Visualization of thrombin receptors on mouse embryo fibroblasts using fluorescein-amine conjugated human...

The localization of thrombin receptors on mouse embryo (ME) cells has been examined by direct fluorescence microscopy using a fluorescein amine-labeled thrombin. Two fluorescein amines, 4-(N-2-aminoethyl thioureal)-fluorescein and 4-(N-5-aminohexyl thioureal)-fluorescein, were synthesized and attached to the carbohydrate moiety of highly purified human alpha-thrombin by periodate oxidation of the carbohydrate and selective reduction of the Schiff's base using sodium cyanoborohydride. Preparations of fluorescent thrombin with from 1 to 4 fluoresceins per molecule of thrombin retained their ability to proteolytically cleave fibrinogen to form fibrin clots, to bind to thrombin receptors on ME cells, and to initiate cell division. After incubating mitogenic concentrations of the fluorescein amine labeled thrombin with ME cells at 4 degrees C, a diffuse fluorescent pattern was observed over the surface of the ME cells. This diffuse pattern was specific: it was not observed on cells from parallel cultures incubated with fluorescent thrombin plus a 20-fold excess of unlabeled thrombin. Thus, thrombin receptors appear to be distributed randomly over the surface of ME cells prior to interaction with thrombin. Increasing the temperature to 37 degrees C following binding at 4 degrees C resulted in a rapid dissociation of the fluorescent pattern from the cells leaving only the autofluorescent vesicles. This result may reflect the unique ability of thrombin to proteolytically cleave its own receptor.