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Single-cell amperometry reveals platelets’ secrets dense-body (␦) granule, which is where sity of North Carolina Chapel Hill, the Platelets are the smallest and simplest of researchers first aspirated a carbon fiber cells, lacking a nucleus and much of the platelets store serotonin that they have into a glass capillary, then drew it under biochemical machinery that drives every taken up from the bloodstream. heat into two microelectrodes with taother cell in the body. But their simplicBy measuring the width of the pulse ity belies their key role in heat half-height, the investigators mostasis, the multistep biodetermined that the release chemical cascade that stops process was completed in just bleeding. In a recent AC paper over 7 ms. Area-under-the(DOI 10.1021/ac8024202), curve measurements and TEM Christy Haynes and colleagues data enabled Haynes and her at the University of Minnesota colleagues to calculate that aldescribe their use of single-cell though the size of an individual amperometry to tease out granule varies, the concentramany of the details of serototion of serotonin in each grannin release from individual ule remains constant at ⬃0.5 plateletsOa key step in triggerM. “It’s always been assumed ing clot formation and vessel that platelets release their repair. “Using carbon-fiber chemical messengers in a fixed Schematic of a typical experimental setup for measuring serotonin microelectrodes, we’ve been concentration just like other release from platelet δ-granules. Each peak in the amperometric able to make the first realsecreting cells such as neurons readout corresponds to the quantized release of serotonin from inditime, dynamic measurements do, but this is the first time that vidual granules. The varying heights of the peaks correspond to the showing that serotonin release we have hard data showing that changing number of serotonin molecules released by granules of from platelets is quantal in nathis is, in fact, what’s actually different sizes. ture,” says Haynes. happening,” says Haynes. Until now, researchers’ The demand from biomedipered tips. After coating the microelechard-won knowledge about serotonin cal researchers for just such a technique trodes with an epoxy resin, they polished has been surprising, according to release from platelets came from using techniques such as HPLC and radiomet- the tips to a 45° angle that maximizes Haynes. “It’s been amazing how many the area of electrode-to-cell contact. To ric assays. But these methods can only people are suggesting experiments that measure the average release over time by conduct amperometric measurements, we should do,” she says. For example, washed platelets were deposited into a a large number of cells. Transmission Haynes is collaborating with Minnesota temperature-controlled chamber. The electron microscopy (TEM) provides colleague James White, an authority on information about the structural changes investigators applied a 700-mV positive blood diseases. Together, they are using potential to the working electrode, posi- single-cell amperometry to study the that occur upon release from individual tioned it tightly onto a platelet, and platelets, but such measurements are biochemistry and biophysics of platelets then activated the platelet using a static, representing just a snapshot in from patients with inherited platelet dis3-second dose of ionomycin administime. eases such as Hermansky⫺Pudlak syntered through a glass pipette positioned In a previous paper published in Biodrome, a type of albinism accompanied near the cell of interest. chemistry (2008, 47, 7020⫺7024), by a tendency to bleed excessively. Activated platelets secrete serotonin Haynes and colleagues described a Haynes’s group is also using the techthat is immediately oxidized by the apmethod for using glass-encased carbonnique to study the way that platelets plied potential, registering as a change in respond to cholesterol, alcohol, and horfiber microelectrodes to conduct ampercurrent flowing between the working ometric measurements of individual mone exposure. In addition, she and her and reference electrodes. On the basis of colleagues are looking at how serotonin platelets. “The incredibly small size of a a 3D random-walk simulation of detecsingle platelet creates a very challenging release from platelets is affected by selection efficiency, Haynes says she is confianalytical problem if you want to meative serotonin reuptake inhibitors, the dent that the detected change in current most commonly used class of antidesure chemical messenger release from represents complete oxidation of every one platelet in real time,” she explains. pressants. “I’ve really become interested molecule of serotonin released. Each Using techniques first developed by in how these drugs influence a very basic current pulse therefore corresponds to Mark Wightman, Haynes’s postdoctoral property of platelets,” she says. —Joe Alper adviser, and Ralph Adams at the Univer- the serotonin released from a single 10.1021/AC900440G 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 03/20/2009
MAY 1, 2009 / ANALYTICAL CHEMISTRY
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