Probing the interaction of coagulation factors with phospholipid vesicle surfaces by surface plasma resonance.
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 N(2)-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.
Wikström
Angelica
A
Department of Applied Physics, Chalmers University of Technology, S-431 90 Göteborg, Sweden.
Deinum
Johanna
J
D001779:Blood Coagulation Factors / Q000737:chemistry* / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
D001779:Blood Coagulation Factors; D010743:Phospholipids; D004247:DNA