Abstract
Purpose::
A cell-based functional assay that measures the cofactor activity of factor H on factor I mediated iC3b formation was used to study the modulation of this activity by heparin, heparan sulfate (HS) and C-reactive protein (CRP). Purified samples of factor H from individuals homozygous for either the major genetic risk factor for age-related macular degeneration (AMD) form (402HH) or normal (402YY) were used to determine whether this amino acid substitution influences the kinetics of the reaction.
Methods::
A functional factor H-cofactor activity assay, which quantitates the inactivation of cell-bound radiolabeled C3b by human factor H, and factor I was used to study iC3b formation as a function of time. iC3b formation was detected by low dose trypsin to release C3c. Purified, isoform specific, factor H was incubated with factor I and cell-bound C3b. In parallel studies, cold, cell-bound, C3b was used with radiolabeled anti-factor H to track the binding of factor H to the cells under similar conditions. Experiments were performed in the presence or absence of heparin, heparan sulfate, or CRP and the results confirmed by immunoblot analysis of factor H in the cell lysate after specific times of incubation.
Results::
Under normal conditions factor H rapidly binds to the cells. In the presence of HS, factor H binding was enhanced after 1 minute of incubation at 37 degrees. Heparin decreased binding of factor H to the cells and CRP had no effect. Results measuring iC3b formation parallel these findings: heparan sulfate increased iC3b formation, heparin decreased it and CRP had no effect. Results of these experiments were identical with either factor H isoform (402HH or the 402YY).
Conclusions::
Heparan sulfate increased factor H mediated proteolytic cleavage of C3b by factor I as evidenced by increased formation of iC3b. This may be due to HS increasing binding of factor H to C3b-coated cells. Heparin decreased the binding of factor H to the C3b-coated cells with concomitant decreased formation of iC3b. CRP had no effect under the conditions used. Understanding the mechanism of action of factor H and the influence of various biological molecules associated with AMD pathology on this reaction may give us an insight into the role of factor H in the pathogenesis of AMD.
Keywords: age-related macular degeneration • immunomodulation/immunoregulation • proteoglycans/glycosaminoglycans