728 Thromb Haemost 2002; 87: 728–34 © 2002 Schattauer GmbH, Stuttgart Brain-derived Neurotrophic Factor Is Stored in Human Platelets and Released by Agonist Stimulation Hironobu Fujimura, C. Anthony Altar 1 , Ruoyan Chen, Takashi Nakamura, Takeshi Nakahashi, Jun-ichi Kambayashi, Bing Sun, Narendra N. Tandon 2 Otsuka Maryland Research Institute, LLC, Rockville, MD, USA Keywords BDNF, platelets, aggregation, secretion, shear stress Summary Brain-derived neurotrophic factor (BDNF), a member of the neuro- trophin family, plays critical roles in the survival, growth, and mainte- nance of brain and peripheral neurons. We report the presence of BDNF protein in human platelets and its release upon agonist stimulation. The BDNF content of washed platelets varied widely, from 3.5 to 67 ng/ 4  10 8 platelets, averaging 25.2 ± 21.2 ng/4  10 8 platelets (mean ± SD). The BDNF concentration in platelet-poor plasma was low (1.7 ± 1.7 ng/ml, n = 11). Thrombin, collagen, the Ca ++ ionophore A23187, and shear stress each induced a rapid release of BDNF from platelets. Up to only half of platelet BDNF was secreted upon agonist stimula- tion, suggesting that platelets may have a non-releasable pool of BDNF, or that the released BDNF binds to a recognition site on the platelet sur- face and is internalized, as occurs with serotonin. However, the cognate BDNF receptor, TrkB, was not detected in platelets. Nevertheless, the ability of BDNF to bind washed platelets was shown by FACS analysis confocal microscopy and by the binding and apparent internalization of [ 125 I]-BDNF by platelets. A very high affinity site (K d = 130  10 -15 M, ~80 sites/platelet) and a moderately high affinity site (K d = 20 nM, ~3750 sites/platelet) were identified. The BDNF content in two mega- karyocytic cell lines, DAMI and Meg-01, was only 0.1% of the content measured in platelets. No BDNF mRNA was detected by Northern blotting in these cell lines or in platelets. The pituitary gland was also ruled out as a source for platelet BDNF, since the BDNF content of rat platelets did not decrease 2 weeks after hypophysectomy. Thus, plate- let BDNF is not acquired from the megakaryocyte or pituitary gland, but is probably acquired from other sources via the blood circulation. Platelets appear to bind, store and release BDNF upon activation at the site of traumatic injury to facilitate the repair of peripheral nerves or other tissues that contain TrkB. Introduction Since the discovery of nerve growth factor (NGF) (1, 2), a family of NGF-related proteins has been found that confers differentiating, functional, and survival-promoting effects on cells of the nervous system. These proteins include NGF, brain-derived neurotrophic factor (BDNF) (3, 4), neurotrophin-3 (NT-3) (5), and neurotrophin-4 (NT-4) (6). These small and highly basic proteins bind to the low affinity NGF receptor, also known as p75 (7, 8). They also bind with higher affinity to the three tropomyosin receptor-related tyrosine kinases, or Trks. These high affinity receptors are designated as TrkA, TrkB, and TrkC (9, 10), and preferentially bind NGF, BDNF and NT-4, and NT-3 re- spectively. TrkB and TrkC also exist as truncated proteins that lack intracellular kinase domains (11). Among the neurotrophins, BDNF has been of particular interest be- cause of its potential role in non-neuronal tissues. BDNF mRNA has been found in the rat aorta (12), kidney, submandibular gland, ovary, dorsal root ganglia (13), heart (14), retina, muscle, lung (15, 16), T and B immune cells (17), endothelial cells and platelets (18). The TrkB re- ceptor is present in peripheral targets including vascular endothelial cells (19), vascular smooth muscle (20), dorsal root ganglia neurons (21), Schwann cells (22), spleen white pulp (23), B (24) and T (25) lym- phocytes, and intestinal ganglion cells, glia (26), and endocrine cells (27). Human platelets have been shown to express BDNF mRNA and contain biological activity ascribed to BDNF protein (18). Interesting- ly, human serum contains BDNF at far greater concentrations than does human plasma (28, 29). The strong correlation between serum seroto- nin, a marker for platelet activation, and serum concentration of BDNF, indicated that BDNF may be released by platelets during clotting pro- cess (29). However, there has been no direct measure of BDNF from platelets, nor there has been an evaluation of BDNF release from plate- lets, the role of classical agonists of platelet activation in this process, the sequestration of BDNF by platelets, or the potential source of BDNF that is stored in platelets. To evaluate these possibilities, plate- lets were activated in the present study by various physiological ago- nists such as ADP, epinephrine, serotonin, collagen, thrombin and shear stress and the resulting BDNF release was measured. We also demonstrated the binding of exogenous BDNF to platelets and exam- ined the potential contribution of several sources to the BDNF that is present in platelets. Materials and Methods Acid-insoluble equine tendon fibrillar collagen (Chrono Par) was obtained from Chrono-Log Corp., (Broomall, PA). [2- 14 C]Serotonin (58 mCi/mmol) was purchased from Amersham Corp. Prostaglandin E 1 (PGE 1 ) was from Cayman Chemicals (Ann Arbor, MI). Kits for the measurement of BDNF, chicken anti- human BDNF IgY, and FITC-anti chicken IgY were purchased from Promega Corp (Madison, WI). Kits for platelet factor 4 (PF4A) were obtained from Diagnostica Stago (France). 125 I-labeled BDNF was obtained from NEN Life Science Products, Inc. (Boston, MA). Human recombinant BDNF was from Biodesign International (Kennebunk, ME) and also was generously provided by Regeneron Pharmaceuticals Inc. (Tarrytown, NY). Peptides Arg-Gly-Asp- Ser (RGDS) and Gly-Pro-Arg were obtained from Penninsula Labs., Inc. (Belmont, CA). All other chemicals were from Sigma (St. Louis, MO). 1 Present address: Gene Discovery Department, Psychiatric Genomics, Inc., 19 Firstfield Road, Gaithersburg, MD 20878, USA 2 Correspondence to: Department of Thrombosis Research, Otsuka Maryland Research Institute, LLC, 9900 Medical Center Drive, Rockville, MD 20850, USA – Tel.: 240-683-3301; Fax: 301-424-9054 ; E-mail: narendrt @otsuka.com For personal or educational use only. 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