Denatured hemoglobin increases human blood mononuclear cell procoagulant effect

Area of Science:

• Biochemistry
• Immunology
• Hematology

Background:

• Cell-free hemoglobins are used as oxygen carriers.
• Thrombotic lesions have been reported in animal studies following hemoglobin administration.
• Common contaminants in hemoglobin solutions include red cell stroma, bacterial endotoxin, and denatured hemoglobin.

Purpose of the Study:

• To investigate the thrombotic potential of common hemoglobin solution contaminants.
• To determine the specific effects of red cell stroma, bacterial endotoxin, and denatured hemoglobin on blood procoagulant activity.
• To elucidate the mechanism behind reported thrombotic lesions after hemoglobin administration.

Main Methods:

• Isolation of human blood mononuclear cells using Ficoll-Hypaque gradients.
• Incubation of mononuclear cells with hemoglobin, red cell stroma, bacterial endotoxin, and denatured hemoglobin, both separately and in combination.
• Assay of mononuclear cell procoagulant activity using a recalcification time assay after cell lysis.

Main Results:

• Bacterial endotoxin and boiled (denatured) hemoglobin significantly increased mononuclear cell procoagulant activity.
• Denatured hemoglobin, even at a low concentration (1:8 ratio with undenatured hemoglobin), elevated procoagulant activity over tenfold compared to undenatured hemoglobin alone.
• Red cell stroma did not significantly affect procoagulant activity.

Conclusions:

• Denatured hemoglobin, not undenatured hemoglobin or red cell stroma, is a primary driver of increased blood procoagulant activity.
• Increased procoagulant activity is a potential marker for macrophage activation, suggesting a mechanism for cell-free hemoglobin toxicity.
• The findings underscore the critical need for sensitive assays to detect hemoglobin denaturation in clinical preparations.