Modulation of P-selectin expression on isolated human platelets y an NO donor assessed by a novel ELISA application
1997 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 200, no 1-2, 135-143 p.Article in journal (Refereed) Published
Adhesion molecules such as P-selectin are potential markers for evaluating platelet activation and studying the role of cell-cell interactions in numerous biological processes related to hemostasis and inflammation. The expression of P-selectin and related molecules has previously been quantified with different techniques. As an alternative to the most common method, flow cytometry, we have developed a useful ELISA method to simultaneously analyse 96 samples for platelet expression of P-selectin. Samples may be stored for at least 7 days at 4°C prior to analysis. The method is simple, reproducible, flexible and requires only standard equipment. Washed platelets (WP) from healthy male volunteers, at a concentration of 1 × 107/microtiter plate well, were stimulated with various known platelet activators and fixed with 0.1% formaldehyde for 10 min. The fixed WP were centrifuged to form a confluent layer in the wells and then incubated with optimal dilutions of primary antibodies (1/2000) directed against P-selectin, CD41, CD9 and secondary antibodies conjugated with alkaline phosphatase. Our results show that P-selectin expression on WP increases significantly upon stimulation with thrombin (0.1–1.0 U/ml), ADP (10 μM) and epinephrine (100 μM). The induction of P-selectin expression by thrombin is fast and has different kinetics depending on the concentration of the agonist. Prior incubation with the nitric oxide donor SNAP (10 μM) inhibits the up-regulation of P-selectin induced by sub-maximal concentrations of thrombin (p < 0.05). This ELISA is suitable for studying the expression and regulation of P-selectin and other surface molecules on human platelets in various pathological states.
Place, publisher, year, edition, pages
1997. Vol. 200, no 1-2, 135-143 p.
Platelet, Adhesion molecule, P-selectin, CD62P, ELISA, Nitric oxide
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:liu:diva-79738DOI: 10.1016/S0022-1759(96)00198-6OAI: oai:DiVA.org:liu-79738DiVA: diva2:544101