Isolation Of Dense Bodies From Human Blood Platelets On Metrizamide Gradient

Abstract

In view of the prominent role of dense bodies in platelet activation suggested by the platelet dysfunctions observed in storage pool diseases, we have developed a method for the isolation of human platelet dense bodies, using mepa- crine to follow the purification.Each step of the purification (washing procedures, lysis and subcellular separation) has been controlled in order to obtain the minimum release of these granules. Platelet lysates were centrifuged on a short two step discontinuous metrizamide gradient which allowed the attainment of a high density pellet. This pellet consisted of isolated mepacrine fluorescent granules which showed the typical appearance of dense bodies by electron microscopy. The granule pellet was relatively free from plasma membranes as estimated by the remaining (3H) -concanavalin A or 125I after labelling the whole platelets before the fractionation. The low contamination by other granule populations was estimated by the different assayed markers, β-glucuronidase, monoamine oxidase and platelet factor 4. The method is simple, reproducible and allows the highest enrichment in dense bodies obtained until now with human platelets(x 170 enrichment in calcium and x 110 enrichment in (14C) 5-HT after labelling the whole platelets as compared to the homogenate). Functional studies performed with the isolated granules showed a rapid accumulation of (14C)-5-HT, and the initial uptake was inhibited by reserpine but remained insensitive to imipramine.The technique can be applied to the study of inherited disorders where the serotonin uptake and release mechanism has to be clarified.