Incorrect threading of the sequence in the published structures of beta-Lg affects four of the nine beta strands. The basic lipocalin fold of the polypeptide chain is unchanged, however. The relative orientation of the monomers in the beta-Lg dimer differs in the two lattices. On raising the pH, there is a rotation of approximately 5 degrees, which breaks a number of intersubunit hydrogen bonds. It is not yet clear, however, why the stability of the structure should depend so heavily upon the external loop around residue 64 or the beta strand with the free thiol, each of which shows genetic variation.

Previous CD measurements of changes in the conformation of beta-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 degrees C. New CD measurements are reported at temperatures up to 80 degrees C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 degrees C with a ZnSe circle cell and a single simplified model of the substructure of the amide I' band was used to give the fractional contents of beta-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of beta-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 degrees C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 degrees C a partly unfolded state is formed, possibly by destabilization of the intermolecular beta-strand I and the loss of the main helix, but it is not a classical molten globule transition.

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We have previously suggested that the human homologue of the Drosophila transient receptor potential protein, TRPC1, is involved in conducting store-operated Ca2+ entry (SOCE) in human platelets since an antibody raised against the pore-forming region of TRPC1 inhibited SOCE. Here we have investigated plasma membrane expression of TRPC1 in human platelets and have probed for the presence of other TRPC proteins in these cells. Biotinylation revealed the presence of TRPC1 in the plasma membrane of resting platelets. Surface expression was not detectibly changed following Ca2+ store depletion or stimulation with thrombin. Western blotting demonstrated the presence of TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 in platelet lysates. TRPC1, TRPC4 and TRPC5 coimmunoprecipitated, as did TRPC3 and TRPC6. TRPC1, TRPC4 and TRPC5 were associated with detergent-resistant platelet membranes, from which they were partially released when the cells were cholesterol-depleted using methyl-beta-cyclodextrin. The distributions of TRPC3 and TRPC6 between soluble and membrane fractions were not affected by methyl-beta-cyclodextrin treatment. These results suggest that TRPC1, TRPC4 and TRPC5 form a heteromultimer associated with platelet lipid raft domains, whereas TRPC3 and TRPC6 associate independently of lipid rafts.

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To cite this article: Harper AGS, Brownlow SL, Sage SO. A role for TRPV1 in agonist-evoked activation of human platelets. J Thromb Haemost 2009; 7: 330-8.Summary. Background: Platelets play a role in a number of inflammatory conditions including atherosclerosis; however, the mechanisms of platelet activation under these conditions are unclear. Objectives: To investigate the presence of the vanilloid receptor, TRPV1, which is stimulated by noxious stimuli and by inflammatory mediators, in human platelets. Methods: Platelets loaded with fura-2 or sodium-binding benzofuran isophalate acetoxymethyl ester (SBFI) were used to monitor cytosolic calcium or sodium concentrations. 5-HT secretion was determined by fluorescence assay after conjugation with o-phthaldialdehyde. ATP secretion was determined using luciferin-luciferase. Results: TRPV1 was identified by Western blotting using a specific anti-hTRPV1 antibody. The TRPV1 agonist, capsaicin, evoked both Ca 2+ influx and Ca 2+release from intracellular stores, responses that were blocked in a dose-dependent manner by the TRPV1 antagonists, 5¢-Iodoresiniferatoxin (5¢-Iodo-RTX) and AMG 9810. Capsaicin also increased platelet cytosolic [Na + ]. Capsaicin-evoked Ca 2+ release was abolished in the absence of extracellular Na + or by the 5-HT 2A receptor antagonist, ketanserin. Capsaicin evoked 5-HT release from platelets, a response abolished in the absence of extracellular Na + or by 5¢-Iodo-RTX. Thus capsaicinevoked Ca 2+ release appeared to be mediated by Na + -dependent 5-HT release. TRPV1-dependent 5-HT release also contributed to ADP-and thrombin-evoked Ca 2+ entry and release. 5¢-Iodo-RTX reduced ADP-and thrombin-evoked Ca 2+ signals, effects not additive with those of ketanserin, and 5¢-Iodo-RTX inhibited agonist-evoked 5-HT and ATP release. Conclusion: These results indicate that TRPV1 is present and functionally important in human platelets. The presence of this receptor may provide a link between inflammatory mediators and platelet activation in conditions such as atherosclerosis.

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