Assembly and GPIIIa content of cytoskeletal core in platelets agglutinated with bovine von Willebrand factor.
The association between occupancy of the von Willebrand factor (vWf) receptor glycoprotein (GP) Ib, agglutination, and the assembly and composition of the cytoskeletal core was studied in 125I-surface-labeled aspirin-treated washed platelets. Binding of ligands to GPIIb-IIIa and platelet aggregation were abolished by addition of EDTA. Platelet agglutination induced by bovine vWf generated a complete cytoskeletal core (Triton-insoluble residue), shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be composed of actin-binding protein (ABP) (260 Kd), 235-Kd protein, myosin heavy chain (200 Kd), alpha-actinin (100 Kd), and actin (43 Kd). In addition, autoradiography of the gels showed a 125I 105-Kd GP, identified by immunoblot as GPIIIa, as well as GPIb, GPIIb, and another band at 87 Kd, probably GPIV. Neither cytoskeletal assembly nor GPIIa incorporation was altered if calpain was inhibited with leupeptin. Platelet suspensions exposed to bovine vWf without stirring (ie, nonagglutinated) or platelets in which agglutination was inhibited with ADP showed smaller cytoskeletons with little ABP, 235 Kd protein, and alpha-actinin. Autoradiographs showed mainly GPIb. Cytochalasin D (CD) and monobromobimane (MB) enhanced agglutination and prevented the inhibitory action of ADP on bovine vWf-induced platelet agglutination. CD markedly inhibited the assembly of the cytoskeletal core as well as GPIIIa retention, whereas MB resulted in a large Triton-insoluble residue which contained GPIIIa. Thus, development of a platelet cytoskeletal core is apparently not required for agglutination, but when a cytoskeletal core is assembled in agglutinated platelets, GPIIIa is retained.
Puszkin
E G
EG
Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10467.
Mauss
E A
EA
Zucker
M B
MB
D000185:Actinin / Q000032:analysis; D000199:Actins / Q000032:analysis; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000328:Adult; D000818:Animals; D001792:Blood Platelets / Q000502:physiology / Q000648:ultrastructure*; D002154:Calpain / Q000037:antagonists & inhibitors; D002417:Cattle; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008840:Microfilament Proteins / Q000032:analysis; D008970:Molecular Weight; D009218:Myosins / Q000032:analysis; D010974:Platelet Aggregation / Q000187:drug effects*; D010980:Platelet Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism*; D014841:von Willebrand Factor / Q000494:pharmacology*
D000199:Actins; D007976:Leupeptins; D008840:Microfilament Proteins; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D000185:Actinin; D000244:Adenosine Diphosphate; D004492:Edetic Acid; D002154:Calpain; D009218:Myosins; C032854:leupeptin