Kinetic studies on the interactions of heparin and complement proteins using surface plasmon resonance

Abstract

Heparin is a naturally occurring polysaccharide known to interact with complement proteins and regulate multiple steps in the complement cascade. Quantitative information, in the form of affinity constants for heparin-complement interactions, is not generally available and there are no reports of a comprehensive analysis using the same interaction method. Such information should improve our understanding of how exogenously administered pharmaceutical heparin and the related endogenous polysaccharide, heparan sulfate, regulate complement activation. The current study provides the first comprehensively analysis of the binding of various complement proteins to heparin using surface plasmon resonance (SPR). Complement proteins C1, C2, C3, C4, C5, C6, C7, C8, C9, C1INH, factor I, factor H, factor B and factor P all bind heparin but exhibit different binding kinetics and dissociation constants (Kd) ranging from 2 to 320 nM. By taking into account these Kd values and the serum concentrations of these complement proteins, the percentage of each binding to exogenously administered heparin was calculated and found to range from 2% to 41%. This study provides essential information required for the rational design of new therapeutic agents capable of regulating the complement activation.

Fig. 1

Current view of heparin effects on the classical and alternative pathways.

Fig. 2

Structure of heparin showing the major and variable disaccharide sequences, where $\mathrm{X}={\text{SO}}_3^{-}$ or H and $\mathrm{Y}={\text{SO}}_3^{-}$, COCH₃ or H.

Fig. 3

Sensorgrams showing the interaction of heparin with: C1 at 6.25, 12.5, 25, 50 and 100 nM; C2 at 75, 100, 150, 200 and 250 nM; C3 at 12.5, 25, 50, and 100 nM.

Fig. 4

Sensorgrams showing the interaction of heparin with: C4 at 50, 100, 200, and 300 nM; C5 at 6.25, 12.5, 25, 50, and 75 nM; C6 at 31.25, 62.5, 125 and 250 nM; C7 at 25, 50, 100, 200 and 300 nM; C8 at 25, 50, 100, 200 and 300 nM; C9 at 50, 100, 200 and 300 nM.

Fig. 5

Sensorgrams showing the interaction of heparin with: C1INH at 100, 200, 400, 600, and 750 nM; factor I at 31.25, 62.5, 125, 250 and 500 nM; factor B at 12.5, 50, 75 and 100 nM); factor P at 20, 30, 40 and 50 nM; factor H at 75, 125, 250, 500 and 700 nM; C1INH and factor I were injected over an heparinized SA sensor chip. Factor B, factor H and factor P were injected over a heparinized PEG-neutravidin sensor chip.