Isolation and Characterization of Plasma Membranes from Human Blood Platelets

TLDR

Human platelets were lysed hypotonicly after glycerol loading and subjected to glycerol gradient centrifugation, yielding a homogeneous membrane fraction; a subsequent density step and sucrose gradient separated two subfractions at densities 1.090 and 1.120 with identical pI 3.9. The isolated platelet membranes are lipoglycoproteins (~7 % carbohydrate) with protein, lipid, and carbohydrate contents of 31.9–40.1 %, 48.2–55.9 %, and 6.9–7.3 % respectively, a cholesterol‑to‑phospholipid ratio of ~0.45–0.49 comparable to liver membranes, enriched for phosphodiesterase, acid phosphatase, and ATPase, and lacking intracellular organelles, confirming that the platelet outer membrane is essentially a plasma membrane derived from megakaryocytes.

Abstract

Abstract Human platelets were subjected to hypotonic lysis after being loaded intracellularly with glycerol (4.3 m) by centrifugation in a glycerol gradient under controlled conditions. A homogeneous membrane fraction, free of intracellular organelles, was isolated after a single density step centrifugation, while continuous sucrose density gradient centrifugation gave two subfractions with densities of 1.090 and 1.120, and with identical isoelectric points (pI 3.9) as determined by isoelectric focusing. Compositional studies indicated that the platelet membrane was a lipoglycoprotein with a carbohydrate content of approximately 7%. The sugar components were identified as glucose, galactose, mannose, hexosamines (GlcN:GalN = 6:1), sialic acid, and fucose. The chemical composition of the platelet membranes isolated at d, 1.090 was protein, 31.9%; lipid, 55.9%; carbohydrate, 7.3%; RNA, 0.3%; DNA, 0.0%; and for those isolated at d, 1.120, it was protein, 40.1%; lipid, 48.2%; carbohydrate, 6.9%; RNA, 0.4%; DNA, 0.0%. The membranes were characterized by a high molar ratio of cholesterol to phospholipid (0.49 and 0.45) that was similar to that found in liver plasma membranes. Phosphodiesterase, acid phosphatase and ATPase were purified 8-, 4-, and 2-fold, respectively, in the membrane fractions, whereas succinic dehydrogenase, esterase, and a variety of β-glycosidases were present only at very low levels. These results demonstrate the absence of intracellular membranes and, together with the chemical analyses, they suggest that the outer membrane of the platelet is similar to plasma membranes of other cells, and they provide biochemical confirmation of its origin from the plasma membrane of the megakaryocyte. The vesicles of the two membrane bands differed in ultrastructure, the lighter (d, 1.090) having an average diameter of 1750 A with numerous concentric double membrane structures, while the heavier (d, 1.120) consisted of single membranes of diameter 700 A.