Underlying mechanism and specific prevention of hemolysis-induced platelet activation.

Thromboembolic complications significantly impair the outcome of hemolytic disorders. We hypothesized that red cell adenosine diphosphate (ADP) release results in significant platelet activation in hemolysis and that this prothrombotic state can be prevented by inhibition of the ADP P2Y12 receptor. In the current study, we therefore sought to investigate the mechanism and inhibition of hemolysis-induced platelet activation. The expression of activated integrin αIIbß3 was determined by flow cytometry, and platelet aggregation was assessed by multiple electrode platelet aggregometry. We demonstrate platelet activation and increased platelet aggregation by adding hemolytic blood (lysates) to whole blood, similarly to that achieved by the platelet agonist ADP. Enhanced platelet activation and reactivity in the presence of hemolytic blood were significantly abolished by apyrase, which catalyzes ADP degradation, and inhibited by blockade of the platelet ADP P2Y12 receptor with cangrelor. Platelets from patients treated with the ADP P2Y12 receptor antagonist clopidogrel showed a reduced response to lysates compared to platelets from healthy controls without antiplatelet treatment. Further, in vitro blood group ABO incompatibility induced hemolysis and led to increased platelet activation. Finally, "spontaneous" platelet aggregation seen in patients with cold agglutinin disease was completely abolished by cangrelor. In conclusion, hemolysis is associated with increased platelet activation and aggregation due to red cell derived ADP, which can be prevented by ADP receptor blockade.