Submitted October 25, 2006
Accepted February 21, 2007
Ligand density dramatically affects integrin 
IIb
3-mediated platelet signaling and spreading
Marketa Jirouskova*, Jyoti K. Jaiswal, and Barry S. Coller
Laboratory of Blood and Vascular Biology, Rockefeller University, New York, NY, United States
Laboratory of Cellular Biophysics, Rockefeller University, New York, NY, United States
* Corresponding author; email: marketa{at}rockefeller.edu.
The impact of ligand density on integrin-mediated cell adhesion and outside-in signaling is not well understood. Using total internal reflection fluorescent microscopy, conformation-specific antibodies and Ca2+ flux measurements, we found that the surface density of fibrinogen affects 
IIb
3-mediated platelet signaling, adhesion, and spreading. Adhesion to fibrinogen immobilized at low-density leads to rapid increases in cytosolic Ca2+ and sequential formation of filopodia and lamellipodia. In contrast, adhesion to high-density fibrinogen results in transient or no increases in Ca2+ and simultaneous formation of filopodia and lamellipodia. IIb3 receptors at the basal surface of platelets engage fibrinogen in a ring-like pattern at the cell edges under both conditions. This engagement is, however, more dynamic and easily reversed on high-density fibrinogen. Src and Rac activity and actin polymerization are important for adhesion to low-density fibrinogen, whereas PKC/PI3 kinases contribute to platelet spreading on high-density fibrinogen. We conclude that two fundamentally different signaling mechanisms can be initiated by a single integrin receptor interacting with the same ligand when it is immobilized at different densities.
