Extracellular Hemoglobin Increases the Binding of the Active Form of Von Willebrand Factor to Glycoprotein Ibα and Platelet Activation Under Shear Stress: Implications for Sickle Cell Disease and Other Hemolytic Disorders
Authors: Cruz Miguel A. , Guchhait Prasenjit , Brown Ryanne Ashley 
Published: October 2019

Abstract
Abstract 2095
Sickle cell disease (SCD) is characterized by a hypercoagulable state that accelerates vaso-occlusive events in microcirculation, leading to acute and chronic organ damage. Studies have indicated that excessive release of hemoglobin from erythrocytes into plasma may contribute to platelet activation and thrombosis. This finding is likely due to the infusion of extracellular hemoglobin (ECHb), which causes platelet aggregation and adhesion on prothrombotic surfaces, and inhibits the metalloprotease ADAMTS13, the enzyme that reduces the size of the constitutively active ultra large (UL) von Willebrand factor (VWF) multimers into a smaller non-active form of VWF. In fact, we have described that the binding of ECHb to the A2 domain of VWF prevents the cleavage of VWF by ADAMTS13. This inhibition from ECHb may raise the levels of hyperreactive VWF multimers in blood, contributing to thrombosis. Interestingly, a recent study from another research group described a direct correlation between the rate of hemolysis and the levels of circulating hyperreactive VWF multimers in plasma from SCD patients. We further analyzed the functional consequences of ECHb binding to active VWF multimers and are now describing a novel role for ECHb in VWF-mediated thrombosis. With increasing concentrations of ECHb (0 – 600 μg/ml) mixed with a constant concentration of endothelial-derived ULVWF, we observed incremental increases in the binding capacity of ULVWF for platelet GPIbα. Maximal binding capacity was obtained at 200 μg/ml of ECHb. Identical results were obtained with plasma-derived VWF using a low dose of ristocetin to activate VWF. This finding is due to the preferential binding of ECHb to the active form of VWF. The anti-A2 domain monoclonal antibody VP-1 effectively inhibited the upregulated effect of ECHb on VWF-GPIbα binding by blocking the interaction between ECHb and the A2 domain in both ULVWF and plasma VWF. We then tested the effect of ECHb on VWF-mediated platelet activation/adhesion to a surface coated with fibrin(ogen) under high shear rates. This assay required the addition of a low dose of ristocetin (0.15 mg/ml) to whole blood prior to perfusion. The effect of ristocetin on platelet activation/adhesion was effectively blocked with either EDTA or antibodies against GPIbα and αIIbβ3. ECHb (200 μg/ml) or buffer was added to whole blood prior to perfusion over the fibrin(ogen)-coated surface. At a shear rate of 1500s−1, the amount of activated/adhered platelets observed with blood containing ECHb was significantly higher (300%) than that of blood containing buffer. Comparable results were obtained when ristocetin was substituted with a gain-of-function recombinant A1A2A3 mutant (R1450E, 100nM), which exposes the A2 domain and exhibits increased GPIbα-binding activity. The number of adherent platelets from blood containing only ECHb without ristocetin was <5% of that from blood containing only ristocetin. Previously, we have demonstrated that the dissociation of A1 and A2 domains in VWF increases the binding of A1 to GPIbα. Therefore, we speculate that the interaction of ECHb with the exposed A2 domain in active VWF influences the structure of the neighboring A1 domain, provoking A1 to adopt a conformation with a higher binding affinity for GPIbα. Furthermore, these results suggest an important role for the interaction between ECHb and hyperreactive VWF in SCD pathology, and most likely other conditions presenting with microangiopathic hemolytic anemia.

Disclosures:
No relevant conflicts of interest to declare.