Interaction between Choline Transporter-like Protein-2 and von Willebrand Factor: Importance to Transfusion-related Acute Lung Injury and Beyond. (LSBR 1503)
Project leader: Dr. Bart J.M. van Vlijmen, Dept. of Thrombosis and Hemostasis, LUMC, Leiden
Postdoc researcher: Chrissta X. Maracle (Sept. 2016 – Aug. 2019)
Transfusion-related acute lung injury (TRALI) is a serious complication of blood transfusion. In TRALI, a 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 occurs after blood transfusion because 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), some of which have variant A and others a B, determined hereditary. People with the A variant can develop antibodies against the B variant and vice versa. The production of the antibody can take place during pregnancy, for example, when a woman with A is a carrier of a child with B. This fortunately does not lead to problems (the occurrence of TRALI) in the unborn child. However, this can cause problems if this woman acts as a blood donor and her anti-HNA-3B antibodies end up upon transfusion in a recipient with variant B. TRALI can now occur in the blood recipient. The exact biological mechanisms underlying the fatal response of the recipient to the antibodies are not fully understood.
Recently, we discovered that the hereditary factor, i.e. the gene encoding HNA-3A or B, also plays a role in the disease venous thrombosis (VT). VT is the occurrence of unwanted blood clotting in the veins, which can may have fatal consequences. Although the same hereditary factor, whose gene confusingly also bears the name “CTL2” or “SLC44A2”, there are no indications that for VT related to this factor unwanted antibody production also plays a role. 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 is therefore what the normal function of this factor is in a healthy person or organism? A second question is whether the 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? By answering these questions, we hope to gain insight into how HNA-3 / CTL2 / SLC44A2 contributes to TRALI and VT, and to find clues for better treatment.
To further investigate the role of HNA-3 / CTL2 / SLC44A2 within the complex interplay of flowing blood and blood vessel wall, we have conducted a number of studies using genetically modified mice lacking this factor, for convenience further referred to as ‘SLC44A2 knockout mice’ . We have shown that the absence of SLC44A2 in these mice leaves normal blood coagulation intact, but is accompanied by a slight reduction in plasma of Von Willebrand Factor (VWF), a protein known for a function in hemostasis. After damage to the blood vessel wall, the formation of blood clots in these mice was found to be abnormal. Also when situations were simulated that lead to the occurrence of VT. In one such situation, we saw that the VT in SLC44A2 knockout mice was abnormal. The blood vessel wall with the endothelium appeared normal after VT in mice without 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, it can be missed, though can be involved in venous thrombosis.
Subsequent experiments with cultured human endothelial cells showed that the reduction in plasma VWF (at in SLC44A2 knockout mice) could not be linked to a different production of VWF with less SLC44A2. Less SLC44A2 also had no effect on functioning for (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 coupled to both VT and TRALI (HNA-3A) was able to bind to VW, while the HNA-3B related to less risk of VT was unable to do so (note: 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 platelets without SLC44A2 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, but different for 2 proteins that are known to play a role in cell-cell interactions, including those between platelets, neutrophils and endothelial cells.
In conclusion, these studies underline the importance of SLC44A2 in disease. We have found that SLC44A2 plays a role in neutrophil-VWF interactions and in platelet biology, two key factors that underlie the pathogenesis of VT and TRALI. These are starting points for further unraveling the role of SLC44A2 in disease and a step towards an alternative, better and safer treatment strategy for TRALI and / or VT.