Transfusion-related acute lung injury (TRALI) is a serious complication of blood transfusion. In TRALI, blood transfusion can cause unwanted activation of the neutrophils circulating in the blood, with consequences for the cells that line the blood vessels of the lungs (the endothelial cells). This causes injury of the lungs, sometimes with fatal consequences. Neutrophil activation may occur upon transfusion when there are antibodies in the donor blood directed against antigens on the surface of the neutrophils of the recipient. This antigen is the human neutrophil antigen 3 (HNA-3), which can occur as the inherited variants A or B. People with the A variant can develop antibodies against the B variant and vice versa. When transfused, such antibodies can lead to TRALI. Recently, we discovered that the gene encoding HNA-3A or B also plays a role in venous thrombosis (VT). There are no indications, however, that unwanted antibody production against HNA-3A, which also bears the name “CTL2” or “SLC44A2”, is involved in VT. Interestingly, in both TRALI and VT, neutrophils, endothelium and perhaps also platelets are involved in the development of disease. Little is known about HNA-3/CTL2/SLC44A2. The first research question was therefore what its normal function is in a healthy person or organism. A second question is whether HNA-3/CTL2/SLC44A2 is really directly involved with VT, and if so, by which mechanism. Third, is there an overlapping role for this factor in the development of TRALI and VT? Answers to these questions should provide insight into how HNA-3/CTL2/SLC44A2 contributes to TRALI and VT, and to find clues for better treatment.

To investigate the role of HNA-3/CTL2/SLC44A2 within the complex interplay of flowing blood and blood vessel wall, we used genetically modified mice lacking this factor, further referred to as ‘SLC44A2 knockout mice’. We found that the absence of SLC44A2 leaves normal blood coagulation intact, but is accompanied by a slight reduction in plasma of Von Willebrand Factor (VWF), a protein involved in hemostasis. Upon vascular injury, the formation of blood clots in these mice was abnormal. The vascular endothelium appeared normal after VT in mice lacking SLC44A2. There were, however, indications of a change in the involvement of neutrophils and VWF. We concluded that SLC44A2 is not of major importance in a healthy organism because it seems dispensable, but nevertheless can be involved in venous thrombosis.

Subsequent experiments with cultured human endothelial cells showed that the reduction in plasma VWF (as in SLC44A2 knockout mice) could not be linked to a reduced production of VWF. Reduced SLC44A2 neither had an effect on function of (mouse) neutrophils. Experiments with cultured human cells that artificially express the HNA-3A or the HNA-3B variant of SLC44A2 on the cell surface showed that the variant that is coupled to both VT and TRALI (HNA-3A) was able to bind to VWF, while the HNA-3B related to lower risk for VT was unable to do so under conditions that mimic the slow venous blood flow. The same was seen with neutrophils obtained from blood from volunteers carrying the HNA-3A or the HNA-3B variant of SLC44A2 on the cell surface. The binding of HNA-3A neutrophils to VWF also led to activation and “spitting out” of the neutrophil DNA, the so-called NETs. These results may explain why SLC44A2 and the HNA-3 variance coincide with VT and possibly also TRALI. In addition to these findings, we observed that the absence of SLC44A2 affected the function of the (mouse) platelets, with a reduced platelet accumulation in blood clots that form after damage to the vascular wall or due to delayed blood flow. These SLC44A2-lacking platelets also had a reduced response to various stimuli. Finally, we analyzed the blood plasma of SLC44A2 knockout mice for hundreds of different proteins. The plasma protein profile was remarkably normal for these mice, except for a few proteins that are known to play a role in cell-cell interaction, including those between platelets, neutrophils and endothelial cells. Collectively, these findings demonstrate that SLC44A2 plays a role in neutrophil-VWF interactions and in platelet biology, two key factors that underlie the pathogenesis of VT and TRALI. Further unraveling the role of SLC44A2 may lead towards an alternative, better and safer treatment strategy for TRALI and/or VT.

Thesis 2021: The role of solute carrier family 44 member 2 in the pathophysiology of venous thrombosis, Julia Tilburg