Density Gradient Centrifugation and Electron Microscopic Characterization of Subcellular Fractions from Human Blood Platelets

Summary

Homogenized human blood platelets have been fractionated by centrifugation in Ficoll and sucrose density gradients. The different fractions were examined by electron microscopy.

Although Ficoll allows for the separation of very distinct zones, its ability to form complexes with cellular components made sucrose the preferable gradient. Sucrose, in spite of its unfavorable osmotic effect, allows for an acceptable fractionation of platelet components.

• References

• 1 Baker R. V, Da Prada M, Pletscher A. The isolation from blood platelets of particles containing 5-hydroxytryptamine and adenosine triphosphate. J. Physiol 149: 55 1959;
• 2 Behnke O. Incomplete microtubules observed in mammalian blood platelets during microtubule polymerization. J. Cell Biol 31: 697 1967;
• 3 Bettex-Galland M, Lüscher E. F. Studies on the metabolism of human blood platelets in relation to clot retraction. Thrombos. Diathes. haemorrh. (Stuttg) 04: 178 1960;
• 4 Bettex-Galland M, Lüscher E. F. Thrombosthenin, the contractile protein from blood platelets and its relation to other contractile proteins. Advanc. Prot. Chem 20: 1 1965;
• 5 Born G. V. R, Ingram G. I, Stacey R. S. The relationship between 5-hydroxytryptamine and adenosine triphosphate in blood platelets. Brit. J. Pharmacol 13: 62 1958;
• 6 Davey M. G, Lüscher E. F. Platelet proteins. In: Kowalski E, Niewiarowski S. (eds.), Biochemistry of blood platelets. Acad. Press; London and New York: 1966
• 7 Day H. J, Holmsen H, Hovig T. Subcellular particles of human platelets. Scand. J. Haemat., Suppl 7 1969;
• 8 Falcão L, Gautier A, Lombardi L, Jean G, Probst M. Analogie entre deux types d’organites. J. Microscopie 03: 519 1964;
• 9 Firkin B. G. The Platelet. Anstr. Ann. Med 12: 261 1965;
• 10 Frasca J. M, Parks V. R. A routine technique for doublestaining ultrathin sections using uranyl and leadsalts. J. Cell Biol 25: 157 1965;
• 11 Holmsen H. Changes in radioactivity of P³²-labelled acid soluble organo-phosphates in blood platelets during collagen-induced and adenosine-induced platelet aggregation. Scand. J. clin. Lab. Invest 17: 537 1965;
• 12 Johnson S. A, Sturrock R. M, Rebuck J. W. Morphological location of platelet factor 3 activity in normal platelets. Proc. VI. Int. Congr. Biochem; Vienna: 1958
• 13 Käser-Glanzmann R, Lüscher E. F. The mechanism of platelet aggregation in relation to hemostasis. Thrombos. Diathes. haemorrh. (Stuttg) 07: 480 1962;
• 14 Luft J. H. Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol 09: 409 1961;
• 15 Marcus A. J, Zucker-Franklin D, Safier L, Ullman H. Studies on human platelet granules and membranes. J. clin. Invest 45: 14 1966;
• 16 Parmeggiani A. Elektronenmikroskopische Beobachtungen an menschlichen Thrombozyten während der viskosen Metamorphose. Thrombos. Diathes. haemorrh. (Stuttg) 06: 517 1961;
• 17 Prentice T. G, Bishop C. Separation of rabbit red cells by density methods and characteristics of separated layers. J. cell. comp. Physiol 65: 113 1965;
• 18 Reynolds E. S. The use of lead citrate of high pH as an electronopaque stain in electron microscopy. J. Cell Biol 17: 208 1963;
• 19 Rodman N. F, Brinkhous K. M. Some pathogenetic mechanisms of white thrombusformation: agglutination and selfdestruction of the platelets. Fed. Proc 22: 1356 1963;
• 20 Schulz H. Thrombozyten und Thrombose im elektronenmikroskopischen Bild. Springer; Berlin-Heidelberg-New York: 1968
• 21 Schulz H, Hiepler E. Über die Lokalisation von gerinnungsphysiologischen Aktivitäten in submikroskopischen Strukturen der Thrombozyten. Klin. Wschr 06: 273 1959;
• 22 Siegel A. Die Isolierung und Charakterisierung der subzellulären Strukturen menschlicher Thrombozyten. Thesis; Berne: 1966
• 23 Siegel A, Lüscher E. F. Non-identity of the α-granules of human blood platelets with typical lysosomes. Nature 215: 745 1967;
• 24 Thomas D. P. Effect of catecholamines on platelet aggregation caused by thrombin. Nature (Lond) 215: 298 1967;
• 25 Tranzer J. P, Da Prada M, Pletscher A. Ultrastructural localization of 5-hydroxytryptamine in blood platelets. Nature 212: 1574 1966;
• 26 Ulutin O. N. The qualitative platelet diseases. In: Johnson S. A, Monto R. W, Rebuck J. W, Horn Jr. R. V. (eds,), Blood Platelets. 553 Little Brown and Co; Boston: 1961
• 27 Wallach D. F. H, Kamat V. B. Plasma and cytoplasmic membrane fragments from Ehrlich ascites carcinoma. Proc. nat. Acad. Sci. (Wash) 52: 721 1964;
• 28 Weber E, Walter E, Morgenstern E, Mondt H, Rose U, Towliati H, Knaudt E. Untersuchungen an fraktionierten Plättchenhomogenaten. Biochem. Pharmacol 19: 1893 1970;
• 29 Zucker M. B, Borrelli J. Relationship of some blood clotting factors to serotonin release from washed platelets. J. appl. Physiol 07: 432 1955;
• 30 Zucker M. B, Borrelli J. A survey of some platelet enzymes and functions. The platelets a source of normal serum acid glycerophosphatase. Ann. N.Y. Acad. Sci 75: 203 1958;