Plasma protein binding properties to immobilized heparin and heparin-albumin conjugate.
Selective adhesion of plasma proteins to immobilized heparin is considered to be beneficial regarding hemocompatibility of foreign materials in contact with blood. Prothrombin, thrombin, antithrombin III (AT3), and fibrinogen were selected for analysis in an experimental model. Biomolecular interaction analysis employing surface plasmon resonance was utilized to record and analyze their binding properties in real time. Biotinylated heparin, heparin-albumin conjugate, and albumin, respectively, were immobilized onto streptavidin-coated sensors as ligands. Prothrombin did not bind to any of the ligand surfaces and no specific binding of any of the plasma proteins to albumin was observed. Binding kinetics of thrombin to heparin and to heparin-albumin conjugate were calculated using two different methods. For heparin, identical K(D)(equilibrium dissociation constant) values of 61 x 10(-9) M were obtained with both methods. For the conjugate, only slightly different K(D) values of 111 x 10(-9) and 104 x 10(-9) M, respectively, were calculated. The affinity of thrombin toward the heparin-coated surface proved to be higher than its affinity toward the heparin conjugate. The binding pattern of AT3 to both heparin and heparin-albumin conjugate, although specific, was biphasic, possibly due to a conformational change during the binding process. Steady-state kinetic analysis revealed a K(D) value of 281 +/- 24 x 10(-9) M for the heparin surface. For the conjugate surface, a K(D) of 53 +/- 5 x 10(-9) M was calculated, indicating a higher affinity toward heparin-albumin conjugate. A high-affinity binding of fibrinogen to high-density surfaces of both heparin and the conjugate was observed. However, as binding to low-density surfaces was considerably reduced, specificity remained uncertain.
Mirow
Nikolas
N
Department of Thoracic and Cardiovascular Surgery, Heart Centre North-Rhine-Westfalia, Ruhr-University of Bochum, Bad Oeynhausen, Germany. nmirow@hdz-nrw.de
Zimmermann
Bastian
B
Maleszka
Ariane
A
Knobl
Hermann
H
Tenderich
Gero
G
Koerfer
Reiner
R
Herberg
Friedrich W
FW
D000327:Adsorption; D000990:Antithrombin III / Q000378:metabolism*; D001672:Biocompatible Materials; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism*; D012709:Serum Albumin / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013499:Surface Properties; D013917:Thrombin / Q000378:metabolism*
D001672:Biocompatible Materials; D012709:Serum Albumin; D000990:Antithrombin III; D011516:Prothrombin; D006493:Heparin; D013917:Thrombin