Decline in Postmortem Serum Tryptase Levels in Anaphylactic Deaths

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To the Editor:
We read the article by Sravan et al1 published in the American Journal of Forensic Medicine and Pathology with considerable interest.1
In this article, the authors described a case of fatal anaphylaxis in which 2 separate tryptase determinations were performed in blood samples taken from the same sampling site (femoral vein) but 24 hours apart. The 2 measurements revealed a substantial decrease in tryptase from 130 μg/L at day 2 postmortem to 84.8 μg/L at day 3.
The authors hypothesized that this decline might be the consequence of molecule degradation and therefore recommended that early blood sample be obtained as soon as possible after death in situations of suspected anaphylaxis because a significant downward trend in blood tryptase levels may be expected with increasing postmortem interval in anaphylactic deaths (in contrast to the increasing trend observed by other authors in nonanaphylactic cases).
We found the conclusions made by Sravan et al1 to be of utmost interest and deserving of some comments.
Firstly, human mast cell (βII)-tryptase stored in the secretory granules of mast cells is a tetrameric, neutral serine protease with a molecular weight of 140 kDa, made up of 4 identical subunits of 30 to 36 kDa. The precursor βII-preprotryptase is converted to the βII-protryptase by removing the 18–amino acid signal peptide. The further activation of βII-protryptase involves 2 proteolytic steps: the first is an autocatalytic intermolecular cleavage that occurs optimally at acidic pH and in the presence of heparin or dextran sulfate. The resulting product is a monomer (β-pro'tryptase), which is approximately 50 times less active than the final tetramer.
The second step involves the removal of the remaining precursor dipeptide by the enzyme dipeptidyl peptidase I, which functions at acidic pH values, to form the mature monomer. In the presence of heparin, the monomers come together on a heparin backbone to form an active tetramer. These events probably take place in the Golgi and post-Golgi secretory progranules in mast cells. The mature, heparin-stabilized, packaged β-tryptase is then stored until mast cell activation causes granular content release.2–4 When tryptase is released, it is secreted as a large-sized active proteoglycan complex (200–250 kDa) that limits diffusion away from mast cell activation sites.5
It has been observed that active tryptase tetramer is stabilized by heparin proteoglycan and other polymers with high anionic charge densities. Indeed, tryptase binds to heparin or other proteoglycans through its cationic groove. In the absence of heparin, the tryptase tetramer rapidly loses activity in a process that is accompanied by tetramer dissociation into inactive monomers. The exact mechanism for the inactivation/regulation of tryptase activity is unknown. The tetrameric structure of β-tryptase with the active site of each of the 4 monomers oriented toward the inner face of a central pore makes it resistant to inactivation by endogenous protease inhibitors of serine proteases. The most probable regulation mechanism is postulated as spontaneous β-tryptase inactivation associated with destabilization/conformational molecule changes and slow β-tryptase dissociation from heparin proteoglycan. This dissociation would be promoted by the neutral pH to which β-tryptase is exposed after its release from mast cells and the consequent, weaker affinity for heparin proteoglycan, mediated by basic proteins such as antithrombin III.2,5,6
β-Tryptase determination as a support of the clinical diagnosis of anaphylaxis has been emphasized as highly valuable if correctly performed and completely useless if blood collection timing is inappropriate. Two elements must be considered for informative serum β-tryptase determination as an anaphylactic marker: the comparison of each patient's peak serum β-tryptase level with his/her own baseline β-tryptase level and the knowledge of serum β-tryptase kinetics.

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