Possible Role of Calpain in Normal Processing of -Amyloid Precursor Protein in Human Platelets Ming Chen,* , † Jacques Durr,‡ , § and Hugo L. Fernandez* ,¶,1 *Neuroscience Research Laboratory; ‡Experimental Nephrology Laboratory, Medical Research and Development Service (151), Bay Pines VA Medical Center, Bay Pines, Florida 33744; and †Department of Pharmacology and Therapeutics; §Department of Internal Medicine, ¶ Department of Neurology and Department of Physiology and Biophysics, University of South Florida College of Medicine, Tampa, Florida, 33612 Received May 15, 2000 Abnormal proteolytic processing of -amyloid pre- cursor protein (APP) underlies the formation of amy- loid plaques in aging and Alzheimer’s disease. The proteases involved in the process have not been iden- tified. Here we found that spontaneous proteolysis of intact APP in detergent-lysed human platelets gener- ated a N-terminal fragment that was immunologically indistinguishable from secreted APP, reminiscent of the action of a putative -secretase. This proteolysis of APP was inhibited by EDTA, suggesting that a metal- dependent protease was involved. Among the several metals tested, calcium was the only one that enhanced APP proteolysis and the reaction was blocked by EGTA as well as by several calpain inhibitors. The APP fragments generated by spontaneous proteolysis in platelet lysates were identical to those produced by exposure of partially purified APP to exogenous cal- pain. Finally, the secretion of APP from intact plate- lets was inhibited by cell-permeable calpain inhibi- tors. Taken together, these results suggest that normal processing of APP in human platelets is mediated by a calcium-dependent protease that exhibits calpain-like properties. © 2000 Academic Press Key Words: Alzheimer; amyloid; calcium; calpain; aging. Alteration in the proteolytic processing of -amyloid precursor protein (APP) leading to amyloid plaques is a prominent feature in patients with Alzheimer’s dis- ease (AD). Amyloid plaques predominantly contain -amyloid peptide (A), a 39 – 43-amino acid peptide derived from APP. Physiologically, APP is mostly cleaved within the A domain at the Lys 16 -Leu 17 bond on the cell surface by an unidentified protease known as -secretase, producing a secreted 100 –120 kDa form of APP (APP s ). Alternatively, APP is cleaved by - and -secretases to produce A (1– 4). Considerable research efforts have been devoted to the identification of the foregoing secretases and a number of proteases have been proposed as potential candidates for putative -secretase. These include mul- ticatalytic protease, gelatinase A, metalloendopepti- dase, proteosome, two yeast proteases (Yap3 and Mkc7), glycosyl-phosphatidylinositol-linked aspartyl proteases, and TNF--converting enzyme (5–11). In most cells, the major product of -secretase, APP s , is much more abundant than A (4). This characteristic may allow -secretase activity to be traced with less difficulty. Previous studies including our own have shown that platelets are the primary circulating repos- itory for APP and A (12–14). Inasmuch as APP pro- cessing in platelets is similar to that in neuronal cells, platelets offer an excellent model for the study of APP processing. MATERIALS AND METHODS Materials Fresh platelets were obtained from the blood of healthy volunteers. Monoclonal antibodies 22C11 (to APP N-terminus) was purchased from Boehringer Mannheim (Indianapolis, IN), Dako (to A8-17) from Dako Co. (Carpinteria, CA), and 4G8 (to A17-24) from Senetek PLC (Maryland Heights, MO). Polyclonal antibody 369 to C-terminal residues 645– 694 of APP 695 was a gift from Dr. Samuel Gandy (Rockefeller University). The epitope-specificities of these antibodies are shown (Fig. 1). Peptides A1-16 and A17-28 were from QCB, Inc. (Hopkinton, MA). Trypsin and protease K were from Boehringer Mannheim (Indianapolis, IN). Calpastatin (recombinant) was from CalBiochem (San Diego, CA). Thrombin, iodoacetate, benzamidine, aprotinin, pepstatin A, calpeptin, E64d, E64c, A23187, calpain (80 kDa subunit of rabbit skeletal muscle m-calpain), calpain inhibitor I and II, and a calpastatin-derived peptide (27 amino acids corre- sponding to residues 162–188 of muscle calpastatin) were all from Sigma (St. Louis, MO). 1 To whom reprint request should be addressed at Neuroscience Research Laboratory, Medical Research & Development Service (151), Department of Veteran Affairs Medical Center, P.O. Box 4125, Bay Pines, FL 33744. E-mail: hugo.fernandez@med.va.gov. Biochemical and Biophysical Research Communications 273, 170 –175 (2000) doi:10.1006/bbrc.2000.2919, available online at http://www.idealibrary.com on 170 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.