Subcellular Particles of Human Platelets

Abstract

Human platelet subcellular particles have been fractionated by three methods. The isolated fractions have been examined by electron microscopy and assayed for protein, serotonin, lactate dehydrogenase, cytochrome c oxidase, β-hydroxybutyrate dehydrogenase, 5′-nucleotidase, acid phosphatase, β-glucu-ronidase, β-galactosidase, β-N-acetylgluco-saminidase, cathepsin, aryl sulphatase and a-mannosidase. The latter two enzymes had low activities. Homogenization with blendor caused great solubilization of serotonin and lysosomal enzymes but not cytochrome c oxidase. The granules in the homogenate were swollen and showed great loss of osmo-philia. The particles were isolated by differential centrifugation and separated into 3 zones after centrifugation on discontinuous sucrose gradients. The upper zone contained proteinaceous material, the second contained mostly vesicular membranes and the lowest one contained α-granules, transected pseudo-pods plus mitochondria with varying degrees of preservation. Acid phosphatase was present in the membranes but the highest activity was found in the upper part of the granular band together with β-galactosidase. β-Glucuronidase, β-N-acetylglucosaminidase and cathepsin were mainly located in the lower part of this band. Homogenization with a ‘no-clearance’ Teflon pestle caused less solubilization than the blendor and gave better organelle preservation. Isolated particles from such homogenates separated into two bands on gradient centrifugation which morphologically and biochemically were equal to those obtained when these homogenates were applied directly on the gradient. The upper particulate band contained membranes and had bound 5′-nucleotidase. The lower band possessed in its upper part well-preserved α-granules and mitochondria whereas in the lower regions more electron dense granules, often with ‘Bull's eye’, were present. Swollen α-granules were present mainly in the lower part of this band. All lysosomal enzymes were localized at the same sucrose density. Probably, the α-gra-nules are the platelet lysosomes, but with pestle homogenization there was not the biochemical heterogenity as found when using the blendor. With pestle homogenization particle bound serotonin was found in fractions of high sucrose density which contained the dense α-granules with the ‘Bull's eye’.