Agonist-induced calcium response in single human platelets assayed in a microfluidic device.
To facilitate drug discovery directed toward platelet-specific targets, we developed a platelet isolation and fluorophore-loading method that yields functionally responsive platelets in which we were able to detect agonist-induced calcium flux using a microfluidics-based screening platform. The platelet preparation protocol was designed to minimize preparation-induced platelet activation and to optimize signal strength. Measurement of platelet activation, as monitored by ratiometric determination of agonist-induced calcium flux in fluor-loaded human platelets, was optimized in a macrosample cuvette format in preparation for detection in a microfluidic chip-based assay. For the microfluidic device used in these studies, a cell density of 1 to 2 x 10(6) platelets per milliliter and a nominal flow rate of 5 to 10 nl per second provided optimal event resolution of 5 to 20 platelets traversing the detection volume per unit time. Platelets responded in a dose-dependent manner to adenosine diphosphate and protease-activating peptide (PAR) 1 thrombin receptor-activating peptide (TRAP). The work presented here constitutes proof-of-principle experiments demonstrating the enabling application of a microfluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets.
Tran
Louie
L
Caliper Technologies, Mountain View, CA 94043, USA.
Farinas
Javier
J
Ruslim-Litrus
Lily
L
Conley
Pamela B
PB
Muir
Craig
C
Munnelly
Kevin
K
Sedlock
David M
DM
Cherbavaz
Diana B
DB
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D046210:Microfluidic Analytical Techniques; D010455:Peptides / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000819:agonists
D010455:Peptides; D044463:Receptor, PAR-1; D000244:Adenosine Diphosphate; D002118:Calcium