Platelet aggregation monitored in a 96 well microplate reader is useful for evaluation of platelet agonists and antagonists.
Optimal conditions for a method to simultaneously measure aggregation in 96 samples using a microplate reader were developed. The temperature of the assay was set at 25 degrees C, the optimal platelet concentration range was determined to be from 1-3 x 10(8) per mL, the assay volume was determined to be best at 100 microL and an agitation rate of setting #5 on the vortex was found to yield the most reliable aggregation response. After these initial assay parameters were established, EC50 values for standard platelet agonists including ADP, thrombin, collagen and thrombin receptor activating peptides were determined using the plate assay and compared to those obtained by measuring light transmittance in an aggregometer. The results were quantitatively similar, and qualitatively the shapes of the aggregations as monitored by both methods were characteristic of those expected for each agonist. The use of this assay was then extended to quantitate the inhibition of aggregation by antagonists of the fibrinogen receptor as well as by an inactive thrombin receptor peptide and by antibodies against the thrombin receptor. This method provided useful data for characterization of both platelet agonists and antagonists and should be useful for future platelet aggregation studies.
Bednar
B
B
Merck Research Laboratories, West Point, PA 19486, USA.
Condra
C
C
Gould
R J
RJ
Connolly
T M
TM
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D003094:Collagen / Q000494:pharmacology; D006405:Hematology / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D010446:Peptide Fragments / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects; D018179:Receptors, Thrombin / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology
D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D018179:Receptors, Thrombin; C082835:thrombin receptor peptide (42-47); D000244:Adenosine Diphosphate; D003094:Collagen; D013917:Thrombin