Abstract 13212: Extensive Reserve Capacity for the Binding and Movement of Fibrinogen on the Surface of Activated Human Platelets
We have previously imaged fibrinogen binding to its integrin αIIb β3 receptor on activated human platelets through labeling with colloidal gold-ligand conjugates followed by light and/or electron microscopy (EM). When αIIb β3 engages cross-linking ligand or antibody, it translocates over the membrane to central areas of spread platelets or platelet aggregates. Centralization of the fibrinogen-receptor complexes clears the outer areas and makes room for additional fibrinogen-receptor interactions. We hypothesized that the number of interactions made possible over time by clearing exceeds the number required to support normal hemostasis. To test this hypothesis, we performed multiple rounds of labeling. First, we exposed surface-activated platelets to two applications of colloidal gold-fibrinogen labels. Live video-DIC microscopy demonstrated labeling in both rounds; however, it was somewhat unclear whether individual platelets bound labels both times. Second, we labeled surface-activated platelets with Alexa Fluor 488-fibrinogen (Alexa-FGN, 1.5 or 0.5 mg/mL) following preincubation with 3 mg/mL unlabeled fibrinogen (plasma concentration). Fluorescence light microscopy demonstrated fibrinogen binding comparable to that observed with buffer preincubation. Third, we tested serial dilutions of Alexa-FGN in 3 mg/mL unlabeled fibrinogen or platelet-poor plasma (PPP), and labeled surface-activated platelets following preincubation with unlabeled fibrinogen or PPP. We observed a dose-dependent decrease in fluorescence intensity, but could clearly detect binding of Alexa-FGN at 32 μg/mL in PPP. Finally, we aggregated gel-filtered platelets with unlabeled fibrinogen, then labeled them with colloidal gold-fibrinogen. Scanning and transmission EM revealed less incorporation of colloidal gold labels into aggregates than when they were formed with labels as the sole fibrinogen source, but labels were still readily found on aggregate surfaces and translocated deep into aggregate interiors. We conclude that the ability of platelets to bind fibrinogen is in great excess of that required for the initial phase of hemostasis, and the excess is likely important to the mechanism of temporally sustained platelet recruitment into thrombi.
- © 2010 by American Heart Association, Inc.