Identification of Membrane Proteins Mediating the Interaction of Human Platelets

• Published in: The Journal of cell biology Vol. 86; no. 1; pp. 77 - 86
• Main Authors: Phillips, David R., Jennings, Lisa K., Edwards, Harold H.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.07.1980; The Rockefeller University Press
• Subjects: Actins; Actins - blood; Aggregation; Blood Platelets - physiology; Blood Platelets - ultrastructure; Cell Adhesion; Cell membranes; Chemical suspensions; Cytoskeleton - physiology; Electrophoresis; Gels; Glycoproteins; Glycoproteins - physiology; Humans; Membrane proteins; Membrane Proteins - blood; Microfilaments; Platelet Aggregation; Platelets; Polyethylene Glycols; Solubility; Thrombin - metabolism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.86.1.77

Membrane glycoproteins that mediate platelet-platelet interaction were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin-aggregated platelets (platelets activated in the presence of mM Ca++) were prepared by first treating platelet suspensions with 1% Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties identical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material ∼1 μm in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26% of the total in pre-extracted platelets) and III (14%), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.