Erythrocytes from Sickle Cell Mice Activate the Endothelium and Blood Cells and Induce Vaso-Occlusion When Transfused into ADAMTS13-Deficient Mice: Role of Lipid Degradation

Background: Sickle cell disease (SCD) is a complex systemic disease caused by homozygous or compound heterozygous inheritance of a single nucleotide mutation in the β-globin gene. This mutation produces myriad downstream effects, including erythrocyte sickling and dehydration, excessive oxidant production, and systemic endothelial activation. Although sickling of the erythrocytes is undoubtedly important in initiating the complex pathophysiology of SCD, it is not clear that it is the most important consequence of the β-globin mutation. For example, sickle erythrocytes spontaneously generate twice as much superoxide, hydrogen peroxide, and hydroxyl radical as normal red cells. These oxidants cause accelerated hemoglobin autooxidation, heme loss, and formation of hemichromes. They also generate high concentrations of oxidized lipids on the erythrocyte membrane. This suggests the possibility that SCD red cells may themselves initiate vasoocclusion (VOC), perhaps by reaction of bioactive oxidation products with other blood cells and endothelium. We tested this possibility by transfusing sickle cells into mice in which proinflammatory vascular phenomena should be readily apparent, mice deficient in the VWF-cleaving protease ADAMTS13.

Methods:

Transfusion and intravital imaging. We used whole blood or isolated red cells (RBC) from Townes SS (sickle cell), AA (control) mice, and WT mice. The RBCs were fluorescently labeled with FITC-conjugated antibody to Ter119. The circulating platelets and leukocytes of the recipient mice (adult male ADAMTS13-KO mice) were labeled with cell-specific fluorescent antibodies. The mice were then transfused with 200 μL of fluorescent-labeled RBCs or whole blood via jugular vein catheter while recording the cremaster microcirculation microscopically. Interactions of fluorescently labeled platelets, leukocytes, and transfused RBCs with the vessel wall were recorded for up to 20 min.

Lipidomics. We analyzed whole blood from Townes SS and Towns AA mice and from a patient with SCD (SS genotype) for free polyunsaturated fatty acids (PUFAs) and lyso phospholipids (lysoPL) by mass spectrometry. PLs were extracted with 80% methanol containing isotope-labeled internal standards and separated on a T3 column. Analytes were detected by multiple reaction monitoring. Each lysoPL with fatty acid chains 16:0, 18:0, and 18:1 was quantified relative to their internal standard analogs (lysoPC 17:1, lysoPE 17:1, lysoPS 17:1, lysoPI 17:1).

Results: Before being transfused, ADAMTS13-KO mice showed a few individual platelets or strings of platelets adhered to the vessel wall and increased rolling leukocytes in venules compared to WT mice. As soon as the transfused red cells from Townes SS reached the region being observed, there was extensive endothelial adhesion of platelets, leukocytes, and infused sickle cells on the cremaster venules. The numbers of adherent cells increased during the observation, with microvessels often occluding completely by about 5 min post-transfusion. Transfused whole blood from Townes SS had a similar thrombotic effect. In contrast, neither RBCs nor whole blood from Townes AA or wild-type mice had a discernable effect on the cremaster microcirculation. These observations suggested that the red cells themselves were acting as potent agonists activating the endothelium, platelets, and leukocytes. Likely culprits are oxidized bioactive membrane lipids. Blood from both SCD mice and an SCD patient had higher concentrations of free arachidonic acid and other PUFAs than controls. Several lysoPLs were more abundant in SCD blood (mouse and human) than control but not in plasma, including lysophosphatidylinositol, lysophosphatidyserine, and lysophosphatidylethanolamine, all of which arise by degradation of glycerophospholipids from the inner plasma membrane leaflet. Lyso-PAF was also more abundant in sickle blood. Lysophosphatidylcholine, which arises from the outer membrane leaflet, was less abundant.

Conclusion(s): We demonstrate that sickle erythrocytes behave as potent agonists to activate endothelial cells and other blood cells, leading to VOC in transfused ADAMTS13-KO mice. This phenomenon is associated with profound changes in the phospholipid and PUFA composition of the blood. Dissecting the relative importance of the different bioactive lipids in VOC should reveal new therapeutic targets.