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. 1984 Aug 15;222(1):235–246. doi: 10.1042/bj2220235

Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes.

N Hack, N Crawford
PMCID: PMC1144165  PMID: 6477508

Abstract

By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Carey F., Menashi S., Crawford N. Localization of cyclo-oxygenase and thromboxane synthetase in human platelet intracellular membranes. Biochem J. 1982 Jun 15;204(3):847–851. doi: 10.1042/bj2040847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Clemetson K. J., Capitanio A., Lüscher E. F. High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes. Biochim Biophys Acta. 1979 May 3;553(1):11–24. doi: 10.1016/0005-2736(79)90027-0. [DOI] [PubMed] [Google Scholar]
  3. Clemetson K. J., McGregor J. L., James E., Dechavanne M., Lüscher E. F. Characterization of the platelet membrane glycoprotein abnormalities in Bernard-Soulier syndrome and comparison with normal by surface-labeling techniques and high-resolution two-dimensional gel electrophoresis. J Clin Invest. 1982 Aug;70(2):304–311. doi: 10.1172/JCI110618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. George J. N. Studies on platelet plasma membranes. IV. Quantitative analysis of platelet membrane glycoproteins by (125I)-diazotized diiodosulfanilic acid labeling and SDS-polyacrylamide gel electrophoresis. J Lab Clin Med. 1978 Sep;92(3):430–446. [PubMed] [Google Scholar]
  5. Gerrard J. M., White J. G. Prostaglandins and thromboxanes: "middlemen" modulating platelet function in hemostasis and thrombosis. Prog Hemost Thromb. 1978;4:87–125. [PubMed] [Google Scholar]
  6. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  7. Lagarde M., Bryon P. A., Guichardant M., Dechavanne M. A simple and efficient method for platelet isolation from their plasma. Thromb Res. 1980 Feb 1;17(3-4):581–588. doi: 10.1016/0049-3848(80)90098-5. [DOI] [PubMed] [Google Scholar]
  8. Lagarde M., Guichardant M., Menashi S., Crawford N. The phospholipid and fatty acid composition of human platelet surface and intracellular membranes isolated by high voltage free flow electrophoresis. J Biol Chem. 1982 Mar 25;257(6):3100–3104. [PubMed] [Google Scholar]
  9. Lagarde M., Menashi S., Crawford N. Localisation of phospholipase A2 and diglyceride lipase activities in human platelet intracellular membranes. FEBS Lett. 1981 Feb 9;124(1):23–26. doi: 10.1016/0014-5793(81)80045-2. [DOI] [PubMed] [Google Scholar]
  10. McGowan E. B., Ding A., Detwiler T. C. Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation. J Biol Chem. 1983 Sep 25;258(18):11243–11248. [PubMed] [Google Scholar]
  11. McGregor J. L., Clemetson K. J., James E., Capitanio A., Greenland T., Lüscher E. F., Dechavanne M. Glycoproteins of platelet membranes from Glanzmann's thrombasthenia. A comparison with normal using carbohydrate-specific or protein-specific labelling techniques and high-resolution two-dimensional gel electrophoresis. Eur J Biochem. 1981 May 15;116(2):379–388. doi: 10.1111/j.1432-1033.1981.tb05346.x. [DOI] [PubMed] [Google Scholar]
  12. Menashi S., Authi K. S., Carey F., Crawford N. Characterization of the calcium-sequestering process associated with human platelet intracellular membranes isolated by free-flow electrophoresis. Biochem J. 1984 Sep 1;222(2):413–417. doi: 10.1042/bj2220413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Menashi S., Davis C., Crawford N. Calcium uptake associated with an intracellular membrane fraction prepared from human blood platelets by high-voltage, free-flow electrophoresis. FEBS Lett. 1982 Apr 19;140(2):298–302. doi: 10.1016/0014-5793(82)80918-6. [DOI] [PubMed] [Google Scholar]
  14. Menashi S., Weintroub H., Crawford N. Characterization of human platelet surface and intracellular membranes isolated by free flow electrophoresis. J Biol Chem. 1981 Apr 25;256(8):4095–4101. [PubMed] [Google Scholar]
  15. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  16. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  17. Rittenhouse-Simmons S., Deykin D. Isolation of membranes from normal and thrombin-treated gel-filtered platelets using a lectin marker. Biochim Biophys Acta. 1976 Apr 5;426(4):688–696. doi: 10.1016/0005-2736(76)90133-4. [DOI] [PubMed] [Google Scholar]
  18. Sixma J. J., Schiphorst M. E., Verhoeckx C., Jockusch B. M. Peripheral and integral proteins of human blood platelet membranes. alpha-Actinin is not identical to glycoprotein III. Biochim Biophys Acta. 1982 Jun 4;704(2):333–344. doi: 10.1016/0167-4838(82)90163-7. [DOI] [PubMed] [Google Scholar]

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