Background: One of the pathophysiological events associated with diseases like sickle cell disease (SCD), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) is hemolysis. Several studies propose different mechanisms for intravascular hemolysis to enhance platelet activation and directly and/or indirectly promote thrombosis. Hemolysis leads to free hemoglobin but also free hemin within the blood stream. Here, we examine the hemin induced platelet activation to further understand the complex pathophysiology of the thrombotic complications in intravascular hemolysis and analyze the direct interactions between cGMP and hemin induced platelets.

Methods: The evaluate the effect of hemin on platelets, we utilized flow cytometry, aggregometry, immunoblot and spectrofluorometric measurements, flow chamber experiments for ex vivo thrombus formation as well as mass spectrometry analysis of the platelet lipidome.

Results: In general, hemin does induce platelet activation, aggregation and ex vivo thrombus formation in a concentration-dependent manner. Low hemin (1 - 6 µM) concentrations do increase CD62P presentation, Integrin aIIb/bIII activity and fibrin binding. Whereas high hemin concentration (> 6 µM) are marked by significant CD62P shedding and loss of CD41 as well as decreased Fibrin binding. Induction of cGMP production by Riociguat and DEA/NO does significantly decrease hemin-induced platelet activation and the corresponding activation dependent Calcium and ATP release. In addition, cGMP induction also decreases platelet aggregation and ex vivo thrombus formation.

Hemin does induce platelet apoptosis, demonstrated by a concentration-dependent increase of Annexin V, increase in reactive oxygen species (ROS) and decrease in Mitochondrial function (TMRE). Again, the apoptotic effect of hemin can be significantly decreased by cGMP induction.

Analysis of the platelet lipidome after 25 µM hemin treatment showed significant changes within the lipidome, such as a cyclooxygenase (COX) mediated increase in Prostaglandin E₂, 12-HHT or Thromboxane B₂ and an increase of the arachidonic acid metabolite 15-HETE. Once more, changes within the lipidome were reversed by cGMP stimulation.

Conclusions: We were able to show that hemin does significantly induce platelet activation and aggregation as well as platelet apoptosis and corresponding changes in the platelet lipidome. All these effects were at least partially reversibly by activation of cGMP production within platelets. These results show that platelet cGMP levels are directly affecting hemin-induced platelet activation and that these interaction does reduce the platelet reactivity to hemin stimulation. Modulation of cGMP may be a therapeutic strategy to control thrombotic events in hemolytic diseases.