Immobilization of heparin oligosaccharides onto radiofrequency plasma modified pyrolytic carbon-coated graphite

Heparin oligosaccharides with different anticoagulant activities were prepared and immobilized onto pyrolytic carbon coated graphite (PC) heart valve materials commonly used in mechanical heart valve prostheses. Prior to immobilization, PC surfaces were modified by radiofrequency plasma polymerized N-vinyl-2-pyrrolidone (PPNVP) thin films (approximately 100 nm) and derivatized to provide surface hydroxyl groups. Cleaved, low affinity heparin (C-heparin) with factor Xa inhibition activity of 107 to 130 IU/mg, was prepared by partial deaminative cleavage of commercial crude heparin, and high-affinity heparin (HA-heparin) with factor Xa inhibition activity of 550 to 1000 IU/mg was prepared by fractionation of C-heparin using agarose-ATIII affinity chromatography. C-heparin and HA-heparin were immobilized to surface modified PC by reductive amination. Anticoagulant activity of the heparin immobilized surfaces was determined by chromogenic assay for the inhibition of factor Xa. Highest surface anticoagulant activity was measured on C-heparin immobilized surfaces (64.0 +/- 7.3 mIU/cm2) compared with HA-heparin immobilized surfaces (27.2 +/- 12.2 mIU/cm2), suggesting higher binding of C-heparin than HA-heparin on the modified PC surfaces. Immobilized surfaces were evaluated under dynamic flow conditions, by subjecting samples to shear stress of up to 206 dyn/cm2 in the presence of 5% albumin solution or human plasma. Anticoagulant activity of the immobilized heparin was retained, although reduced, and the modified surfaces showed evidence for protein resistance.