Identification of Dp71 isoforms in the platelet membrane cytoskeleton. Potential role in thrombin-mediated platelet adhesion.
Utrophin is a component of the platelet membrane cytoskeleton and participates in cytoskeletal reorganization (Earnest, J. P., Santos, G. F., Zuerbig, S., and Fox, J. E. B. (1995) J. Biol. Chem. 270, 27259-27265). Although platelets do not contain dystrophin, the identification of smaller C-terminal isoforms of dystrophin, including Dp71, which are expressed in a wide range of nonmuscle tissues and cell lines, has not been investigated. In this report, we have identified Dp71 protein variants of 55-60 kDa (designated Dp71Delta(110)) in the membrane cytoskeleton of human platelets. Both Dp71Delta(110) and utrophin sediment from lysed platelets along with the high speed detergent-insoluble pellet, which contains components of the membrane cytoskeleton. Like the membrane cytoskeletal proteins vinculin and spectrin, Dp71Delta(110) and utrophin redistributed from the high speed detergent-insoluble pellet to the integrin-rich low speed pellet of thrombin-stimulated platelets. Immunoelectron microscopy provided further evidence that Dp71Delta(110) was localized to the submembranous cytoskeleton. In addition to Dp71Delta(110), platelets contained several components of the dystrophin-associated protein complex, including beta-dystroglycan and syntrophin. To better understand the potential function of Dp71Delta(110), collagen adhesion assays were performed on platelets isolated from wild-type or Dp71-deficient (mdx(3cv)) mice. Adhesion to collagen in response to thrombin was significantly decreased in platelets isolated from mdx(3cv) mice, compared with wild-type platelets. Collectively, our results provide evidence that Dp71Delta(110) is a component of the platelet membrane cytoskeleton, is involved in cytoskeletal reorganization and/or signaling, and plays a role in thrombin-mediated platelet adhesion.
Austin
Richard C
RC
Department of Pathology, McMaster University and the Henderson Research Centre, Hamilton, Ontario L8V 1C3, Canada. raustin@thrombosis.hhscr.org
Fox
Joan E B
JE
Werstuck
Geoff H
GH
Stafford
Alan R
AR
Bulman
Dennis E
DE
Dally
Ghassan Y
GY
Ackerley
Cameron A
CA
Weitz
Jeffrey I
JI
Ray
Peter N
PN
D000818:Animals; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D016189:Dystrophin / Q000031:analogs & derivatives* / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D016253:Microscopy, Immunoelectron; D009132:Muscles / Q000648:ultrastructure; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013049:Spectrin / Q000737:chemistry; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors; D049411:Utrophin; D016596:Vinculin / Q000737:chemistry
D003598:Cytoskeletal Proteins; D016189:Dystrophin; D008565:Membrane Proteins; D020033:Protein Isoforms; C483600:Utrn protein, mouse; D049411:Utrophin; C085339:apo-dystrophin 1; D016596:Vinculin; D013049:Spectrin; D003094:Collagen; D013917:Thrombin