A novel role of acellular hemoglobin in hemolytic thrombosis

Hemolytic thrombosis has been associated with acellular hemoglobin released from damaged red blood cells during hemolysis. However, the precise molecular mechanism underlying acellular hemoglobin-induced thrombosis remains arguable. In this study, we examined the interaction between hemoglobin and the A1 domain of von Willebrand factor (VWF), which is a critical mediator of platelet activation.

Previous studies have suggested that the interaction between hemoglobin and the A1 domain of VWF enhances VWF's hemostatic activity. We employed a multidisciplinary investigation to re-examine this interaction, and identified significant differences in binding affinity between the active and inactive forms of A1.

We found that hemoglobin binds more strongly to the active A1 than the inactive form. Using hydrogen‑deuterium exchange mass spectrometry, we identified the specific residues involved in this interaction, which are located on the α1-β2 and β3-α2 loops that are typically covered by the "autoinhibitory module" in the inactive A1. This observation provides a structural explanation for the differential binding affinity between the active and inactive forms of A1. We demonstrated that the binding of hemoglobin to A1 blocks the interaction between GPIbα and VWF, and inhibits VWF-mediated thrombosis in vivo. Furthermore, we found that administration of hemoglobin led to similar levels of thrombocytopenia and microthrombosis in both wildtype and VWF-deficient mice, indicating that the mechanism underlying acellular hemoglobin-induced thrombosis is VWF-independent.

These findings challenge the previous theory that hemoglobin-induced thrombosis occurs solely through binding with VWF, and provide evidence supporting a novel role for hemoglobin in hemolytic thrombosis.