Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood.
Platelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or alphaIIbbeta3 and GPIbalpha were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.
Laboratory for Thrombosis Research, KU Leuven-Campus Kortrijk, Kortrijk, Belgium. i.salles@imperial.ac.uk
Isabelle
I
Salles
Katleen
K
Broos
Alexandre
A
Fontayne
Tímea
T
Szántó
Changgeng
C
Ruan
Alan T
AT
Nurden
Karen
K
Vanhoorelbeke
Hans
H
Deckmyn
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000244:Adenosine Diphosphate; D000527:Alprostadil / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D016718:Arachidonic Acid; D001792:Blood Platelets / Q000187:drug effects / Q000276:immunology / Q000502:physiology; D002352:Carrier Proteins; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006023:Glycoproteins / Q000276:immunology; D057166:High-Throughput Screening Assays; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology; D039421:Integrin alpha2 / Q000276:immunology; D039661:Integrin beta3 / Q000276:immunology; D015588:Observer Variation; D019007:P-Selectin / Q000276:immunology; D010455:Peptides; D015539:Platelet Activation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010979:Platelet Function Tests; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology
C048566:A1BG protein, human; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D006023:Glycoproteins; C056564:ITGA2B protein, human; C061248:ITGB3 protein, human; D007136:Immunoglobulins; D039421:Integrin alpha2; D039661:Integrin beta3; D019007:P-Selectin; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; C435908:collagen-related peptide; D016718:Arachidonic Acid; D000244:Adenosine Diphosphate; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D026423:S-Nitroso-N-Acetylpenicillamine; D000527:Alprostadil