Development of a whole platelet ELISA to detect circulating activated platelets.

Abstract

P-selectin is a granule membrane protein that is expressed on the surface of activated endothelial cells and platelets. Flow cytometry has been used as a means of detecting activated platelets in the circulation by using antibodies to P-selectin and other surface markers. In the study reported here, we developed a whole platelet ELISA for measuring P-selectin on platelets in platelet-rich plasma. Platelet-rich plasma samples for analysis were isolated from fresh blood by centrifugation, fixed with 1.0% paraformaldehyde, and used within 3 hours or after storage at -70 degrees C for up to 10 months. Paraformaldehyde-fixed, phorbol myristate acetate-activated or thrombin receptor peptide-activated platelets were used to construct a standard calibration curve. These platelets were stable after 10 months of storage at -70 degrees C. Interassay variability showed a high degree of correlation, with r = 0.98 +/- 0.03 (n = 12). The accuracy and specificity of the ELISA was verified by using fluorescence-activated flow cytometric analysis and is as sensitive (< or = 0.5%) as flow cytometry for detecting P-selectin expression on platelets. To assess the ability of the platelet ELISA to detect platelet activation in the systemic circulation, we examined 24 patients with unstable angina and 12 age-matched control subjects. Patients with unstable angina demonstrated significantly higher levels of circulating activated platelets than did age-matched control subjects. Although storage-dependent differences in absolute platelet activation levels were found, platelet ELISA results of samples evaluated within either 3 hours or after 10 months of storage were comparable to results obtained by fluorescence-activated flow cytometric analysis.(ABSTRACT TRUNCATED AT 250 WORDS)