Thrombin induces broad spectrum proteolysis in human serum samples

Abstract

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. Clin Chem Lab Med 2009;47:685-93.

Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum

“…This correlates to earlier studies profiling proteolytic activities using internally quenched fluorogenic probes [32,33]. Different metabolic markers resulting from thrombin assisted protein degradation, e.g., FPA and its metabolites, provide some information about the coagulation progress and the proteolytic activity in serum and plasma [34,35]. Indeed, FPA was absent in blood immediately after sample collection and the quantities of FPA, FPA(2–16), and FPA(3–16) were different after one hour than in serum prepared accordingly.…”

Brain-Derived Neurotrophic Factor in Patients with Huntington's Disease

“…The aspect of protein instability is serum is well known to enzymologists and biochemists [29], [30]. Proteolytic enzymes in the clotting cascade may greatly, and randomly, affect the level of a given protein in serum samples.…”

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“…However, serum is a difficult matrix with high intrinsic proteolytic activity caused by different endoproteases e.g. from the coagulation cascade and the complement system [14,31,32] as well as a multitude of exoproteases [33]. Furthermore, the proteolytic profile in blood specimens is not only altered in malignant disease but also under non-malignant conditions e.g.…”