Role of extracellular hemoglobin in thrombosis and vascular occlusion in patients with sickle cell anemia

Abstract

Sickle cell anemia (SCA) is a common hemolytic disorder caused by a gene mutation in the β-globin subunit of hemoglobin (Hb) and affects millions of people. The intravascular hemolysis releases excessive amount of extracellular hemoglobin (ECHb) into plasma that causes many cellular dysfunctions in patients with SCA. ECHb scavenges NO which promotes crisis events such as vasoconstriction, thrombosis and hypercoagulation. ECHb and its degradation product, heme, are known to cause oxidative damage to the vessel wall and stimulate the expression of adhesive protein ligands on vascular endothelium. Our study shows that ECHb binds potently to VWF-largest multimeric glycoprotein in circulation-through the A2-domain, and significantly inhibits its cleavage by the metalloprotease ADAMTS13. Furthermore, a subpopulation of VWF multimers bound to ECHb exists in significant amount, accounting for about 14% of total plasma VWF, in SCD patients. The Hb-bound VWF multimers are resistant to ADAMTS13, and are hyperactive in aggregating platelets. Thus, the data suggest that Hb-bound VWF multimers are ultralarge and hyperactive because they are resistant to the protease. The Hb-bound VWF multimers are elevated parallely with the level of ECHb in patients' plasma, and is associated with the pathogenesis of thrombosis and vascular occlusion in SCA.

Figure 1

Hb-bound VWF multimers are prevalent in SCD patients and are hyperactive. (a): the VWF was purified from plasma (1 mL) of SCD patients or normal individuals by a Superose gel filtration column. The 1st protein peak (at 280 nm UV) was collected for VWF. Further, the Hb-bound VWF was isolated from above VWF fraction using an Ni-NTA affinity column. The VWF antigen level was measured using a commercial kit. The Hb VWF multimers existed as about 14% of total VWF in patients compared to only about 3.5% in normals (n = 5). (b): the ristocetin cofactor VWF activity assay shows that the Hb-bound VWF multimers are 35% more activity than the Hb-free counterparts. (c), The collagen binding assay also shows that the Hb-bound VWF multimers are 33% more adhesive to collagen than the Hb-free multimers.

Figure 2

The schematic diagram shows that upon activation of the vascular endothelium, ultralarge VWF is released and is cleaved by ADAMTS13 to smaller fragments that circulate in plasma as an inactive form. Under pathophysiological conditions such as SCA, excessive extracellular Hb blocks the cleavage of a subpopulation of VWF multimers. The uncleaved Hb-bound VWF multimers accumulate in plasma, which are hyperactive in binding to platelets or sickled/fragmented-RBCs to promote cell adhesion and events such as thrombosis and vascular occlusion.