High-resolution probing heparan sulfate–antithrombin interaction on a single endothelial cell surface: single-molecule AFM studies

Abstract

Heparan sulfate (HS) plays diverse functions in multiple biological processes by interacting with a wide range of important protein ligands, such as the key anticoagulant factor, antithrombin (AT). The specific interaction of HS with a protein ligand is determined mainly by the sulfation patterns on HS chain. Here, we reported the probing single-molecule interaction of AT and HS (both wild type and mutated) expressed on endothelial cell surface under near-physiological condition by atomic force microscopy (AFM). Functional AFM imaging revealed the uneven distribution of HS on endothelial cell surface though they are highly expressed. Force spectroscopy measurement with an AT-functionalized AFM tip revealed AT interacts with endothelial HS on the cell surface through multiple binding sites. The interaction essentially requires HS to be N-, 2-O- and/or 6-O-sulfated. This work provides a new tool to probe the HS-protein ligand interaction at a single-molecular level on cell surface to elucidate the functional roles of HS.

A “turn on” fluorescent probe for heparin and its oversulfated chondroitin sulfate contaminant

“… 1 It can bind antithrombin with a high affinity, and the binding significantly enhances antithrombin's inhibition activity towards thrombin and other coagulation factors. 2 Thus, Hep has been widely used in clinics as both prophylactic and therapeutic agents, especially as an anticoagulant in surgery. 3 The Hep therapeutic dosage should be maintained within a range of 2–8 U mL –1 for cardiovascular surgery, and a range of 0.2–1.2 U mL –1 for post-operative and long-term therapy.…”

Rupture Forces among Human Blood Platelets at different Degrees of Activation

“…Single-cell force spectroscopy (SCFS) experiments measure adhesive interaction forces and binding kinetics under physiologically relevant conditions at single cell regimes and contribution of single or a few molecules to such interactions 32 . SCFS is increasingly used to study ligand-receptor interactions in living cells 33 34 35 , microbial cell adhesion 36 , integrin and glycocalyx mediated contributions to cell adhesion 37 , as well as single cell-cell interaction 32 38 . For measurement of interaction forces between two cells with SCFS, a single cell is immobilized at the end of a cantilever and another cell is fixed on a solid substrate.…”

Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix

“…The cleavage of perlecan’s domain I GAG chains with heparinases, but not chondroitinase, completely prevented binding to collagen I (PMID 15738663). Single molecule approaches have been used to measure the binding strength of heparan sulfate, with one study finding that as high as 310 pN was required to rupture a multivalent heparan sulfate-ligand interaction [48]. This high strength of GAG-ligand interaction may explain our observations that perlecan with GAG showed a shorter persistent length L p (1.2 nm vs. 22 nm) and a higher stretch elasticity K (2000 vs. 890 pN) than perlecan devoid of GAG (Fig.…”