Reaction of Factor VIIa (VIIa) with Plasma Inhibitors Ex Vivo and In Vivo.

Activated proteases are typically short-lived in the circulation where they react with plasma inhibitors and are removed as complexes by scavenger receptors. In contrast VIIa is characterized by a relatively long in vivo clearance half-life of 2–3 hr. Free VIIa exists in a latent state that is fully activated only upon binding to tissue factor (TF). Ex vivo VIIa is described to react predominantly with anti-thrombin (AT) in a slow reaction enhanced by heparin and by TF. Free VIIa is assumed to be rather inert towards plasma inhibitors also in vivo, and this may account for its slow clearance. Still it is not known whether the reaction with AT or other plasma inhibitors is an obligatory step in the in vivo clearance of VIIa. To clarify this question we studied the interaction between VIIa and plasma inhibitors in human and murine serum and plasma, and also studied this interaction during in vivo clearance of VIIa in mice. The VIIa-inhibitor complexes formed after spiking of serum with VIIa for 24 h were purified by affinity chromatography and subjected to SDS-PAGE and characterized by MALDI analysis. In the absence of heparin we identified VIIa complexes with alpha 2-macroglobulin (alpha 2M), AT, alpha 2-plasmin inhibitor and inter-alpha-trypsin inhibitor with the complex with alpha 2M as the most prominent component, whereas the AT complex was pre-dominant in the presence of heparin. The interaction between VIIa and plasma inhibitors might be differently affected by contact with the vasculature in the circulation. We therefore next studied VIIa complex formation in vivo in mice after i.v. injection of ¹²⁵I-VIIa. Complex formation was followed by SDS PAGE and autoradiography, and clearance was followed by measurements of the VII antigen level. The data suggest that alpha 2M and AT complexes are formed in vivo, however, without affecting VIIa antigen clearance. This was confirmed by studies of in vivo clearance of VIIa or a preformed VIIa-AT complex. Measurements of VIIa clot activity, and of VII- and VIIa-AT antigen concentrations suggested

• that in vivo clearance of VIIa from circulation does not include complex formation with a plasma inhibitor as an obligatory intermediate,

• that the VIIa-AT complex is cleared with the same rate as VIIa and

• that a transient VIIa-AT complex is formed subsequent to VIIa bolus injection which may account for a faster clearance of VIIa when measured by clot activity relative to VII clearance when this is measured by antigen concentration.