STIMULUS RESPONSES AND AMYLOID PRECURSOR PROTEIN PROCESSING IN DAMI MEGAKARYOCYTES

Platelets, when released as anuclear cells by their precursor megakary ocytes, already carry soluble proteolytic fragments of the amyloid pre cursor protein (APP) within their alpha-granules and intact APP in the alpha-granule membranes. In response to activation signals elicited b y physiologic stimuli such as thrombin, platelets release their granul es' soluble contents and translocate granule membrane-bound proteins t o the plasma membrane. Because platelets carry >90% of the circulation 's APP, activated platelets have been implicated as origins of the bet a-amyloid peptide fragment of APP (A beta), whose deposition in the ce rebrovasculature is characteristic of Alzheimer's disease, We have the refore studied the APP contents and proteolytic processing in resting DAMI human megakaryocytic cells, along with the consequences of the ac tivation of these cells by thrombin, comparing the results in each cas e to those with human platelets. Resting and PMA-differentiated DAMI c ell contents were examined by Western blotting, immunoprecipitation, o r metabolic labeling with sulfur 35-labeled methionine during culture, while plasma membrane-bound APP was evaluated by flow cytometry, Acti vation was followed by changes in cytoplasmic calcium concentration (( Ca++)(in)) and in membrane potential. Like platelets, DAMI cells exhib ited a thrombin dose-dependent Delta(Ca++)(in), and membrane potential change; in contrast to the surface of a platelet, the surface of an a granular resting DAMI cell expresses granule-membrane proteins (APP an d CD63) that appear on platelets only after activation. DAMI cell cult ure with S-35-labeled methionine confirmed that megakaryocytes synthes ize large amounts of APP, of slightly higher molecular weight, and deg rade their APP extensively before platelets are formed.