P-selectin dimers are present on the surface of activated platelets.

Resting (−) and TRAP (+T)- or A23187 (+A)-activated platelets were treated with DPDPB and a non–membrane permeable biotinylation reagent. The cells were then lysed in buffer containing alkylating agents, and P-selectin was immunoprecipitated with a polyclonal P-selectin antibody. (A) Samples were electrophoresed under nonreducing conditions and immunoblotted with a monoclonal antibody to P-selectin AC1.2 (left) or biotin (right). The positions of the dimer (d) and monomer (m) are indicated. The cross-linked species migrating faster than the dimer in TRAP-activated platelets is identified with an asterisk (*). (B) Gel strips of TRAP-activated (left) andA23187-activated (right) platelet samples electrophoresed in the first dimension were reduced and placed on top of a second 10% polyacrylamide gel (as shown), then subjected to electrophoresis from top to bottom and immunoblotted with a monoclonal antibody to biotin to determine the composition of the oligomers. A broad band detected between molecular-weight markers 134 and 209 migrated at the position for P-selectin. The biotinylated protein of approximately 81 kd present in TRAP- but not in A23187-activated platelet samples is indicated by an arrowhead. (C) Two gel strips of TRAP-activated platelet samples were subjected to electrophoresis in the second dimension, and gels were immunoblotted with either the monoclonal antibiotin antibody (left, as in B) or a polyclonal antibody to P-selectin (right). The biotinylated band of approximately 81 kd (arrowhead at left) was not detected in gels immunoblotted with P-selectin antibody (right).