Denatured hemoglobin increases human blood mononuclear cell procoagulant effect

To study the effects of the common contaminants of hemoglobin solutions, red cell stroma, bacterial endotoxin, and denatured hemoglobin on the causes of the thrombotic lesions which have been reported in animal experiments after hemoglobin administration.

Human blood mononuclear cells were isolated on Ficoll-Hypaque gradients and incubated with hemoglobin from the LAIR production facility, red cell stroma, bacterial endotoxin (E. coli, Wittaker Bioproducts), and hemoglobin denatured by boiling. Incubations were performed separately and in combinations. Mononuclear cells were then lysed and assayed for procoagulant activity in a recalcification time assay.

Only bacterial endotoxin and hemoglobin denatured by boiling increased the procoagulant activity of human blood mononuclear cells. Denatured hemoglobin mixed one part in eight with undenatured hemoglobin increased mononuclear cell procoagulant activity by more than ten-fold that of the undenatured hemoglobin control.

The study suggests that denatured but not undenatured hemoglobin causes increased blood procoagulant activity which is thought to be a marker of macrophage activation. These findings suggest a possible mechanism of toxicity of cell-free hemoglobins and the need for sensitive measures of hemoglobin denaturation.