IDENTIFICATION OF THE CYTOSKELETAL PROTEIN ALPHA-ACTININ AS A PLATELET THROMBOSPONDIN-BINDING PROTEIN

Binding of the alpha-granular thrombospondin (TSP) to the plasma membr ane of activated platelets has long been documented, yet the molecular mechanism involved in its secretion and surface expression have not b een elucidated, Using a ligand blot binding assay where electrophoreti cally separated platelet proteins were incubated with purified I-125-l abeled TSP, we observed a strong interaction of [I-125]TSP with a 100 kDa single chain protein. On performing a platelet subfractionation, t he 100 kDa protein was predominantly localized in the cytosol from whi ch it was purified by preparative electrophoresis and was identified b y amino acid sequencing to the cytoskeletal protein, alpha-actinin. We further demonstrated that [I-125]TSP interacts with alpha-actinin in a specific manner and with a high affinity (K-d = 6.6 nM) in a solid-p hase binding assay.