Rap1B and Rap2B translocation to the cytoskeleton by von Willebrand factor involves FcgammaII receptor-mediated protein tyrosine phosphorylation.
Stimulation of human platelets with von Willebrand factor (vWF) induced the translocation of the small GTPases Rap1B and Rap2B to the cytoskeleton. This effect was specifically prevented by an anti-glycoprotein Ib monoclonal antibody or by the omission of stirring, but was not affected by the peptide RGDS, which antagonizes binding of adhesive proteins to platelet integrins. Association of Rap2B with the cytoskeleton was very rapid, while translocation of Rap1B occurred in a later phase of platelet activation and was totally inhibited by cytochalasin D. vWF also induced the rapid tyrosine phosphorylation of several proteins that was prevented by the tyrosine kinases inhibitor genistein and by cAMP-increasing agents. Under these conditions, also the association of Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the FcgammaII receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of selected substrates with molecular masses of about 75, 95, and 150 kDa. Three of these substrates were identified as the tyrosine kinase pp72(syk), the phospholipase Cgamma2, and the inositol 5-phosphatase SHIP. Our results indicate that translocation of Rap1B and Rap2B to the cytoskeleton is regulated by tyrosine kinases and suggest a novel role for the FcgammaII receptor in the mechanism of platelet activation by vWF.
Torti
M
M
Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy. mtorti@unipv.it
Bertoni
A
A
Canobbio
I
I
Sinigaglia
F
F
Lapetina
E G
EG
Balduini
C
C
D001792:Blood Platelets / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D017452:Receptors, IgG / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D020668:rap GTP-Binding Proteins; D014841:von Willebrand Factor / Q000378:metabolism*
D010980:Platelet Membrane Glycoproteins; D017452:Receptors, IgG; D014841:von Willebrand Factor; D014443:Tyrosine; D019204:GTP-Binding Proteins; C065562:RAP2B protein, human; D020668:rap GTP-Binding Proteins