Evaluation of a novel hirudin-coated polyester graft to physiologic flow conditions: hirudin bioavailability and thrombin uptake.

Abstract

Our laboratory has developed methods required to covalently bind recombinant hirudin (rHir) to the surface of polyester vascular grafts. Using alkaline hydrolysis of the polyester surface, carboxyl-binding sites are created on the outer periphery of each fiber. A series of static, in vitro experiments have demonstrated that surface-bound rHir rapidly removes and inhibits activated human alpha-thrombin from the reaction system; however, the performance of this modified graft material under physiologic flow conditions was undefined. An in vitro flow loop was used to evaluate structural stability of the 125I-rHir and 131I-albumin covalently bound to the surface of 6 mm interior diameter crimped polyester grafts exposed to either constant flow (n = 4; shear rate, 300 sec(-1)) or pulsatile flow (n = 4; maximum shear rate, 780 sec(-1)) conditions for a 7-day period. In a separate series of experiments, the kinetics of thrombin-rHir interaction were evaluated through perfusion of 125I-rHir-coated grafts (n = 6) with 131I-thrombin for a 27-hour period under constant flow conditions. Identically prepared 125I-albumin-coated grafts (n = 3) were used as controls. Results of the stability experiments were independent of flow conditions, demonstrating moderate loss of both proteins, with rHir and albumin losses of 52.1% and 19.9% under constant flow and 49.1% and 21.6% under pulsatile flow, respectively. With results comparable with those of previous static experiments, rHir-coated grafts were significantly more effective at removing thrombin from the perfusion stream with 131I-thrombin binding densities of 3.08 +/- 0.61 and 0.64 +/- 0.04 NIHU/cm2 (p < 0.01) for rHir-coated and albumin-coated grafts, respectively. Estimates of the total amount of thrombin inactivated during the perfusion period similarly demonstrated a marked difference between the rHir-coated and control graft segments (125 +/- 8 vs. 3 +/- 14 NIHU; p < 0.005). These in vitro flow results illustrate that polyester grafts with covalently bound rHir can provide significant reductions in local thrombin concentration under physiologic flow conditions, and can serve as a foundation with which to understand the performance of these grafts when implanted in vivo under physiologic flow and shear rates.