Factor Xa inhibition by immobilized recombinant tissue factor pathway inhibitor

Abstract

The recombinant form of the endogenous anticoagulant, tissue factor pathway inhibitor (rTFPI), is a potent inhibitor of Factor Xa (FXa) and the tissue factor-factor VIIa (TF:VIIa) complex. Surface-immobilized rTFPI reduces the thrombogenicity and intimal hyperplasia associated with synthetic vascular grafts in animal models and specifically reduces fibrin deposition on collagen-impregnated Dacron grafts from native blood in an in vitro flow model. The FXa inhibitory capacity of rTFPI in the bulk phase has been demonstrated in static systems and immobilized rTFPI reduces fibrin deposition in whole blood in vitro and animal studies; however, the specific mode of this anticoagulation has not been studied. Therefore, a comparison was made between the FXa binding capacity of two forms of immobilized rTFPI, i.e., passively adsorbed and covalently bound. The rTFPI-coated surfaces were evaluated using a parallel-plate flow reactor and comparing the amount of FXa exiting the flow chamber after exposure to an rTFPI-coated versus an uncoated plate. The results demonstrate that adsorbed rTFPI exhibits increased binding capacity (1.5-3.6 times) the expected stoichiometry via interactions with the C-terminus, whereas covalently-bound rTFPI interacts with FXa in a 1:1 stoichiometry. Thus, the results imply that specific FXa inhibition is a key component of the anticoagulant effect of rTFPI-coated surfaces and that passive adsorption of rTFPI to glass surfaces produces a more effective coating than covalent binding of rTFPI.

Computational Modeling of Factor Xa Inhibition by Immobilized Tissue Factor Pathway Inhibitor

“…This range of parameters includes the experimental conditions of Chandiwal 6 , where the experiments were carried out at wall-shear rates of 100, 400, and 1200 s )1 for an inlet FXa concentration of 20 nM.…”

“…6 The density of the flowing fluid was 1000 kg m )3 and the kinematic viscosity was 1 Â 10 )6 m 2 s )1 . The diffusion coefficient of FXa was 5 Â 10 )11 m 2 s )1 .…”

“…The initial surface density of rTFPI was fixed at the value of 12.3 nM m )2 corresponding to the experiments. 6 …”

“…6 The device possesses an inherent advantage to study heterogeneous biological reactions and provides an excellent control over the wall-shear rate, however, no local measurements are possible and the progress of the reaction has to be measured on the basis of the reduction in concentration of FXa in the discharge stream. Thus, the experiments yield an average decrease in circulating FXa over the entire reaction surface.…”

A Computational Analysis of an In Vitro Vessel Wall Injury Model

“…4 In addition, a higher than the expected one-to-one binding ratio between FXa molecules and the passively adsorbed rTFPI was also found in previously published experiments. 3 It is possible that at low shear rates the conformation of TFPI is such that FXa binds not only to the K2 domain but also to the C-terminus region. As the shear rate is increased the TFPI conformation changes and the second binding site becomes unavailable resulting in decreased inhibition.…”