P10 – Probing the interaction of coagulation factors with phospholipid vesicle surfaces by surface plasma resonance

Journal

Analytical Biochemistry, Volume 362, Issue 1, 1 March 2007, Pages 98-107 (Full article)

Authors

Angelica Wikström^a and Johanna Deinum^bOrganization

Organisation

^aDepartment of Applied Physics, Chalmers University of Technology, S-431 90 Göteborg, Sweden

^bDepartment of Molecular Pharmacology, AstraZeneca R&D, S-431 83 Mölndal, Sweden

Abstract

The dynamics of the binding of human coagulation factor Xa (FXa) and prothrombin to small unilamellar vesicles (25% phosphatidylserine, 75% phosphatidylcholine) were compared and quantified by Biacore, using two immobilization techniques. The vesicles were either tagged with different molar ratios of cholesterol–DNA and attached on Au chips or fused directly on L1 chips. The diameter in solution was 145 nm, but the more DNA tags/vesicle the more compressed the immobilized vesicles became; with 30 DNA tags the calculated thickness was 88 nm and with 1 DNA tag it was 138 nm. In both models the affinity for the vesicles was higher for the activated coagulation factors than for the corresponding zymogens. FXa and prothrombin had the highest affinities. The affinity was dependent on the vesicle preparation since overall K_D values were up to 10 times lower for N₂-dried than for vacuum-dried phospholipids, although with apparently fewer binding sites. However, compression of the vesicles had no effect on the K_D. In contrast, the rate constants were dependent on the number of DNA tags; thus deformation of the vesicles was observed. The k_a and k_d for FXa were similar for vesicles attached with 30 DNA tags or fused on the L1 chip but higher with fewer tags and approximately 10 times higher if attached with 1 tag. Thus for controlled kinetic studies immobilized DNA-tagged vesicles should be used.

Published 2006-12-29, at 11:49.