Multielectrode Arrays as a Means to Study Exocytosis in Human Platelets

Abstract

Platelets are probably the most accessible human cells to study exocytosis by amperometry. These cell fragments accumulate biological amines, serotonin in particular, using similar if not the same mechanisms as those employed by sympathetic, serotoninergic, and histaminergic neurons. Thus, platelets have been widely recognized as a model system to study certain neurological and psychiatric diseases. Platelets release serotonin by exocytosis, a process that entails the fusion of a secretory vesicle to the plasma membrane and that can be monitored directly by classic single cell amperometry using carbon fiber electrodes. However, this is a tedious technique because any given platelet releases only 4–8 secretory d-granules. Here, we introduce and validate a diamond-based multielectrode array (MEA) device for the high-throughput study of exocytosis by human platelets. This is probably the first reported study of human tissue using an MEA, demonstrating that they are very interesting laboratory tools to assess alterations to exocytosis in neuropsychiatric diseases. Moreover, these devices constitute a valuable platform for the rapid testing of novel drugs that act on secretory pathways in human tissues.

Molecular Design of SERTlight: A Fluorescent Serotonin Probe for Neuronal Labeling in the Brain

“…In addition to brain imaging studies, SERTlight may also find use in other applications. A recent discovery indicated that platelets in a subset of Parkinson’s patients show reduced SERT expression levels. , Moreover, a link between platelets and depression has been postulated but remains poorly characterized . Platelets are recognized as 5HT storage units and models for studying 5HT uptake and release .…”

Analytical Determination of Serotonin Exocytosis in Human Platelets with BDD-on-Quartz MEA Devices

“…Human platelet isolation was conducted by modifying the Abcam ® method (Cambridge, UK) [16,17]. Briefly, nine milliliters of blood were taken by venipuncture from 10 participating volunteers.…”

“…Blood samples were subjected to serial centrifugation at room temperature with no brake [17]: (i) 200× g for 20 min; separating two-thirds of the supernatant or PRP (platelet-rich plasma); (ii) the PRP was mixed in a 1:1 ratio with a solution of HEPES buffer supplemented with prostaglandin E 1 (1 µM, final concentration). The mixture was centrifuged at 100× g for 15 min; (iii) the supernatant resulting from this second centrifugation was transferred to a sterile tube and centrifuged at 800× g for 20 min.…”

“…(iv) This pellet was resuspended in 5 mL of buffer 2. The final density of the platelet suspension was adjusted to 10 5 /µL with buffer 2 and was tested by turbidimetry and hemocytometer counting [17].…”

“…They do not synthesize serotonin but avidly uptake it from blood; therefore, over 90% of the blood serotonin is in platelets. Serotonin is mainly stored and released through exocytosis by the δ-granules [16,17]. Amperometry has been applied for monitoring serotonin exocytosis in platelets, including humans, notably by the group of Haynes using single-cell recordings with conventional glass-encapsulated carbon fiber amperometry [18][19][20][21].…”