Interaction of heparin with two synthetic peptides that neutralize the anticoagulant activity of heparin

Abstract

Two synthetic analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein (Ac-SRGKAKVKAKVKDQTK-NH2) and the all-d-amino acid version of the same peptide (l-HIPAP and d-HIPAP, respectively) were synthesized, and their efficacy as agents for neutralization of the anticoagulant activity of heparin was assayed. The two analogue peptides were found to be equally effective for neutralization of the anticoagulant activity of heparin, as measured by restoration of the activity of serine protease factor Xa by the Coatest heparin method. The finding that l-HIPAP and d-HIPAP are equally effective suggests that d-amino acid peptides show promise as proteolytically stable therapeutic agents for neutralization of the anticoagulant activity of heparin. The interaction of l-HIPAP and d-HIPAP with heparin was characterized by 1H NMR, isothermal titration calorimetry (ITC), and heparin affinity chromatography. The two peptides were found to interact identically with heparin. Analysis of the dependence of heparin-peptide binding constants on Na+ concentration by counterion condensation theory indicates that, on average, 2.35 Na+ ions are displaced from heparin per peptide molecule bound and one peptide molecule binds per hexasaccharide segment of heparin. The analysis also indicates that both ionic and nonionic interactions contribute to the binding constant, with the ionic contribution decreasing as the Na+ concentration increases.

Figure 1

Restoration of the activity of FXa as a function of the concentration of L-HIPAP and D-HIPAP.

Figure 2

(A) Composite chemical shift-pH titration curves for the lysine C_εH₂ protons and (B) chemical shift-pH data for the arginine C_δH₂ protons of L-HIPAP in the absence and presence of heparin. The open circles are for free peptide, and the open squares are for peptide plus heparin, both at 25 °C. The peptide concentration was 5 mM and the heparin disaccharide-to-peptide ratio was 4:1.

Figure 3

CD spectra of L-HIPAP and D-HIPAP, free and bound by heparin.

Figure 4

Binding isotherm for the interaction of L-HIPAP with heparin. The peptide (0.18 mM in the cell) was titrated with 0.57 mM heparin (in the syringe). In the top figure, the peaks indicate the heat absorbed after each addition of heparin. The peaks were integrated and the total heat per injection (peak area) is plotted as a function of molar ratio in the bottom figure. The line through the points represents the fit of the ITC data that gave the thermodynamic parameters reported in Table 2.

Figure 5

The change in chemical shift of the backbone amide NH resonances of L-HIPAP upon binding to heparin. Chemical shift difference = δ_{free peptide} - δ_{with heparin}