Abstract
The major clinical manifestations of sickle cell anemia are due to a vaso-occlusive process that leads to severe pain crises, tissue infarction, and in some instances, death. The pathophysiology of vaso-occlusion is extremely complex, the proximate cause appearing to be an increase in the adhesive interaction between sickle erythrocytes and vascular endothelium. There is ample evidence that it is the highest molecular weight forms of VWF, the ultra-large multimers (ULVWF), expressed on endothelium support sickle erythrocyte adhesion most potently. We postulate that variation in the activity of the plasma metalloprotease, ADAMTS-13, which processes ULVWF to the VWF form normally found in plasma will be an important determinant of the propensity for individuals with sickle cell anemia to develop vaso-occlusive crises. It has also been postulated that increased production of reactive oxygen species (ROS) also contributes to the pathology of sickle cell disease. ROS have been implicated in diverse cellular processes, acting through oxidation of cysteine residues. ADAMTS-13 is a cysteine-rich protein; we therefore hypothesized that its function could be affected by ROS. We measured ADAMTS-13 activity in the plasmas of sickle cell disease patients, both in the “steady state” and at various times during vaso-occlusive crises. We used three assays: a static assay that measures the ability of patient plasma to reduce the size of endothelial-derived ULVWF, a static assay that assesses the ability of patient plasma to cleave a recombinant VWF A2 fragment, and an assay performed under flow that evaluates the ability of patient plasma to cleave ULVWF attached to the surfaces of histamine-stimulated human umbilical vein endothelial cells (HUVECs). Five of nine patients with sickle cell anemia had significantly lower ADAMTS-13 activity than measured in normal controls during the chronic phase of the illness, some with activity measuring below 50% of normal levels. We also evaluated activity in two patients during acute vaso-occlusive crisis. Both patients had levels on the day of hospitalization that were indistinguishable from the levels found in patients with thrombotic thrombocytopenic purpura. Recovery of activity correlated with clinical recovery, to the point that in both cases activity was normal upon hospital discharge. We then compared superoxide generation facilitated by sickle erythrocytes with that generated by normal erythrocytes and found, as others have a significantly higher generation by sickle erythrocytes. We also found that superoxide (produced using a hypoxanthine-xanthine oxidase system) significantly decreased the activity of both plasma and recombinant ADAMTS-13 in vitro. Superoxide was also able to stimulate release of ULVWF and cell-membrane expression from cultured HUVEC. These results suggest that the enhanced production of superoxide in patients with sickle cell disease plays an important role in promoting erythrocyte adhesion to the endothelium by both stimulating release of ULVWF and inhibiting its cleavage by ADAMTS-13.
