The role of the adapter molecule SLP-76 in platelet function and hemostasis.

摘要

Adapter proteins lack intrinsic effector functions, but contain distinct modular domains which mediate protein interactions. These molecules thus serve as the scaffolding around which effectors and their substrates assemble. SLP76 is a hematopoietic specific adapter composed of three modular domains; an amino-terminal acidic region containing key tyrosines, a central proline-rich region and a carboxy-terminal SH2 domain. Upon phosphorylation the amino-terminal tyrosine residues of SLP76 bind Vav, Nck, and Itk. The adapter SLAP130(Fyb) inducibly associates with the SH2 domain of SLP76. The proline-rich region of SLP76 binds constitutively to Gads. Following T cells antigen recognition, Gads associates with another adapter, LAT, bringing SLP76 to glycolipid-enriched microdomains (GEMS), specialized regions within the membrane in which signal transduction complexes form.; Mice deficient in SLP76 demonstrate a failure of T cell development and fetal hemorrhage. To study the structure/function of SLP76 in vivo , I adopted a retroviral transduction and bone marrow reconstitution system. This system makes use of a bicistronic retroviral vector to co-express variants of SLP76 and GFP in transduced SLP76−/− stem cells which are then used to reconstitute lethally irradiated hosts. Whereas WTSLP76 expression rescues T cell development, hemostasis and platelet function, animals reconstituted with the Y3FSLP76 mutant have a phenotype similar to those mice reconstituted with GFP alone. These findings indicate that the phosphorylated tyrosines of SLP76 are likely critical for both T cell and platelet function. In contrast, reconstitution with the Δ224244SLP76 mutant results in a severe defect in T cell development, but rescue of the bleeding diathesis. Analysis of platelets expressing Δ224244SLP76 revealed a restoration of responses (e.g. increased P-selectin expression and spreading on a fibrinogen matrix) to collagen receptor agonists suggesting the SLP76-Gads-LAT association is not essential. Consistent with this, LAT deficient platelets aggregate fully in response to high concentrations of collagen. In addition, unlike SLP76, LAT is not phosphorylated or required during signaling through integrin αIIbβ3. Collectively, these results indicate that there is a differential requirement of the functional domains of SLP76 during platelet versus T cell activation.