Isolation, characterization and chemical composition of the membrane from sheep platelets

A procedure of the isolation of platelets from the blood of adult sheep (Ovis aries L. var domestica) is reported. This procedure is based on differential centrifugation and a specific lysis for elimination of erythrocytes. We have obtained platelets with a purity at least 99% and a relative high yield (2.3 +/- 0.4 g wet/l whole blood identical to 235 +/- 40 mg platelet proteins/l whole blood). After disruption, homogenisation and ultracentrifugation onto a discontinuous sucrose gradient (1.6 M, 1.1 M, 1.0 M and 0.6 M sucrose), four fractions were obtained. We have separated, for the first time, a particulate preparation enriched in the whole sheep plasma membrane. This fraction was characterized by: (i) the typical membrane morphology as shown by electron micrographs; (ii) the highest activities in membrane marker enzymes such as bis(p-nitrophenyl)phosphate phosphodiesterase (EC 3.1.16.1) and 5'-dTMP-p-nitrophenyl ester phosphodiesterase (EC 3.1.3.35), and the relatively low activity for marker enzymes associated to other subcellular fractions; (iii) the highest sialic acid, cholesterol and phospholipid concentrations. The chemical composition of the platelet membrane isolated is: total proteins, 49%; lipids, 47%; carbohydrates, congruent to 3.4% (the content of hexoses is twice as high as that of hexosamines and sialic acid). The similarities and differences of this preparation with others from several sources are discussed.