Platelet cytoskeleton alpha-actinin in normal and thrombasthenic platelets: distribution and immunologic characterization.
The subcellular localization of alpha-actinin (Mr 100,000) in human skeletal muscle is restricted to the Z line, in which it is believed to anchor actin filaments. Recently, this protein was identified in normal and thrombasthenic human platelets by its antigenic cross-reaction with antibodies to chicken gizzard alpha-actinin. In our study, the biochemical interaction between purified platelet alpha-actinin and striated muscle F-actin was examined by electron microscopy of negatively stained preparations. Like its muscle counterpart, platelet alpha-actinin promotes the cross-linking and bundling of actin filaments. Antibodies prepared to human platelet alpha-actinin cross-reacted with chicken gizzard alpha-actinin as shown by immunoelectrophoresis and the western blotting technique. Immunoblots prepared with normal and thrombasthenic platelets with antibodies to human platelet alpha-actinin revealed that this protein is susceptible to proteolysis. Extracts of freshly drawn platelets showed a protein band of 100 K. When the platelet extracts were incubated at 37 degrees C for various times, the immunoblots showed protein bands of 100 and 80 K. The proportion of the 80 K protein band increased with incubation time. This proteolysis can be prevented by chelating agents such as EDTA or the protease inhibitor leupeptin. Indirect immunofluorescent studies of human skin fibroblasts with antibodies to chicken gizzard actin and human skeletal muscle, chicken gizzard, and platelet alpha-actinin revealed the staining pattern characteristic of each protein. The distribution of alpha-actinin in normal and thrombasthenic platelets was assessed by ferritin-labeled immunoelectron microscopy. Ferritin particles were found in the cytoplasm immediately below the membrane and in some granules. There was no labeling associated with the mitochondria.
D000185:Actinin / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D001791:Blood Platelet Disorders / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D003429:Cross Reactions; D004492:Edetic Acid / Q000494:pharmacology; D005347:Fibroblasts / Q000032:analysis; D006801:Humans; D007122:Immunoelectrophoresis; D007976:Leupeptins / Q000494:pharmacology; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000378:metabolism; D011817:Rabbits / Q000276:immunology; D012867:Skin / Q000032:analysis
D007976:Leupeptins; D000185:Actinin; D004492:Edetic Acid; C032854:leupeptin