Hemoglobin Bound Von Willebrand Factor (HbVWF) Multimers Are Hyperactive and More Prevalent in Patients with Sickle Cell Disease.

Abstract 3058

Poster Board II-1034

Elevated levels of von Willebrand factor (VWF), especially the ultra-large multimers, play a significant role in the pathogenesis of vascular occlusion in sickle cell disease (SCD) by promoting cell adhesion to the endothelium. Investigating the pathophysiology of vaso-occlusion and thrombosis in SCD, we have recently observed that excessive extracellular-hemoglobin (Hb) in plasma significantly inhibited ADAMTS-13 proteolysis of VWF by binding directly to the enzyme cleavage-site on VWF. Here, we further show that subpopulations of VWF multimers, which are bound to extracellular-Hb, exist in plasma. We have successfully isolated the Hb-bound VWF (HbVWF) multimers from SCD patients' plasma using the Ni-NTA column and quantified by commercial kit. The HbVWF multimers exist in 5 to 6-times less quantity than the Hb-free multimers as measured in SCD patients. Purified HbVWF multimers are mostly uncleavable by recombinant ADAMTS-13 in vitro. These HbVWF multimers are hyper active in agglutinating platelets as detected by ristocetin cofactor (RCof) activity assay, and also hyper adhesive to collagen type-III compared to the Hb-free multimers. The HbVWF multimers exists in about 2-fold more quantity in SCD patients than normal individuals [mean percent level ± SE, 8.1±1.8 (individual mean 6 – 11) vs. 16.6±3 (12 – 21), P <0.001; n=10]. Using another sandwich-ELISA assay we have reexamined the HbVWF levels, which showed a similar pattern as above. Further, the increased level of HbVWF multimers exists parallely with an elevated RCof activity of plasma VWF [mean percent activity ± SE, 100.4±15.1 (78 – 124) vs. 132.9±11.4, (109 – 149), P <0.001] and high extracellular-Hb levels [mean mg/L ± SE, 59±6.5 (42 – 96) vs. 281.5±71.7 (184 – 410), P <0.001] in plasma of SCD patients compared to normal individuals. Therefore, we believe that these hyperactive HbVWF multimers play a crucial role in cell adhesion, vascular occlusion and thrombosis in SCD. Also, we speculate that this mechanism is not only limited in SCD, but also occurred in other pathophysiological conditions associated with severe intravascular hemolysis.