Alpha 2-macroglobulin binds and inhibits activated protein C

Area of Science:

• Biochemistry
• Hematology
• Proteomics

Background:

• Previous studies identified a high molecular weight complex of activated Protein C (APC) with an unknown binding protein in primate models.
• This complex formation was also observed in vitro in human plasma.

Purpose of the Study:

• To identify the unknown APC binding protein.
• To characterize the interaction between APC and the binding protein.
• To determine the physiological relevance of this interaction.

Main Methods:

• Purification of the APC binding protein from human plasma using a multi-step chromatographic approach.
• Identification of the purified protein using SDS-PAGE and immunochemical methods.
• Characterization of complex formation using EDTA, citrate, and active site inhibitors (PPACK).
• Assay of APC anticoagulant activity following incubation with alpha 2M.

Main Results:

• The purified APC binding protein was identified as alpha 2-macroglobulin (alpha 2M).
• Complex formation between alpha 2M and APC was inhibited by EDTA and, to a lesser extent, by citrate.
• Active site inhibition of APC or methylamine treatment of alpha 2M prevented complex formation.
• Incubation of APC with alpha 2M led to time-dependent inhibition of APC's anticoagulant activity.
• In vitro formed complexes comigrated with in vivo complexes.

Conclusions:

• Alpha 2-macroglobulin (alpha 2M) binds and inhibits activated Protein C (APC) in vitro.
• The interaction is dependent on metal ions and the active site of alpha 2M.
• Alpha 2M is suggested to be a physiologically relevant inhibitor involved in APC processing in vivo.