Biphasic response of complement to heparin: fluid-phase generation of neoantigens in human serum and in a reconstituted alternative pathway amplification cycle.

We describe a study of the effects of heparin on complement activation through the use of assays for fragment C4d, fragment Bb, and the S-C5b-9 complex (S-MAC). In sera from healthy volunteers, virtually no change was observed in C4d either as a function of time or of heparin concentration, whereas changes in Bb and S-MAC were biphasic. This observation was explored in greater detail in the heparin concentration range 0.001-5.0 u/ml (5 x 10(-3) to 25 micrograms/ml). For both Bb and S-MAC, a significant (P < 0.05) increase in production was noted in the heparin concentration range, 0.01-0.5 u/ml (5 x 10(-2) to 2.5 micrograms/ml). At higher heparin concentrations, Bb and S-MAC production decreased markedly (P < 0.05). We reconstituted the alternative pathway amplification cycle (C3, factor B, and factor D) and studied Bb generation. With reactants at concentrations one tenth those of normal serum, we observed a maximum generation of 13.2 micrograms/ml Bb. Control and heparin at 5 x 10(-4) micrograms/ml generated Bb concentrations of 6.8 and 6.1 micrograms/ml, respectively, for a 2-min incubation; at 5 x 10(-3) micrograms/ml heparin, Bb was increased to 9.8 micrograms/ml. Using isoelectric focusing to study anionic pI shifts in heparin-bound factors B and D, it was found that factor B bound heparin only at the highest heparin concentration studied, i.e., 50 micrograms/ml; factor D, however, bound heparin at a much lower concentration (0.05 micrograms/ml). We conclude that, at low concentrations, heparin activates complement due to potentiation of the alternative pathway amplification cycle in the fluid phase.