MODULATION OF P-SELECTIN EXPRESSION ON ISOLATED HUMAN PLATELETS BY ANNO DONOR ASSESSED BY A NOVEL ELISA APPLICATION

Adhesion molecules such as P-selectin are potential markers for evalua ting platelet activation and studying the role of cell-cell interactio ns in numerous biological processes related to hemostasis and inflamma tion. The expression of P-selectin and related molecules has previousl y been quantified with different techniques. As an alternative to the most common method, flow cytometry, we have developed a useful ELISA m ethod to simultaneously analyse 96 samples for platelet expression of P-selectin. Samples may be stored for at least 7 days at 4 degrees C p rior to analysis. The method is simple, reproducible, flexible and req uires only standard equipment. Washed platelets (WP) from healthy male volunteers, at a concentration of 1 x 10(7)/microtiter plate well, we re stimulated with various known platelet activators and fixed with 0. 1% formaldehyde for 10 min. The fixed WP were centrifuged to form a co nfluent layer in the wells and then incubated with optimal dilutions o f primary antibodies (1/2000) directed against P-selectin, CD41, CD9 a nd secondary antibodies conjugated with alkaline phosphatase. Our resu lts show that P-selectin expression on WP increases significantly upon stimulation with thrombin (0.1-1.0 U/ml), ADP (10 mu M) and epinephri ne (100 mu M). The induction of P-selectin expression by thrombin is f ast and has different kinetics depending on the concentration of the a gonist. Prior incubation with the nitric oxide donor SNAP (10 mu M) in hibits the up-regulation of P-selectin induced by sub-maximal concentr ations of thrombin (p < 0.05). This ELISA is suitable for studying the expression and regulation of P-selectin and other surface molecules o n human platelets in various pathological states.