Rapid phosphorylation and selective dephosphorylation of P-selectin accompanies platelet activation.
P-selectin, a receptor for neutrophils and monocytes, is an adhesion molecule on the surface of activated platelets that resides in the alpha granule membrane of unstimulated platelets. To determine whether phosphorylation of P-selectin might accompany platelet activation, P-selectin in resting and thrombin-stimulated platelets labeled with o-[32P]phosphate was immunoprecipitated with the monoclonal antibody AC1.2 directed against P-selectin. SDS-gel electrophoresis of the immunoprecipitates indicated about 10-20-fold higher levels of 32P incorporated into P-selectin from thrombin-activated platelets than in resting platelets, although both sets of platelets contained equivalent amounts of P-selectin. The lower limits of the molar ratio of phosphate to P-selectin in activated platelets is about 0.52 +/- 0.08. Other platelet agonists, including the thrombin receptor peptide (SFLLR), epinephrine, ADP, and collagen, similarly stimulated phosphorylation of P-selectin. The kinetics of P-selectin phosphorylation following thrombin stimulation was rapid, with maximum phosphorylation observed at 15-30 s. Phosphoamino acid analysis of the phosphorylated P-selectin revealed the rapid synthesis of phosphoserine, phosphothreonine, and phosphotyrosine, but 80-90% of the phosphotyrosine and phosphothreonine disappeared within 5 min of platelet activation while the maximal level of phosphoserine remained stable. The rapid phosphorylation and selective dephosphorylation of specific amino acids in P-selectin following platelet activation may be important for P-selectin function and signal transduction within platelets.
Crovello
C S
CS
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, Massachusetts 02111.
Furie
B C
BC
Furie
B
B
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D019007:P-Selectin; D010766:Phosphorylation; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D015815:Cell Adhesion Molecules; D009842:Oligopeptides; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D000244:Adenosine Diphosphate; D003094:Collagen; D013917:Thrombin; D004837:Epinephrine