Oxidation Of Von Willebrand Factor and ADAMTS13 In Patients With Sickle Cell Disease

Sickle cell disease (SCD) is a hemoglobinopathy characterized by vaso-occlusive episodes and hemolysis. We previously showed that SCD patients at disease baseline have elevated levels of von Willebrand factor (VWF) and enhanced VWF adhesive activity (Chen et al. Blood, 2011, 117:3680-3683). The total active VWF (quantity times relative adhesive activity compared to pooled plasma) correlated with the degree of hemolysis. VWF is an adhesive protein capable of binding platelets, erythrocytes, and leukocytes, especially in its newly released form, a portion of which remains attached to the endothelium until it is cleaved off by the plasma metalloprotease ADAMTS13. Here, we explored the mechanisms that could account for increased total active VWF in SCD patients, including increased endothelial secretion, ADAMTS13 inhibition, and VWF oxidation. Previously, we showed that the neutrophil oxidant hypochlorous acid (HOCl), oxidized VWF at the ADAMTS13 cleavage site (Met1606) rendering it uncleavable, and at other sites that increased its platelet-binding activity (Chen et al. Blood, 2010, 115:706-712 and Fu et al. Blood, 2011, 118: 5283-5291). We have also found that HOCl can inactivate ADAMTS13 by oxidizing Met249 in the Met-turn of the metalloprotease domain. We first examined whether patient plasma could activate endothelial cells to secrete VWF strings using plasma from 8 patients at disease baseline. Patient plasma was incubated with monolayers of human umbilical cord vein endothelial cells (HUVECs) in a parallel-plate flow chambers for 20 min at 37°C before fixed platelets were perfused through the chamber to decorate the VWF strings. HUVECs incubated with either normal pooled plasma or phorbol myristate acetate were the negative and positive controls, respectively. The data are expressed as a percent of the strings seen in the positive control. SCD plasma activated HUVECs to secrete more VWF strings than did normal plasma (11% – 31% for patient plasma compared to 1% – 6% for normal plasma). We also measured the concentration of myeloperoxidase (MPO) in the plasma from these patients. Almost all (16 of 17) had elevated MPO concentration, ranging from 1.3 to 16.2 times the control. MPO released from activated neutrophils converts hydrogen peroxide to HOCl in the presence of chloride ion. We therefore have begun to evaluate the extent of VWF and ADAMTS13 oxidation in patient plasma using tandem mass spectrometry. In the one patient examined for VWF oxidation, we found 2.8% and 4.6% % oxidation at M1606 and M1385, respectively, versus 0.2% and 0.5% in the control. Although this is only a small percent of all the vulnerable Met residues, this extent of oxidation was accompanied by a marked defect in the ability of the patient’s endogenous ADAMTS13 to cleave endogenous VWF, even though the ADAMTS13 activity was normal when tested with small A2 peptide substrate. We have also examined ADAMTS13 oxidation in two other SCD patients at disease baseline. Here too, oxidation of Met249 was increased compared to control (4.0% and 4.8% vs 2.5% in the control).

In summary, in studies of several patients with SCD at disease baseline, we have found: elevated levels of VWF and ADAMTS13 oxidation, defective cleavage of endogenous VWF by endogenous ADAMTS13, and activation of endothelial cells with release of VWF by patient plasma. These findings all suggest that oxidative stress associated with SCD contributes to worsening the vaso-occlusive and hemolytic aspects of the disease and increases the risk for thrombosis. We are expanding these studies to include more patients at baseline, and patients in acute crisis, where we expect the oxidative signature to be even higher.