Dual surface modification with PEG and corn trypsin inhibitor: Effect of PEG:CTI ratio on protein resistance and anticoagulant properties

Abstract

The objective of this study was to investigate the bioactivity and protein-resistant properties of dual functioning surfaces modified with PEG for protein resistance and corn trypsin inhibitor (CTI) for anticoagulant effect. Surfaces on gold substrate were prepared with varying ratios of free PEG to CTI-conjugated PEG. Two methods designated, respectively, "sequential" and "direct" were used. For sequential surfaces, PEG was first immobilized on gold and the surfaces were incubated with CTI at varying concentration. For direct surfaces, a PEG-CTI conjugate was synthesized and gold surfaces were modified using solutions of the conjugate of varying concentration. The CTI density on these surfaces was measured using radiolabeled CTI. Water contact angles were measured and the thickness of PEG-CTI layers was determined by ellipsometry. Fibrinogen adsorption from buffer and human plasma, and adsorption from binary solutions of fibrinogen and α-lactalbumin were investigated using radiolabeling methods. Bioactivity of the surfaces was evaluated via their effects on FXIIa inhibition and plasma clotting time. It was found that as the ratio of CTI-conjugated PEG to free PEG increased, bioactivity increased but protein resistance was relatively constant. It is concluded that on these surfaces conjugation of PEG to CTI does not greatly compromise the protein resistance of the PEG but results in improved interactions between the CTI and the "target" protein FXIIa. At the same CTI density, sequential surfaces were more effective in terms of inhibiting FXIIa and prolonging clotting time.

Corn trypsin inhibitor coating attenuates the prothrombotic properties of catheters in vitro and in vivo

“…Based on these observations, we first examined the effect of CTI immobilization on model surfaces. When attached via a PEG spacer, immobilized CTI reduced adsorption of fibrinogen and fXII and attenuated procoagulant activity [19,[22][23][24]. In this study, we set out to determine whether similar results could be obtained with CTI immobilization on catheters and whether reduced in vitro prothrombotic activity would translate into attenuated catheter thrombosis in rabbits.…”

“…In contrast, CTI-coated catheters prolonged the clotting time by 3.5-fold, yielding clotting times significantly (p < 0.05) longer than those obtained in the absence of catheters or in the presence of ovalbumin-coated catheters (p < 0.05). CTI-coated catheters prolonged the clotting time to a greater extent than CTI immobilized on gold or polyurethane surfaces [22][23][24], likely reflecting the greater activity of catheter-bound CTI as evidenced by enhanced fXIIa binding. Thus, like the findings with model surfaces, immobilization of CTI on the catheter surface markedly attenuates their prothrombotic activity.…”

“…We elected to immobilize preformed conjugates because our previous work with model surfaces revealed greater surface density of CTI with this method than with a sequential method wherein initial PEG immobilization was followed by subsequent attachment of CTI [24]. A time course was performed to assess the influence of incubation time on the efficiency of 125 I-CTI or 125 I-ovalbumin immobilization on the catheter surface.…”

“…As first steps to explore the utility of CTI coating, polyethylene glycol (PEG) was conjugated with CTI and then immobilized onto gold or polyurethane model surfaces [22][23][24]. On both surfaces, immobilization of CTI conjugates attenuated protein adsorption from buffer or plasma and prolonged the plasma clotting time.…”

Selective depletion of factor XI or factor XII with antisense oligonucleotides attenuates catheter thrombosis in rabbits

“…Alternatively, surface modification using CTI, an fXIIa inhibitor, provides a method for rendering catheters and other blood-contacting devices less thrombogenic. 12,[40][41][42] The utility of these agents for the prevention and treatment of catheter thrombosis requires further investigation.…”