BIOSYNTHETIC PATHWAYS OF THE PLATELET FIBRINOGEN RECEPTOR IN HUMAN MEGAKARYOCYTES
Authors: Marquerie G , Duperray A , Uzan G , Berthier R 
Published: August 2018

Interaction between cells and between cells and extracellular matrices are critical for a number of biological processes, including organ development, cell differenciation, cell motility, and the inimune' response. These interactions are mediated by a family of adhesion receptors that recognize short sequences such as Arg-Gly-Asp (RGD). These receptors share similar structural properties. They are heterodimers composed of a and B subunits and sometime express common epitopes. This suggests that the structural and functional relationship of these receptors may result from the transcription of related genes or may arise from cell specific post-transcriptional events. Thus, analysis of the biosynthesis and processing of these receptors would provide valuable insights into the molecular mechanism which control their expression at the surface,of different cells. Platelet membrane glycoprotein (GP) IIb-IIIa is a member of this receptor family. This protein is a non covalent heterodimer composed of two distinct polypeptides, Glib which consists of two subunits Ilba and IlbB (Mr = 116 kD, Mr = 25 kD) and GPIIIa (Mr = 100 kD, reduced). GPIIb-IIIa functions at site of platelet aggregation and serves as receptor for RGD containing factors including fibrinogen, fibronectin and von Willebrand factor. We report here on the investigation of the biosynthetic pathways of this RGD receptor in human megakaryocytes. High number of megakaryocytic cells from the megakaryoblastic stage to the polyploid mature megakaryocyte were obtained from liquid culture of cryopreserved leukocyte stem cell concentrates from patients with chronic myelogenous leukemia (CML). After sorting, using a FACS IV and indirect immunofluores-cent labeling with monoclonal antibodies anti-GPIIb-IIIa, 95 % of the cells in culture were of the megakaryocytic lineage. These megakarocytes represented an excellent tool to delineate at the molecular level events associated with the biosynthesis of GPIIb-IIIa.

Metabolic labeling and pulse-chase experiments indicated that GPIIb and GPIIIa are synthesized from separate mRNA and that the two subunits of GPIIb derive from a common precursor. This was further confirmed by cell-free translation of megakaryocyte mRNA and the identification of separate cDNA containing sequences coding for the pro-GPIIb and for GPIIIa. These cDNA were isolated from a Xgt11 expression library constructed with purified megakaryocyte RNA, and were used to size the messengers coding for the two polypeptides. A single mRNA species of 3.9 kB was found to encode the pro-GPIIb, whereas two different mRNA species of 2.9 kB and 4. 1 kB were identified with the GPIIIa cDNA.

The newly synthesized GPIIIa associates early with the pro-GPIIb in the rough endoplasmic reticulum. Examination of the glycosylation pathways with endoglycosidase H, tunicamycin and monensin indicated that high mannose oligosaccharides are added to the GPIIIa and pro-GPIIb polypeptide backbone. The pro-GPIIb is then processed with conversion of high mannose to the complex type carbohydrate, whereas GPIIIa remains endoH sensitive. Glycosylation of pro-GPIIb-IIIa and processing of oligosaccharides are prerequisite for proteolytic maturation of pro-GPIIb and the expression of the mature complex at the surface of the cell. Thus post-translational processing of GPIIb-IIIa requires an early assembly of the complex. This may have important implications in the maturation of megakaryocyte granules and in the molecular mechanism underlying the Glanzmann thrombastenic disease.