A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation.

Abstract

We have previously characterized a monoclonal antibody, $12, that binds only to activated platelets (McEver, R. P., and M. N. Martin, 1984, J. Biol. Chem., 259:9799-9804). It identifies a platelet membrane protein of Mr 140,000, which we have designated as GMP-140. Using immunocytochemical techniques we have now localized this protein in unstimulated and thrombin-stimulated platelets. Polyclonal antibodies to purified GMP-140 were used to enhance the sensitivity of detection. Nonpermeabilized, unstimulated platelets, incubated with anti-GMP-140 antibodies, and then with IgG-gold probes, showed very little label for GMP-140 along their plasma membranes. In contrast, thrombin-stimulated platelets exhibited at least a 50-fold increase in the amount of label along the plasma membrane. On frozen thin sections of unstimulated platelets we observed immunogold label along the alpha-granule membranes. We also employed the more sensitive technique of permeabilizing with saponin unstimulated platelets in suspension, and then incubating the cells with polyclonal anti-GMP-140 antibodies and Fab-peroxidase conjugate. Alpha-granule membranes showed heavy reaction product, but no other intracellular organelles were specifically labeled. These results demonstrate that GMP-140 is an alpha-granule membrane protein that is expressed on the platelet plasma membrane during degranulation.

Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway.

“…Colocalization of ACTH in the matrix with tissue factor on the membranes was observed in the dense core granules ( Figure 8B). These data confirm that the chimeric construct 3 is sorted into dense core granules that store ACTH and indicate that the cy- (Stenberg et al, 1985;Berman et al, 1986;Bonfanti et al, 1989;Hattori et al, 1989;McEver et al, 1989). Our results indicate that this membrane protein is also efficiently transported to storage granules in AtT20 cells, suggesting that the machinery for regulated secretion is conserved across cell types and species.…”

“…Table 1 summarizes the surface and intracellular protein distributions for P-selectin and for other constructs used in this study. When platelets or endothelial cells are stimulated by agonists that induce degranulation, P-selectin is redistributed to the plasma membrane (Stenberg et al, 1985;Berman et al, 1986;McEver et al, 1989;Hattori et al, 1989). To determine whether agonist-induced degranulation of transfected AtT20 cells would lead to increased surface expression of P-selectin, cells were incubated with [125I]S12 in the presence or absence of an agonist, 8-Br-cAMP, that induces secretion of ACTH from storage granules (Gumbiner and Kelly, 1982).…”

“…Recently, the primary structures of some of these proteins have been determined by cDNA cloning (ohnston et al, 1989a;Perin et al, 1988Perin et al, , 1991. One of these molecules is Pselectin (CD62, formerly known as GMP-140 or PAD-GEM), a 140-kDa membrane glycoprotein identified in storage granules of two cell types: the a granules of platelets (Stenberg et al, 1985;Berman et al, 1986) and the Weibel-Palade bodies of endothelial cells (Bonfanti et al, 1989;Hattori et al, 1989;McEver et al, 1989). Pselectin is a member of the selectin family of lectin-like Figure 1.…”

Application of whole‐exome sequencing to direct the specific functional testing and diagnosis of rare inherited bleeding disorders in patients from the Öresund Region, Scandinavia

“…Assessment of platelet activation by FC was performed as previously described (Nishibori et al , 1993; Stenberg et al , 1985; Shattil et al , 1985). Briefly, platelet activation and granule release were investigated by FC using markers for CD63, CD62P and activated GPIIb/IIIa.…”

Methods for evaluation of platelet function

“…Platelets become activated during preparation and storage of PCs for transfusion. Platelet activation in PCs can be measured by release of P-selectin [14] (soluble or surface-bound), the active conformation of GPIIb/IIIa and GPIb expression. Surface expression of GPIIb/IIIa, Pselectin and GPIb can be measured by flow cytometry.…”