P-SELECTIN MESSENGER-RNA IS EXPRESSED AT A LATER PHASE OF MEGAKARYOCYTE MATURATION THAN MESSENGER-RNAS FOR VONWILLEBRAND-FACTOR AND GLYCOPROTEIN IB-ALPHA

The assembly of alpha-granules occurs exclusively in megakaryocytes be cause platelets have limited capacity for the synthesis of macromolecu les. Thus far, alpha-granule development in megakaryocytes has been pr imarily evaluated by ultrastructural studies. The aim of the study was to obtain molecular and biochemical evidence for the expression of se lected alpha-granule proteins in megakaryocytes. Guinea pig megakaryoc ytes were purified and separated into subgroups at different phases of maturation by the Celsep procedure (Schick et al. Blood 1989;73:1801- 8). Guinea pig-specific probes for P-selectin, von Willebrand factor ( vWF), glycoprotein lb-alpha (Gplb-alpha), and phosphoglycerate kinase were prepared by using the polymerase chain reaction. By Northern blot analysis, P-selectin messenger ribonucleic acid (mRNA) was primarily expressed in the mature megakaryocyte Celsep subgroup, whereas vWF and Gplb-alpha mRNA were expressed at all phases of megakaryocyte maturat ion. In situ hybridization confirmed that P-selectin mRNA was primaril y expressed at later stages of cytoplasmic maturation: 14% +/- 6.2% of stage I, 35.5% +/- 6.1% of stage II, 72% - 5.2% of stage III, and 47. 0% +/- 3.3% of stage IV megakaryocytes expressed P-selectin mRNA. Thus , the expression of mRNA for P-selectin appeared to peak in stage III cells. In contrast vWF mRNA was expressed in immature megakaryocytes a nd persisted throughout megakaryocyte maturation. In situ hybridizatio n did not demonstrate a relationship between the expression of mRNA fo r P-selectin or vWF with megakaryocyte ploidy. The study indicates tha t mRNA for P-selectin, an alpha-granule membrane protein, was expresse d at a later phase of maturation than vWF, a protein packaged in alpha -granules, and Gplb-alpha, a protein located primarily in the plasma m embrane. The study provides new information that broadens our understa nding of platelet biosynthesis and assembly in megakaryocytes and prov ides biochemical markers of megakaryocyte maturation.