title: Proteasome proteolysis supports stimulated platelet function and thrombosis.
abstract: OBJECTIVE
  Proteasome inhibitors used in the treatment of hematologic cancers also reduce thrombosis. Whether the proteasome participates in platelet activation or function is unclear because little is known of the proteasome in these terminally differentiated cells.
  
  
  RESULTS
  Platelets displayed all 3 primary proteasome protease activities, which MG132 and bortezomib (Velcade) inhibited. Proteasome substrates are marked by ubiquitin, and platelets contained a functional ubiquitination system that modified the proteome by monoubiquitination and polyubiquitination. Systemic MG132 strongly suppressed the formation of occlusive, platelet-rich thrombi in FeCl3-damaged carotid arteries. Transfusion of platelets treated ex vivo with MG132 and washed before transfusion into thrombocytopenic mice also reduced carotid artery thrombosis. Proteasome inhibition reduced platelet aggregation by low thrombin concentrations and ristocetin-stimulated agglutination through the glycoprotein Ib-IX-V complex. This receptor was not appropriately internalized after proteasome inhibition in stimulated platelets, and spreading and clot retraction after MG132 exposure also were decreased. The effects of proteasome inhibitors were not confined to a single receptor as MG132 suppressed thrombin-stimulated, ADP-stimulated, and lipopolysaccharide-stimulated microparticle shedding. Proteasome inhibition increased ubiquitin decoration of cytoplasmic proteins, including the cytoskeletal proteins Filamin A and Talin-1. Mass spectrometry revealed a single MG132-sensitive tryptic cleavage after R1745 in an extended Filamin A loop, which would separate its actin-binding domain from its carboxy terminal glycoprotein Ibα-binding domain.
  
  
  CONCLUSIONS
  Platelets contain a ubiquitin/proteasome system that marks cytoskeletal proteins for proteolytic modification to promote productive platelet-platelet and platelet-wall interactions.
authors.affiliation: From the Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland, OH (N.G., W.L., B.W., R.L.S., T.M.M.); and Department of Biological Geological and Environmental Sciences, Cleveland State University, Cleveland, OH (N.G., T.M.M.).
authors.forename: Nilaksh
authors.initials: N
authors.lastname: Gupta
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Belinda
authors.initials: B
authors.lastname: Willard
authors.forename: Roy L
authors.initials: RL
authors.lastname: Silverstein
authors.forename: Thomas M
authors.initials: TM
authors.lastname: McIntyre
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D055252:Cell-Derived Microparticles / Q000378:metabolism; D002712:Chlorides; D003598:Cytoskeletal Proteins / Q000097:blood*; D004195:Disease Models, Animal; D005290:Ferric Compounds; D005343:Fibrinolytic Agents / Q000494:pharmacology; D064448:Filamins / Q000097:blood; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D015539:Platelet Activation / Q000187:drug effects; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D017713:Platelet Transfusion; D046988:Proteasome Endopeptidase Complex / Q000097:blood*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D059748:Proteolysis; D016608:Talin / Q000097:blood; D013917:Thrombin / Q000494:pharmacology; D013927:Thrombosis / Q000097:blood / Q000139:chemically induced / Q000201:enzymology* / Q000517:prevention & control; D013997:Time Factors; D054875:Ubiquitination
chemical_list: D002712:Chlorides; D003598:Cytoskeletal Proteins; D005290:Ferric Compounds; D005343:Fibrinolytic Agents; D064448:Filamins; D008070:Lipopolysaccharides; D019038:Platelet Glycoprotein GPIb-IX Complex; D061988:Proteasome Inhibitors; D016608:Talin; D000244:Adenosine Diphosphate; D013917:Thrombin; D046988:Proteasome Endopeptidase Complex; C024555:ferric chloride
keywords: cell-derived microparticles; cytoskeletal proteins; proteasome endopeptidase complex; thrombin