To investigate systemic serotonin (5-HT) metabolism in migraine, we determined platelet and platelet-free plasma concentrations of 5-HT, its precursors tryptophan and 5-hydroxytryptophan, and its main metabolite 5-hydroxyindoleacetic acid (5-HIAA), as well as the activities of the platelet enzymes monoamine oxidase and phenolsulfotransferase in classic and common migraineurs. Between attacks, migraineurs had lower plasma 5-HT and higher 5-HIAA levels than did healthy controls and patients with tension headache. During migraine attacks, plasma 5-HT levels were substantially higher than during attack-free periods, while 5-HIAA concentrations and platelet enzyme activities were lower. Platelet 5-HT was reduced only during common, but not classic, migraine attacks. We hypothesize that systemic 5-HT metabolism is enhanced in migraineurs during headache-free periods and transiently decreases during attacks, presumably due to a fall in enzymatic degradation. Furthermore, platelet behavior differs during migraine attacks with and without aura, and release of platelet 5-HT cannot (exclusively) be held accountable for the rise of plasma 5-HT during migraine attacks.

No abstract

The importance of substrate residues P2' and P3' on thrombin catalysis has been investigated by comparing the hydrolysis of a series of fluorescence-quenched substrates. Each consisted of a 10-residue peptide, carrying a 2-aminobenzoyl (Abz) group at the N-terminus, and a penultimate 2,4-dinitrophenyl (Dnp) derivatized lysine. Cleavage of such a peptide relieves the intramolecularly-quenched fluorescence, allowing determination of the kinetic parameters. The nature of the P2' residue was found to have a major influence on the rate of cleavage: the Kcat/Km value for the hydrolysis of the Arg-Ser bond in Abz-Val-Gly-Pro-Arg-Ser-Phe-Leu-Leu-Lys(Dnp)-Asp-OH was nearly 3 orders of magnitude higher than that for the hydrolysis of the same substrate with aspartate instead of phenylalanine at the P2' position. Comparatively, the P3' side chain was less important: the kcat/Km value for the substrate with the least effective residue (aspartate) was only 33 times lower than that of the substrate with the most favorable amino acid (lysine). The role of thrombin residues Arg35, Lys36, Glu39 and Lys60f in the putative P2' and P3' binding sites was also examined. Replacement of Lys60f by glutamine improved the rate of cleavage for peptides with P2' lysine or leucine. Compared with thrombin, mutants E39K and E39Q hydrolyzed faster substrates with an acidic residue in P2' or P3', but slightly slower those with a lysine at either position. Mutations R35Q and K36Q only improved the hydrolysis of substrates with an acidic P2' residue. Overall, thrombin prefers bulky hydrophobic side chains in subsite S2' and positively charged residues in S3', whereas acidic residues are markedly antagonistic to both subsites.

For use as an antithrombotic agent, a thrombin inhibitor must be potent and specific, i.e., it should not significantly inhibit the proteases of the anticoagulation (activated protein C) and fibrinolytic systems (plasminogen activator and plasmin). Previous evaluation of potency and specificity has been based on inhibition constants (Ki values). However, consideration of the kinetic parameters for natural plasma serine protease inhibitors indicates that a low Ki value with thrombin is not sufficient; the inhibited complex must also form rapidly. Moreover, potent inhibition of activated protein C and plasmin could be tolerated providing the inhibited complex only forms slowly. An ideal profile of kinetic parameters with thrombin, activated protein C and plasmin is formulated and discussed in relation to various classes of thrombin inhibitors. Examination of kinetic data for thrombin inhibitors currently in clinical trials (hirudin and hirulog) indicates that they possess this ideal profile of kinetic parameters.