Isolation and partial characterization of platelet α-granule membranes

Abstract

Porcine alpha-granules, prepared by a modification of pre-existing methods, were found to be essentially homogeneous by transmission electron microscopy. Freeze-fractured samples of isolated granules revealed numerous intramembranous particles on the EF (exoplasmic fracture) surface and to a lesser extent on the PF (protoplasmic fracture) surface whereas the PS (protoplasmic) surface was relatively smooth. The granules appear to be sealed, as evidenced by: a) the retention of their electron dense core material; b) the inability of impermeant labels to react with the granule contents, and c) the finding that the intragranular proteins are refractory to mild hydrolysis by externally added proteases. Membranes were isolated by alkali extraction of the granules and used for biochemical characterization. Approximately 87% of the protein, but only insignificant amounts of phospholipid were removed by this procedure, which yielded membrane vesicles devoid of the dense core. The membranes contain one major and several minor polypeptides of molecular weights ranging from 28,000 to 230,000, as determined by polyacrylamide gel electrophoresis. The major polypeptide contains carbohydrate residues. The exposure of specific proteins on the cytoplasmic surface of the granule membrane was determined by a combination of surface-specific labeling and proteolysis of intact granules, followed by membrane isolation and analysis. In sealed granules, only a limited number of bands are modified by the reagents whereas most of them are affected following granule lysis, indicating asymmetry in their transmembrane disposition. The fraction eluted by alkali extraction was also analyzed and found to contain nine major polypeptides of molecular weights ranging from 230,000 to 43,000. These are compared to the weights of the macromolecules believed to be secreted from alpha-granules, as determined by radioimmunological techniques.

Association of a 24‐kDa GTP‐binding protein, Gn24, with human platelet α‐granule membranes

“…In our preparation, maximal '2SI-labelled Concanavalin A binding (cpm/mg protein) was found in fraction I (comprising bands 1 and 2) (Fig. 2), consistent with previous observations that this low density fraction is enriched in plasma membrane [22,32]. Relative enrichment over lysate was 2.4-fold in a single experiment in which pelleted fractions I-III were simultaneously prepared for other marker studies.…”

“…B4 and B5 showed comparable enrichment in this marker (4.4-and 4.2fold, respectively in a separate single experiment) and hence were routinely pooled as fraction III. The activity of the mitochondrial marker enzyme, cytochrome c oxidase, was detectable in the material applied to the gradient (lo,1 -C l&9), fraction I (20.48 + 6.6) and fraction II (41.25, J-10.7) (@no1 productlmg/min, mean fSEM, n=3-4) but not in fraction III or in whole platelet lysate (using 50-75 ,ug total protein per assay), This is consistent with the expected localization on sucrose density gradients of mitochondria from both pig [22] and human [32,33] platelet subcellular fractions was assessed using [a-"PJGTP binding on nitrocellulose blots containing polypeptides separated by SDS-polyacrylamide gel electrophoresis. This procedure has been used previously for the dletection of GTP-binding proteins (G,-proteins) in platelets and other cells [ 11.…”

“…Human platelet sub-fractionation was carried out using minor modifications to a protocol previously used for pig platelet [22]. The platelet suspension was adjusted to 5 mM in 2-deoxy-D-glucose and 20pM rotenone (from 10 mM stock in DMSO) and incubated at 37°C for 40 min.…”

“…To study a-granules for the presence of low molecular mass GTP-binding proteins we sub-fractionated human platelets by applying, with minor modifications, a method we had previously successfully applied to pig platelets [22]. To improve &granule yield, ATP-deplction (using 2-deoxy-2.D-glucose and rotenone), and limited proteolysis of the intact platelet (using chymotrypsin) were included in the procedure as these two steps have been shown to contribute towards greater recovery of secretory yranulcs during platclct sub"fractionation [32].…”

“…B4 and I35 were pooled (see below) and designated as fraction III. A pellet, presumed to be enriched in dense granules [22,32], was observed in most preparations but was not recovered due to the small amount of material present.…”