Ph.D. Thesis Defense by Bryan Marshall
Wednesday, March 27, 2002

(Dr. Cheng Zhu, advisor)

"Force and Bond Lifetime Relationship of the P-selectin/PSGL-1 Interaction"

Abstract

P-selectin is a cell adhesion molecule that plays a critical role in the immune system response.  P-selectin initiates the tethering and rolling interactions between leukocytes and the venular endothelium by interacting with its ligand PSGL-1.  The ability of these molecules to function in this mechanically stressful environment has been attributed to the rapid on and off rates and a bond lifetime that is only slightly affected by changes in force.?

Using an atomic force microscope, the relationship between bond lifetime and force was explored.  The results showed that the P-selectin/PSGL-1 interaction displayed a biphasic relationship between force and bond lifetime with the bond lifetime initially increasing with force and then decreasing.  This counter intuitive behavior confirms the existence of catch bonds.

Both a dimeric wild type and a monomeric recombinant form of PSGL-1, were examined.  While the lifetime versus force data for both molecules had qualitatively similar biphasic shapes, the P-selectin/PSGL-1 curve was shifted relative to the P-selectin/rPSGL-1 curve towards larger forces and longer lifetimes.  The formation of a dimeric bond between wild type PSGL-1 and P-selectin explains the discrepancies between the two data sets.

The mechanical properties of the receptor ligand complex were studied with two separate methods.  Both methods yielded similar results.  The molecular complex displayed a linear relationship between force and molecular extension.  P-selectin accounted for the mechanical compliance of the molecular complex.

Dynamic Force Spectroscopy (DFS) measures bond rupture force as a function of loading rate to reveal energy barriers along the unbinding pathway.  The P-selectin/PSGL-1 interaction was investigated with this technique.  The DFS analysis predicted very short bond lifetimes and no catch bond.  These results were in direct contrast with the bond lifetime measurements.  The current underlying theory, which predicts that both of these assays should predict the same relationship, assumes that off rate is a function of force only.  A new paradigm must be considered, where off rate is considered a path dependent variable.