Thrombin induces broad spectrum proteolysis in human serum samples.

Area of Science:

• Biochemistry
• Proteomics
• Enzymology

Background:

• Thrombin cleaves fibrinogen to release fibrinopeptide A (FPA) during clotting.
• FPA fragments in serum, analyzed by MALDI-TOF MS, show time-dependent variations.
• Elevated serum proteolytic activity, potentially from thrombin, suggests broader enzymatic roles.

Purpose of the Study:

• To investigate the proteolytic and peptidolytic activity in serum.
• To identify thrombin-susceptible proteins beyond fibrinogen.
• To understand the contribution of coagulation enzymes to serum protein destabilization.

Main Methods:

• Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for FPA fragment analysis.
• Incubation of serum to observe time-dependent changes in FPA fragments.
• Extrinsic addition of thrombin to plasma proteins (3-30 kDa) to induce and analyze proteolysis.

Main Results:

• Observed multiple, progressively shorter fragments of serum FPA.
• Identified widespread digestion of non-coagulation proteins by extrinsic thrombin.
• Confirmed thrombin digestion of hemopexin, demonstrating a broader substrate range.

Conclusions:

• Serum exhibits broad proteolytic activity, degrading proteins beyond the typical fibrinogen cleavage site.
• Thrombin demonstrates a lack of bias for specific amino acids following R/K residues in cleavage sites.
• Coagulation enzymes, particularly thrombin, contribute to the destabilization of serum protein and peptide content.