CD63 Modulates Spreading and Tyrosine Phosphorylation of Platelets on Immobilized Fibrinogen.

CD63 is a member of the tetraspanin superfamily of integral membrane proteins. Present on a variety of cells, tetraspanins form lateral associations with integrins and may act as ‘organizers’ of multimolecular networks that modulate integrin-mediated signaling, cell morphology, motility and migration. In resting platelets CD63 is present on the membranes of dense granules and lysosomes. Following platelet activation and exocytosis, CD63 relocates to the plasma membrane where it associates with the platelet integrin αIIbβ3-CD9 complex, and with the actin cytoskeleton in an αIIbβ3-dependent manner. In the current study, D545, a monoclonal antibody directed at the second extracellular loop of CD63, was used to investigate the role of CD63 in platelet adhesion, spreading and tyrosine phosphorylation, as determined by immunofluorescence microscopy, confocal imaging and immunoblotting. As previously demonstrated, D545 did not alter adhesion of platelets to fibrinogen, but did affect platelet spreading. Thrombin-activated platelets showed marked spreading, F-actin reorganization, redistribution of vinculin and extensive tyrosine phosphorylation, all of which were inhibited by D545. Similarly D545 inhibited the phosphorylation of focal adhesion kinase in thrombin-activated adherent platelets. These results suggest that CD63 may modulate αIIbβ3-dependent cytoskeletal reorganization.

In some malignant cell lines CD63 has been shown to associate with phosphatidylinositol (PI) kinase. To address a similar association in platelets, lipid kinase assays were performed on D545 immunoprecipitates, using PI as substrate. CD63 co-immunoprecipitated with a lipid kinase that was stimulated by non-ionic detergents, inhibited by adenosine, but unaffected by wortmannin. These enzymatic properties are consistent with a PI 4-kinase type II. The CD63-PI 4-kinase complex was co-purified from both resting and activated platelets and therefore was not activation-dependent. The linkage of CD63 with PI 4-kinase may result in the recruitment of the signaling enzyme to specific membrane locations and influence phosphoinositide-dependent signaling.