Nucleosome-releasing factor: a new role for factor VII-activating protease (FSAP)

Area of Science:

• Biochemistry
• Cell Biology
• Immunology

Background:

• Plasma proteins, including early complement components and IgM, aid in clearing late apoptotic or secondary necrotic (sn) cells.
• A previously identified plasma protease, inhibited by aprotinin, is essential for removing nucleosomes from sn cells.
• Plasmin exhibits nucleosome-releasing factor (NRF) activity, but NRF activity persists in plasma even after plasminogen depletion, suggesting another protease is involved.

Purpose of the Study:

• To purify and identify the specific protease responsible for NRF activity in plasma.
• To characterize the role of this protease in the removal of nucleosomes from sn cells.
• To investigate the potential function of this protease in maintaining cellular homeostasis.

Main Methods:

• Size-exclusion and anion-exchange chromatography were employed to purify the NRF protease from plasma.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze protein bands with NRF activity.
• Sequence analysis identified the protein band as factor VII-activating protease (FSAP).
• Monoclonal antibodies against FSAP were developed to assess their inhibitory effect on NRF activity.
• Affinity chromatography was utilized to purify single-chain FSAP (scFSAP) from recalcified plasma (r-plasma).

Main Results:

• Chromatographic purification and SDS-PAGE identified FSAP as the protein responsible for NRF activity.
• Monoclonal antibodies targeting FSAP completely inhibited NRF activity in plasma.
• Purified single-chain FSAP (scFSAP) demonstrated efficient removal of nucleosomes from sn cells.
• FSAP was identified as the key protease responsible for nucleosome release from sn cells.

Conclusions:

• Factor VII-activating protease (FSAP) is the primary protease responsible for releasing nucleosomes from secondary necrotic cells.
• FSAP plays a significant role in cellular homeostasis by facilitating the clearance of nucleosomes from sn cells.
• The findings identify FSAP as a critical component in the innate immune system's response to cellular damage and debris.