Background: Disseminated intravascular coagulation is an established complication of acute hemolytic transfusion reactions, particularly those involving the ABO red cell (RBC) antigen system. In addition, peripheral blood white cells, particularly monocytes, have demonstrated expression of procoagulant activity (PCA) in response to inflammatory stimuli. To better define the activation of coagulation in immune hemolysis, in vitro experiments were conducted to investigate the expression of PCA by peripheral blood white cells in ABO RBC incompatibility.
Study design and methods: Fresh group O heparinized whole blood was incubated with washed, packed group A or O RBCs. White cells were separated, washed, and lysed before assay of PCA, which was measured by a one-stage recalcified clotting time assay. Units of activity were calculated on the basis of a rabbit brain thromboplastin standard curve. Mechanisms of coagulation activation were investigated by using specific coagulation factor-deficient plasmas, blocking antibodies to tissue factor, and anti-CD11b.
Results: Significant levels of white cell-associated PCA were found at 2 to 6 hours in response to incompatible (group A) RBCs, but not in response to compatible (group O) RBCs. PCA was not correlated with numbers of platelets in whole blood. Nonimmune lysis of compatible RBCs did not induce PCA. When whole blood reconstituted from washed cells and heat-inactivated plasma was incubated with incompatible RBCs, PCA and hemolysis were abrogated, which suggests that complement is a required intermediate. Protein synthesis inhibition by the addition of cycloheximide (5 mg/mL) to whole blood incubated with RBCs prevented the expression of PCA. Substitution of factor VIII-deficient plasma for normal plasma in the recalcified clotting time assay had no effect, whereas PCA was reduced by 68 percent with factor VII-deficient plasma and was unmeasurable with factor X-deficient plasma. PCA was restored by a 1-to-1 mix of normal and factor VII-deficient plasma. Incubation of samples in the PCA assay with tissue factor antibodies resulted in up to 86-percent inhibition of measured PCA. Titration of the response to the amount of tissue factor antibodies added demonstrated that maximal inhibition occurred with 0.45 mg per mL, above which no further inhibition took place. However, the addition of anti-CD11b (0.75 mg/mL) concomitantly with anti-tissue factor abolished measurable activity. This effect was independent of the amount of added protein, and anti-CD11b alone had no effect on measured activity. The addition to whole blood concomitantly with RBCs of polyclonal antibodies to tumor necrosis factor, sufficient to neutralize 2000 pg per mL, did not alter PCA expression.
Conclusion: These results indicate that white cell-associated PCA is generated in whole blood in response to ABO RBC incompatibility and may contribute to disseminated intravascular coagulation in acute hemolytic transfusion reactions. Two possible cellular mechanisms are suggested, which involve tissue factor expression and the activation of factor X by a CD11b-dependent mechanism.