title: Calpain-controlled detachment of major glycoproteins from the cytoskeleton regulates adhesive properties of activated phosphatidylserine-positive platelets.
abstract: In resting platelets, adhesive membrane glycoproteins are attached to the cytoskeleton. On strong activation, phosphatidylserine(PS)-positive and -negative platelet subpopulations are formed. Platelet activation is accompanied by cytoskeletal rearrangement, although the glycoprotein attachment status in these two subpopulations is not clear. We developed a new, flow cytometry-based, single-cell approach to investigate attachment of membrane glycoproteins to the cytoskeleton in cell subpopulations. In PS-negative platelets, adhesive glycoproteins integrin αIIbβ3, glycoprotein Ib and, as shown for the first time, P-selectin were associated with the cytoskeleton. In contrast, this attachment was disrupted in PS-positive platelets; it was retained to some extent only in the small convex regions or 'caps'. It correlated with the degradation of talin and filamin observed only in PS-positive platelets. Calpain inhibitors essentially prevented the disruption of membrane glycoprotein attachment in PS-positive platelets, as well as talin and filamin degradation. With the suggestion that detachment of glycoproteins from the cytoskeleton may affect platelet adhesive properties, we investigated the ability of PS-positive platelets to resist shear-induced breakaway from the immobilized fibrinogen. Shear rates of 500/s caused PS-positive platelet breakaway, but their adhesion stability increased more than 10-fold after pretreatment of the platelets with calpain inhibitor. In contrast, the ability of PS-positive platelets to adhere to immobilized von Willebrand's factor at 100/s was low, but this was not affected by the preincubation of platelets with a calpain inhibitor. Our data suggest that calpain-controlled detachment of membrane glycoproteins is a new mechanism that is responsible for the loss of ability of the procoagulant platelets to resist detachment from thrombi by high shear stress.
authors.affiliation: Laboratory of Molecular Mechanisms of Haemostasis, Centre for Theoretical Problems of Physicochemical Pharmacology, 4 Kosygina Str., Moscow 119334, Russia Cellular Haemostasis and Thrombosis, Federal Research and Clinical Centre of Paediatric Haematology, Oncology and Immunology, Moscow, 1 Samory Mashela Str., Moscow 117198, Russia.
authors.forename: Elena O
authors.initials: EO
authors.lastname: Artemenko
authors.affiliation: Laboratory of Molecular Mechanisms of Haemostasis, Centre for Theoretical Problems of Physicochemical Pharmacology, 4 Kosygina Str., Moscow 119334, Russia Cellular Haemostasis and Thrombosis, Federal Research and Clinical Centre of Paediatric Haematology, Oncology and Immunology, Moscow, 1 Samory Mashela Str., Moscow 117198, Russia.
authors.forename: Alena O
authors.initials: AO
authors.lastname: Yakimenko
authors.affiliation: Laboratory of Immunity Stimulation, Institute of Immunology FMBA of Russia, 24 Kashirskoye sh., Moscow 115478, Russia.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Pichugin
authors.affiliation: Cellular Haemostasis and Thrombosis, Federal Research and Clinical Centre of Paediatric Haematology, Oncology and Immunology, Moscow, 1 Samory Mashela Str., Moscow 117198, Russia Laboratory of Cell Biophysics, Centre for Theoretical Problems of Physicochemical Pharmacology, 4 Kosygina Str., Moscow 119334, Russia Faculty of Physics, Moscow State University, Vorobievy Gory, GSP-1, Moscow 119991, Russia Faculty of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russia.
authors.forename: Fazly I
authors.initials: FI
authors.lastname: Ataullakhanov
authors.affiliation: Laboratory of Molecular Mechanisms of Haemostasis, Centre for Theoretical Problems of Physicochemical Pharmacology, 4 Kosygina Str., Moscow 119334, Russia Cellular Haemostasis and Thrombosis, Federal Research and Clinical Centre of Paediatric Haematology, Oncology and Immunology, Moscow, 1 Samory Mashela Str., Moscow 117198, Russia Faculty of Physics, Moscow State University, Vorobievy Gory, GSP-1, Moscow 119991, Russia Faculty of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russia Therapeutic Faculty, Pirogov Russian National Research Medical University, Moscow 117997, Russia mapanteleev@yandex.ru.
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Panteleev
mesh_terms: D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002154:Calpain / Q000502:physiology*; D002448:Cell Adhesion; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D003598:Cytoskeletal Proteins; D006023:Glycoproteins; D010718:Phosphatidylserines; D002154:Calpain
keywords: adhesion; adhesive glycoproteins; calpain; cytoskeleton; platelet; shear