Association of the low molecular weight GTP-binding protein rap2B with the cytoskeleton during platelet aggregation.
The intracellular distribution of the low-molecular-weight GTP-binding protein rap2B was investigated in resting and agonist-activated human platelets. In both cases, platelets were lysed by Triton X-100, and cell fractions were obtained by differential centrifugations. Using a specific polyclonal antiserum, we found that rap2B in resting platelets was completely detergent-soluble. When platelets were aggregated with thrombin, the thromboxane analogue U46619, or the Ca(2+)-ATPase inhibitor thapsigargin, a significant amount of rap2B became associated with the cytoskeleton. This association was paralleled by a decrease of rap2B in the Triton X-100-soluble fraction. Translocation of rap2B to the cytoskeleton strictly depended on platelet aggregation, and maximal incorporation was found when approximately 50% aggregation was measured. Inhibition of fibrinogen binding to the glycoprotein IIb-IIIa complex completely prevented the interaction of rap2B with the cytoskeleton. These results clearly demonstrate that changes in the intracellular localization of rap2B occur during platelet activation and represent evidence that this low molecular weight GTP-binding protein may be involved in platelet function.
Torti
M
M
Department of Biochemistry, University of Pavia, Italy.
Ramaschi
G
G
Sinigaglia
F
F
Lapetina
E G
EG
Balduini
C
C
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000097:blood* / Q000302:isolation & purification; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D010974:Platelet Aggregation; D013917:Thrombin / Q000494:pharmacology; D020668:rap GTP-Binding Proteins
D003598:Cytoskeletal Proteins; D013917:Thrombin; D019204:GTP-Binding Proteins; C065562:RAP2B protein, human; D020668:rap GTP-Binding Proteins