Surface modification with polyethylene glycol-corn trypsin inhibitor conjugate to inhibit the contact factor pathway...

Blood contacting surfaces bind plasma proteins and trigger coagulation by activating factor XII (FXII). The objective of this work was to develop blood contacting surfaces having the dual properties of protein resistance and inhibition of coagulation. Gold was used as a model substrate because it is amenable to facile modification using gold-thiol chemistry and to detailed surface characterization. The gold was modified with both polyethylene glycol (PEG) and corn trypsin inhibitor (CTI), a potent and specific inhibitor of activated FXII (FXIIa). Two methods of surface modification were developed; sequential and direct. In the sequential method PEG was first chemisorbed on gold; CTI was then attached to the PEG. In the direct method a conjugate of PEG and CTI was first prepared; the conjugate was then immobilized on gold. The surfaces were characterized by water contact angle and XPS. Biointeractions with the modified surfaces were assessed by measuring fibrinogen adsorption from buffer and plasma and by immunoblot analysis of eluted proteins after plasma exposure. Inhibition of FXIIa, autoactivation of FXII, and clotting times of plasma in contact with the surfaces were also measured. Both the sequential and direct surfaces showed reduced protein adsorption, increased FXIIa inhibition and longer clotting times compared with controls. Although the CTI density was lower on surfaces prepared using the sequential method, surfaces so prepared exhibited greater CTI activity than those generated by the direct method. It is concluded that the activity of immobilized PEG-CTI depends on the method of attachment and that immobilized CTI may be useful in rendering biomaterials more blood compatible.