Stimulus responses and amyloid precursor protein processing in DAMI megakaryocytes.

Platelets, when released as anuclear cells by their precursor megakaryocytes, already carry soluble proteolytic fragments of the amyloid precursor protein (APP) within their alpha-granules and intact APP in the alpha-granule membranes. In response to activation signals elicited by physiologic stimuli such as thrombin, platelets release their granules' soluble contents and translocate granule membrane-bound proteins to the plasma membrane. Because platelets carry >90% of the circulation's APP, activated platelets have been implicated as origins of the beta-amyloid peptide fragment of APP (A beta), whose deposition in the cerebrovasculature is characteristic of Alzheimer's disease. We have therefore studied the APP contents and proteolytic processing in resting DAMI human megakaryocytic cells, along with the consequences of the activation of these cells by thrombin, comparing the results in each case to those with human platelets. Resting and PMA-differentiated DAMI cell contents were examined by Western blotting, immunoprecipitation, or metabolic labeling with sulfur 35-labeled methionine during culture, while plasma membrane-bound APP was evaluated by flow cytometry. Activation was followed by changes in cytoplasmic calcium concentration ((Ca++)in) and in membrane potential. Like platelets, DAMI cells exhibited a thrombin dose-dependent delta(Ca++)in, and membrane potential change; in contrast to the surface of a platelet, the surface of an agranular resting DAMI cell expresses granule-membrane proteins (APP and CD63) that appear on platelets only after activation. DAMI cell culture with 35S-labeled methionine confirmed that megakaryocytes synthesize large amounts of APP, of slightly higher molecular weight, and degrade their APP extensively before platelets are formed.

Moderate and advanced Alzheimer's patients exhibit platelet activation differences.

We previously reported that platelets from advanced sporadic Alzheimer's disease (AD) patients exhibit two defects: first, an aberrant signal transduction presenting as a thrombin-induced hyperacidification, which is more severe for donors with the apolipoprotein E4 allele (apoE4), and second, an AD-specific Amyloid Precursor Protein (APP) processing defect that presents as retention of APP on the activated platelets' surface and in independent of the apo E allele. This retention of membrane APP correlates with decreased release of soluble APP. To determine at what stage in the disease progression these defects appear, we performed signal transduction and secretion studies on moderate AD patients. Thrombin-activated platelets from these patients do not exhibit either hyperacidification or APP retention; their APP processing and secretion are normal by Western blotting, suggesting that the two platelet defects appear in the advanced stages of AD.

Activated Alzheimer disease platelets retain more beta amyloid precursor protein.

Upon activation, platelet alpha-granules' soluble contents are secreted and membrane-bound contents are translocated to the plasma membrane. Membrane-bound proteins include the beta-amyloid precursor protein (APP) from which the beta-amyloid (A beta) deposits found surrounding the cerebrovasculature of patients with Alzheimer's Disease (AD) may originate. We show here that activated platelets from AD patients exhibit less APP processing, retain more of the protein on their surface, and secrete less as soluble fragments than do controls. Surface labeling demonstrated that there is little APP or CD62 on the surface of resting platelets. Upon activation, control platelets exhibited more of both proteins on their surface, while advanced AD patients exhibited similar amounts of CD62 as controls, but retained significantly more surface APP. AD platelets secreted similar amounts of most soluble alpha-granule contents as controls, but less APP fragments. Together these results suggest a processing defect that may account for greater deposition of A beta-containing products in the vasculature to which activated platelets adhere.