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

Abstract

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.

Low doses and high doses of heparin have different effects on osteoblast‐like Saos‐2 cells in vitro

“…Signaling in bone is dependent on the presence of LDL receptor-related protein 5 (LRP5), and mutations in this protein have recently been shown to be responsible for specific forms of high-bonemass trait and osteopenia, respectively [Boyden et al, 2002;Kato et al, 2002;Little et al, 2002]. Biphasic effects are not uncommon for heparin [Chevreuil et al, 1993;Keil et al, 1995;LaRochelle et al, 1999]. Regarding a single ligand-receptor system, low concentrations of heparin may facilitate the encounter of the growth factor with its signaling receptor by a ''reduced dimensionality'' mechanism [Lander, 1999], thus promoting the growth factor effect, whereas higher concentrations may be inhibitory due to a saturation of heparin-binding sites.…”

Effects of platelet release products on neutrophilic activity in human whole blood

“…However, there are limitations with in vitro studies. Most anticoagulants such as Calcium chelators like ethylenediaminetetraacetic acid (EDTA), citrate, and heparin interact with complement activation as well as other biologic processes [22,23]. Recently, some authors reported using the thrombin-specific hirudin analog lepirudin as anticoagulant to study the role of complement in the inflammation [24].…”

Candidate inhibitors of porcine complement

“…The model allows crosstalk between all the inflammatory systems in whole blood, with the exception of thrombin which is inhibited by the anticoagulant, lepirudin (64). The advantage of lepirudin is that it does not interfere with other biological systems in particular not the complement system (64), in contrast to other anticoagulants like EDTA, citrate and heparin (169). The whole blood in vitro model allowed studies on cytokine production and inhibition (study II and III), complement activation and inhibition (study II and III), upregulation and inhibition of a granulocyte marker (study II), and studies on bacterial growth and inhibition of the growth (study II).…”