Immunofluorescent visualization of specifically bound thrombin reveals cellular heterogeneity in number and density of preclustered receptors.

Thrombin binding to formaldehyde-fixed mouse embryo (ME) cells was visualized by indirect immunofluorescence as a dot-like pattern with dots of approximately 500 nm diameter located over the entire cell surface. Experiments comparing the binding of 125I-thrombin and dot appearance on parallel cultures indicate that the immunofluorescent pattern is specific for thrombin-binding to high-affinity receptors. Similar patterns were observed on cells fixed in ethanol or glutaraldehyde prior to thrombin binding and on cells maintained at 4 degrees C. These patterns were also observed in a number of established cell lines. Thus, thrombin receptors may be clustered prior to thrombin binding on all cells with these receptors. Comparing the amount of 125I-thrombin bound to CHO cells with the number of fluorescent dots per cell indicated that each dot represents a cluster of over 1000 receptors. On ME cells, the number of thrombin receptor clusters per cell ranged from fewer than 50 to over 5,000. Based on previous studies, this indicates that on ME cells each cluster contains an average of approximately 200 thrombin binding sites with some cells having few, if any, receptors and others having more than a million receptors per cell.