Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib.
Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3H]borohydride method and lysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) Ib. Approximately 70% of the platelet GP Ib was present in this skeleton. Several other minor glycoproteins, including greater than 50% of the GP Ia and small amounts of three unidentified glycoproteins of Mr greater than 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actin-binding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and co-isolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP Ib were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 Mr on the plasma membrane.
Fox
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JE
D008841:Actin Cytoskeleton / Q000032:analysis; D000199:Actins / Q000302:isolation & purification; D000328:Adult; D001792:Blood Platelets / Q000032:analysis / Q000648:ultrastructure*; D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure*; D002498:Centrifugation; D003599:Cytoskeleton / Q000032:analysis / Q000648:ultrastructure*; D003850:Deoxyribonuclease I; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification*; D008840:Microfilament Proteins / Q000302:isolation & purification; D008970:Molecular Weight; D017830:Octoxynol; D010980:Platelet Membrane Glycoproteins; D011092:Polyethylene Glycols; D012995:Solubility
D000199:Actins; D006023:Glycoproteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010980:Platelet Membrane Glycoproteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D003850:Deoxyribonuclease I