Ultrastructural studies of endothelial and platelet receptor binding of thrombin-colloidal gold probes

Endothelial cells and platelets are reported to have receptors for alpha-thrombin. To visualize the binding of alpha-thrombin to these cells, we developed a method to label thrombin with colloidal gold. Formed by electrostatic adsorption of thrombin to the negatively charged gold, the resulting probe is stable for weeks and consists of approximately 30 thrombin molecules adsorbed to each 16.5 nm gold particle. The probe retained about 10% of the enzymatic activity (fibrinogen clotting) of the unlabeled native thrombin and 20% of the ability of the native thrombin to aggregate platelets in platelet-rich plasma (PRP). In PRP, approximately 90% of the observed probes were bound to fibrin strands, with the remaining probes (650 per cell) attached to activated platelets. In contrast, washed, paraformaldehyde-fixed human platelets exhibited a marked increase in probe density (4900 per cell). Time-dependent ultrastructural studies (2-240 min) of binding of the thrombin-gold probe to confluent cultures of porcine aortic endothelial cells revealed that the initial binding (7300 probes per cell) occurred randomly at the cell surface. A limited number (25%) of the probes clustered at coated-pit regions and were internalized (60-240 min). The probe induced a limited amount of cellular retraction similar to that achieved with unlabeled thrombin. These results suggest that the thrombin-gold probe is suitable for investigations of the localization of thrombin receptors on cell surfaces and the interaction of thrombin with these receptors during thrombotic events.