibited factor VIIa binds tissue factor with higher affinity than factor VIIa, primarily due to enhanced binding to non-functional sites.
- Structural differences in the protease domain contribute to the altered binding characteristics.
- These findings suggest that active site-inhibited factor VIIa may have distinct physiological roles and therapeutic potential in modulating coagulation and thrombosis.