title: Evaluation of a novel hirudin-coated polyester graft to physiologic flow conditions: hirudin bioavailability and thrombin uptake.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Berceli
authors.forename: M D
authors.initials: MD
authors.lastname: Phaneuf
authors.forename: F W
authors.initials: FW
authors.lastname: LoGerfo
mesh_terms: D001672:Biocompatible Materials; D001682:Biological Availability; D001807:Blood Vessel Prosthesis; D004347:Drug Interactions; D004355:Drug Stability; D006439:Hemodynamics; D006629:Hirudins / Q000493:pharmacokinetics*; D006801:Humans; D066298:In Vitro Techniques; D010477:Perfusion / Q000295:instrumentation / Q000379:methods; D011091:Polyesters; D011994:Recombinant Proteins / Q000493:pharmacokinetics; D013499:Surface Properties; D013917:Thrombin / Q000493:pharmacokinetics*
chemical_list: D001672:Biocompatible Materials; D006629:Hirudins; D011091:Polyesters; D011994:Recombinant Proteins; D013917:Thrombin