Human platelet membranes

Figure 3. Effect of UV light ( 3 10 nra) on incorporation of [3H]-2-nitroimipramine into human platelet membranes, as shown by SDS-PAGE and fluorography.

Figure il. Photolabeling of human platelet membranes with NI in the presence or absence of 10 M imipramine. Proteins were separated by SDS-PAGE, slices were solubilized in NCS/toluene and counted by scintillation spectrometry. 

Figure 5. Dose response inhibition by imipramine of photolabeling of the 30 KDalton fraction in human platelet membranes in the presence or absence of varying concentrations of imipramine as indicated, followed by solubilization and SDS-PAGE/fluorography.

Human-platelet membranes have been extracted with lithium 3,5-diiodosalicylate, and the soluble glycoproteins separated on O-phosphonocellulose.849 The purified, membrane glycoprotein, molecular weight 100,000, was immunochemically identical to a sample purified from platelet-membrane extract by means of con A-Sepharose affinity chromatography. No further characterization was reported,... 

Figure 2. Two-dimensional BAC/SDS-PAGE of platelet membrane proteins. While not only large gels are recommended for complex samples, utilizing tube gels for the first dimension furthermore provides better resolution and more efficient transfer of proteins to the second dimension (less vertical smearing). (A) Small slab gel in the first dimension (7x7 cm). (B) Large tube gel in the first dimension (1 mm x 15 cm). (C) Summary of the human platelet membrane proteome study (Moebius et al. 2005). 158 proteins were exclusively identified from 2-DB gels, 65 from ID-PAGE. An overlap of 75 proteins was identified from both types of gels.

In a comprehensive study of the human platelet membrane proteome we demonstrated the need for a combined application of 2-DB and one-dimensional SDS-PAGE for maximizing the amount of identified proteins. Since both techniques address different subproteomes (Figure 2C) they may be utilized in a complementary way. In 2-DB, a higher resolution increases the local protein concentration and facilitates identification. However, in SDS-PAGE, whole lanes can be cut into equidistant slices, eliminating the need for protein visualization prior to excision. Thereby, even proteins, which cannot be visualized by staining procedures and therefore will escape detection after 2-DB, can be identified by subsequent MS. 

Moebius, J., Zahedi, R.P., Lewandrowski, U., Berger, C., Walter, U. and Sickmann, A. (2005) The human platelet membrane proteome reveals several new potential membrane proteins. Mol. Cell Proteomics 4, 1754-1761. 

Bayon Y, Croset M, Daveloose D, Guerbette F, Chirouze V, Viret J, Kader J-C, Lagarde M. Effect of spedfic phospholipid molecular spedes incorporated in human platelet membranes on thromboxane... 

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Varani, K, Gessi, S, Dalpiaz, A, Borea, PA, Pharmacological and biochemical diaracterization of purified A adoiosine recqjtors in human platelet membranes by pH]CGS 21680, J Pharmacol 1996 117,1693-1701. 

ShulmanS,Kaq>addnS. Grossed immunoeleGtth esis of human platelet membranes Diminution of major antigens in Glananann s thrombasthenia and Bernard Soulier Syndrome. J Biol Chem 1980 255 4320-4327. 

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SAUSSY DL Jr, MaIS DE, Burch RM, HALUSHKA PV. identification of a putative thromboxane Aj/prostaglandin Hj receptor in human platelet membranes, JBiol Chem 261 3025-3029, 1986. 

Suzuki T, BaNNO Y, NOZAWA Y. Partial purification and cfaaracterizaticHi of two forms of pho hatidylinositol 4-phosphate 5-kinase from human platelet membrane Thromb Res 64 45-56,1991. 

BayonY, Onset M, Daveloose D, Guohette F, OurouzeV, Viret J,KaderJC, and Lagarde M. (1995). Effect of qrecific phospholipid molecular species incorporated in human platelet membranes on thromboxane A2/prostaglandin H2 receptors. J. Lipid Res. 36,47-56. 

Nurden AT Pcdymoiphisnis of human platelet membrane glyccqiroteins Structure and clinical significance. Thromb Haemostat 74 345-351,1995. 

Rock, C. 0-, and Jackowski, S. (1987). Thrombin- and nucleotide-activated phospha tidy linos tol 4,5-bisphosphate phospholipase C in human platelet membranes- /. Biol. Oiem. 261, 5492-5498. 

Preliminary studies indicate nonenzymatic glycosylation of human platelet membranes (S2) whether there is any connection with the abnormalities in platelet function in diabetes mellitus is under investigation (M37). 

Sampietro, T., Lenzi, S., Cecchetti, P., and Navalesi, R., In vitro nonenzymatic glycosylation of human platelet membranes. Diabetologia 27, 326A, Abstr. 456 (1984). 

Potentiation of hormone responses In membranes has been more difficult to observe. Forskolln can potentiate prostaglandin stimulation of adenylate cyclase in human platelet membranes nd Isoproterenol stimulation of adenylate cyclase In membranes from wild type S49 lymphoma cells and rat adipocytes.These potentlatlons are not nearly as large as those seen in Intact cells and tissues. 

However when high micromolar concentrations (200 micro M) of 5-HT or of 5-HT uptake inhibitors were used in determining the dissociation half life of H-imipramine, H-paroxetine and H-dtalopram dissociating from human platelet membrane preparations it appears that not only the dissociation half life was prolonged but also that the prolongation was different for each of the three labeled uptake inhibitors [24]. This led to the conclusion that the three labeled ligands each bind to a different domain on the 5-HT transporter. 

Hung, S.C., Ghali, N.I., Venton, D.L. and LeBreton, G.C. (1983). Specific binding of the thromboxane A antagonist 13-azaprostanoic acid to human platelet membranes. Biochim. Biophys. Acta, 728, 171-178... 

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Lester, H.A., Steer, M.L. and Levitzki, A. (1982). Prostaglandin-stimulated GTP hydrolysis associated with activation of adenylate cyclase in human platelet membranes. Proc. Natl Acad. Sci. USA, 79, 719-723... 

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