CD63 associates with the alpha(IIb)beta(3) integrin-CD9 complex on the surface of activated platelets

The tetraspanins an integral membrane proteins expressed on cell surface an d granular membranes of hematopoietic cells and have been identified in mul ti-molecular complexes with specific integrins. In resting platelets, CD63, a member of the tetraspanin superfamily, is present in dense granule and l ysosomal membranes and, following platelet activation, translocates to the plasma membrane. In the present study, platelet activation by thrombin lead s to incorporation of CD63 into the Triton-insoluble actin cytoskeletal fra ction. This incorporation was inhibited by preincubation of platelets with RGDS or EGTA and did not occur in platelets from a patient with Glanzmann's thrombasthenia, suggesting that it was dependent upon alpha (IIb)beta (3). In activated platelets, the anti-CD63 MoAb, D545, co-immunoprecipitated CD 63 with other surface-labeled proteins, including alpha (IIb)beta (3) and a nother tetraspanin, CD9. The association of CD63 with CD9 and alpha (IIb)be ta (3) was not inhibited by preincubation of platelets with RGDS or EGTA. D 545 did not inhibit the adhesion of activated platelets to purified extrace llular matrix proteins, but significantly decreased adhesion of thrombin-ac tivated platelets to neutrophils in a resetting assay. D545 also caused dis aggregation of platelets stimulated by ADP, but had no effect on aggregatio n induced by other agonists. These results are consistent with the proposal that CD63 becomes part of an alpha (IIb)beta (3)-CD9-CD63 integrin-tetrasp anin complex in activated platelets - an association that may modulate the function of ol,alpha (IIb)beta (3)-dependent interaction with other cells s uch as neutrophils.