Human hemoglobin increases the biological activity of bacterial lipopolysaccharides in activation of Limulus amebocyte lysate and stimulation of tissue factor production by endothelial cells in vitro

Abstract

Previous studies have demonstrated that hemoglobin (Hb) and bacterial endotoxin (lipopolysaccharide, LPS) form stable complexes and result in disaggregation of macromolecular LPS. To examine the effect of complex formation on LPS biological activity, we investigated the ability of Hb to alter LPS-induced activation of the coagulation cascade of Limulus amebocyte lysate (LAL) and expression of tissue factor from human endothelial cells. Both native HbAo and derivatized (covalently cross-linked) hemoglobin resulted in prominent enhancement of LAL activation and endothelial cell tissue factor production by Proteus mirabilis LPS. No substantial differences were observed between the enhancement effect of Hb on P. mirabilis smooth and rough LPS, indicating a dominant role for the lipid A component of LPS. Rough (Re) Salmonella minnesota 595 LPS also demonstrated both enhanced activation of LAL and stimulation of endothelial cell tissue factor in the presence of Hb. In contrast, neither lipid A nor singly dephosphorylated or partially deacylated Re LPS manifested significant enhancement of LAL activation by Hb, and partially deacylated Re LPS showed no enhancement of endothelial cell tissue factor by Hb. These results suggest that the Kdo moieties, as well as the phosphate residues and fatty acyl moieties of lipid A, may be involved in the interaction of Hb with LPS. Comparison of Hb with other endotoxin binding proteins for ability to cause enhancement of LPS biological activity demonstrated more prominent enhancement with lipopolysaccharide binding protein (LBP) than that observed with Hb, lesser enhancement with albumin, and no enhancement effect with IgG or transferrin.

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