Recombinant Kunitz Protease Inhibitory Domain of the Amyloid β-Protein Precursor as an Anticoagulant in Venovenous Extracorporeal Circulation in Rabbits

Summary

Investigations were performed to characterize a recombinant Kunitz protease inhibitory domain of the amyloid β-protein precursor (rKPI) as anticoagulants. After a single intravenous infusion of wild type rKPI into dogs, its elimination fit a two compartment model with a t_½α and t_½β of 5 and 77 min, respectively. Further investigations determined if a variant form of rKPI with 178-fold more potent anti-factor Xa activity (rKPI-DD₁₃₅, K_i = 0.9 nM) could serve as an anticoagulant in a rabbit model of extracorporeal circulation using a venovenous shunt. A prospective investigation was initiated to compare standard heparin (n = 8) at 400 U/kg with different infusion concentrations of rKPI-DD₁₃₅. After a single intravenous infusion of 1.89 mg/kg of rKPI-DD₁₃₅ followed by a constant infusion at 0.003 (n = 3), 0.03 (n = 7), or 0.3 (n = 5) mg/kg/min, the anti-factor Xa activity of the animals’ plasma rapidly reaches a steady state for the two lower infusion concentrations of the agent. All infusions of rKPI-DD₁₃₅ prolong the activated clotting time with less variation than that seen with heparin administration. rKPI-DD₁₃₅ anticoagulation does not prevent a drop in the platelet counts. Fibrinogen levels decrease only slightly when the circuit is anticoagulated with rKPI-DD₁₃₅. rKPI-DD₁₃₅ markedly prolongs the APTT, has little effect on the PT, and reduces plasma prekallikrein and plasminogen activation. The 0.3 mg/kg/min infusion concentration of rKPI-DD₁₃₅ results in reduced deposition of ¹¹¹Indium-labeled platelets on the circuit when compared to heparin. Last, after a steady state level is achieved, 60% of the plasma anti-factor Xa activity of rKPI-DD₁₃₅ is eliminated within 60 min after stopping the infusion. These data show the rKPI-DD₁₃₅ can provide single agent anticoagulation in a rabbit extracorporeal circuit. Development of short acting factor Xa inhibitors may be useful anticoagulants for cardiopulmonary bypass.

Abbreviations: CPB: cardiopulmonary bypass; KPI: Kunitz protease inhibitor domain; rKPI_WT: recombinant wild type Kunitz protease inhibitory domain of the amyloid β-protein precursor; rKPI-DD₁₃₅: recombinant Kunitz protease inhibitory domain of the amyloid β-protein which is a better inhibitor of factor Xa; ECC: extracorporeal circulation; CDP: citrate phosphate dextrose anti-coagulant; PRP: platelet-rich plasma; ACT: activated clotting time; PT: prothrombin time; APTT: activated partial thromboplastin time; PPP: platelet-poor plasma.

Key words

Amyloid β-protein precursor - factor Xa inhibitor - anticoagulant - ECC

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