Thrombin induces broad spectrum proteolysis in human  serum samples.
BACKGROUND
During clotting, a thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of a thrombin by heating the serum sample appears to minimize this variation. These observations suggest that intrinsic proteolytic and peptidolytic activity is elevated in serum and perhaps originates from the coagulation cascade enzymes themselves, especially a thrombin.


METHODS
Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal a thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by a thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion.


RESULTS
Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins results in wide-spread digestion of proteins unrelated to coagulation, revealing a substrate range encompassing more than fibrinogen. Direct digestion of one of these proteins, hemopexin, by a thrombin confirms these observations.


CONCLUSIONS
The resulting peptides indicate broad tolerance beyond the consensus R-G cleavage site of fibrinogen; in fact, there appears to be no bias for the amino acid following the R/K residue. These data support our hypothesis that the enzymatic activities inherent to coagulation, or at least to thrombin, contribute to destabilization of the protein and peptide content of serum.
O'Mullan
Patrick
P
BD Diagnostics, Franklin Lakes, NJ 07417, USA. patrick_o'mullan@bd.com
Craft
David
D
Yi
Jizu
J
Gelfand
Craig A
CA
D000595:Amino Acid Sequence; D001777:Blood Coagulation / Q000502:physiology; D005340:Fibrinogen / Q000378:metabolism; D005344:Fibrinopeptide A / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006466:Hemopexin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010949:Plasma; D011480:Protease Inhibitors / Q000494:pharmacology; D055550:Protein Stability; D040901:Proteomics / Q000379:methods*; D044967:Serum; D013048:Specimen Handling; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013917:Thrombin / Q000378:metabolism / Q000502:physiology*
D010446:Peptide Fragments; D011480:Protease Inhibitors; D005344:Fibrinopeptide A; D005340:Fibrinogen; D006466:Hemopexin; D010447:Peptide Hydrolases; D013917:Thrombin