Thrombin induces broad spectrum proteolysis in human serum samples
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Patrick O'Mullan
Abstract
Background: During clotting, α 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 α 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 α thrombin.
Methods: Extrinsic addition of α thrombin to a subset (3–30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal α thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by α thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion.
Results: Extrinsic addition of α 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 α 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.
©2009 by Walter de Gruyter Berlin New York
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Articles in the same Issue
- Editorial
- A focus on recent advances in proteomics – one step closer to entrance into the clinical arena
- Reviews
- Proteomics in protein misfolding diseases
- Multidimensional protein identification technology for clinical proteomic analysis
- Technical advances in proteomics mass spectrometry: identification of post-translational modifications
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- Research Papers
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- Proteomics of exhaled breath: methodological nuances and pitfalls
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- Increased CA 19-9 level in patients without malignant disease
- General Clinical Chemistry and Laboratory Medicine
- Quantitation of cerebrospinal fluid lactic acid in infectious and non-infectious neurological diseases
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- Reference Values and Biological Variations
- Influence of tourniquet application on venous blood sampling for serum chemistry, hematological parameters, leukocyte activation and erythrocyte mechanical properties
- Systemic cytokine response following exercise-induced muscle damage in humans
- Letters to the Editor
- CACNA1A gene non-synonymous single nucleotide polymorphisms and common migraine in Italy: a case-control association study with a micro-array technology
- Proficiency test of plasma free and total metanephrines: report from a study group
