Modulation of P-selectin expression on isolated human platelets by an NO donor assessed by a novel ELISA application.
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 degrees 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 x 10(7)/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 microM) and epinephrine (100 microM). 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 microM) 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.
Whiss
P A
PA
Department of Pharmacology, Faculty of Health Sciences, Linköping, Sweden. per.whiss@hul.liu.se
Andersson
R G
RG
Srinivas
U
U
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis; D001792:Blood Platelets / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D004837:Epinephrine / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins; D009569:Nitric Oxide / Q000502:physiology*; D009599:Nitroprusside / Q000494:pharmacology*; D019007:P-Selectin / Q000032:analysis*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis; D060245:Tetraspanin 29; D013917:Thrombin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C554869:CD9 protein, human; D008562:Membrane Glycoproteins; D019007:P-Selectin; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D060245:Tetraspanin 29; D009599:Nitroprusside; D009569:Nitric Oxide; D000244:Adenosine Diphosphate; D006632:Histamine; D013917:Thrombin; D004837:Epinephrine