scholarly journals Dynamics of von Willebrand factor reactivity in sickle cell disease during vaso‐occlusive crisis and steady state

2017 ◽  
Vol 15 (7) ◽  
pp. 1392-1402 ◽  
Author(s):  
J. W. R. Sins ◽  
M. Schimmel ◽  
B. M. Luken ◽  
E. Nur ◽  
S. S. Zeerleder ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Von Willebrand Factor ◽  
Cell Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Hemoglobin Blocks Von Willebrand Factor Proteolysis by ADAMTS-13: A Mechanism Associated with Acquired ADAMTS-13 Deficiency in Patients with Sickle Cell Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3919-3919
Author(s):  
Zhou Zhou ◽  
Han Hyojeong ◽  
Miguel A. Cruz ◽  
Jose A. Lopez ◽  
Jing-fei Dong ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Von Willebrand Factor ◽  
Elevated Level ◽  
Dependent Manner ◽  
Cell Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract One of the hallmark events of sickle cell disease (SCD) is vasoocclusion and episodic pain crisis. Although the mechanism of vascular occlusion is very complicated, processes like thrombosis and thromboembolism have been recognized to play an important role in the development of such clinical manifestation in SCD. Studies have shown that the von Willebrand factor (VWF), especially the ultra-large (UL) multimers play a major role in vasoocclusion, which clearly indicates a possible impairment of the VWF-cleaving metalloproteae ADAMTS-13 in these patients with SCD. In a recent work, indeed we have mentioned that the plasma ADAMTS-13 in patients with SCD having normal antigen level showed 35% less protease activity than the normal. There may be several plasma factors responsible for the acquired deficiency of ADAMTS-13 in SCD. Since, the increasing evidences suggest that the elevated level of extracellular hemoglobin (Hb) in plasma parallely associated with the pathogenesis of SCD, we investigated the effects of extracellular Hb on VWF proteolysis by ADAMTS-13. We observed that purified Hb dose-dependently inhibited the ADAMTS-13 cleavage of recombinant(r) VWF and endothelial ULVWF multimers under static and flow conditions. Hb bound to VWF multimers in a saturation-dependent manner and more potently to the rVWFA2 domain (affinity Kd~24nM), which contains the cleavage site for ADAMTS-13. Hb bound also to the ADAMTS-13 (Kd~65nM), with 2.7 times less affinity than to VWFA2. The bindings were neither calcium-dependent nor affected by haptoglobin. However, it is the Hb-binding to VWF that prevented the substrate from being cleaved by ADAMTS-13. These in vitro findings are consistent with the in vivo observations in patients with SCD. An elevated level of extracellular Hb in plasma was inversely correlated (linear regression, r2 =0.6354) with the low activity of ADAMTS-13 in a cohort of ten adult patients with SCD (mean±SE, Hb 346±138 mg/l; activity 33.3±30%) compared to age and gender-matched normal individuals (n=10; Hb 24±8 mg/l; activity 76.2±16%). The data together suggest that patients with SCD suffer from acquired ADAMTS-13 deficiency, primarily because Hb competitively binds and inhibits the proteolysis of VWF multimers, leading to ULVWF accumulation on vascular endothelium and in circulation. The Hb-VWF interaction may therefore be considered as a therapeutic target for reducing thrombotic and vasoocclusive complications in patients with severe hemolysis such as those with SCD.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3058-3058
Author(s):  
Zhou Zhou ◽  
Hyojeong Han ◽  
Mark M. Udden ◽  
Miguel A. Cruz ◽  
Jing-fei Dong ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Von Willebrand Factor ◽  
Percent Level ◽  
Vascular Occlusion ◽  
Cell Disease ◽  
Normal Individuals ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract 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. Disclosures No relevant conflicts of interest to declare.

Haemoglobin blocks von Willebrand factor proteolysis by ADAMTS-13: A mechanism associated with sickle cell disease

2009 ◽  
Vol 101 (06) ◽  
pp. 1070-1077 ◽  
Author(s):  
Zhou Zhou ◽  
Hyojeong Han ◽  
Miguel Cruz ◽  
José López ◽  
Jing-Fei Dong ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Von Willebrand Factor ◽  
Vascular Occlusion ◽  
Dependent Manner ◽  
Cell Disease ◽  
Small Pool ◽  
High Level ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVascular occlusion, thromboembolism and strokes are hallmark events in sickle cell disease (SCD). The von Willebrand factor (VWF), largest adhesive protein in circulation, has been implicated as major component in these processes. In SCD, a high level of extracellular haemoglobin (Hb) in plasma has been shown parallely associated with the disease pathogenesis. Investigating the effect of Hb we observed that purified Hb significantly inhibited the ADAMTS-13 cleavage of VWF under static and flow conditions. Hb bound potently to VWF specifically VWFA2 in a saturation-dependent manner with half-maximal binding 24 nM. Inversely, VWFA2 also bound potently to Hb and binding was inhibited by VP1 antibody, which binds to ADAMTS-13 cleavage site on VWF. Microscopic observation also shows that Hb bound specifically to endothelial VWF under flow. Furthermore, the Hb-bound VWF multimers were isolated from plasma. Though, Hb bound also to ADAMTS-13, it is the Hb binding to VWFA2 that prevented the substrate being cleaved by ADAMTS-13. In an observation in a small pool of patients with SCD, high Hb in plasma was inversely correlated with low proteolytic activity of ADAMTS-13. Thus, the observations suggest that the patients with SCD suffer from an acquired ADAMTS-13 deficiency primarily because Hb competitively bound and blocked the proteolysis of VWF, leading to the accumulation of ultra-large VWF multimers in circulation and on endothelium. Therefore, the Hb-VWF interaction may be considered as a therapeutic target for treating thrombotic and vaso-occlusive complications in patients with severe intravascular haemolysis such as those with SCD.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2304-2304 ◽  
Author(s):  
Junmei Chen ◽  
Yi Wang ◽  
Tahsin Ozpolat ◽  
Colette Norby ◽  
Xiaoyun Fu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Human Umbilical Cord ◽  
Cell Disease ◽  
Normal Plasma ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract 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. Disclosures: No relevant conflicts of interest to declare.

Extracellular Hemoglobin Regulation of von Willebrand Factor Activity in Plasma of Patients with Sickle Cell Disease

2011 ◽  
Vol 07 (02) ◽  
pp. 150
Author(s):  
Zhou Zhou ◽  
Prasenjit Guchhait ◽  
◽  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Von Willebrand Factor ◽  
Vascular Occlusion ◽  
Cell Disease ◽  
Molecular Pathophysiology ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Elevated levels of ultralarge (UL) von Willebrand factor (VWF) multimers in plasma play an important role in cell adhesion and vascular occlusion in sickle cell disease (SCD). Recently, we have shown that the binding of extracellular hemoglobin (ECHb) to the A2 domain of VWF significantly blocks VWF cleavage by the metalloprotease ADAMTS13in vitro. We speculated that on release from the inflamed endothelium, the VWF multimers maintain the UL structure in plasma if ECHb prevents their cleavage. We observed that a subpopulation of VWF multimers that are bound to ECHb (HbVWF), which accounted for about 14 % of the total VWF in the plasma of SCD patients. The plasma HbVWF level is parallely correlated with the ECHb and VWF-antigen levels. The HbVWF multimers are resistant to the metalloprotease ADAMTS13in vitroand are more adhesive to platelets and collagen compared with their Hb-free counterpart. Therefore, we speculate that the HbVWF, which are probably UL multimers, play an important role in tethering and stably adhering blood cells to the vascular endothelium and culminate in vascular occlusion/thrombosis/strokes in SCD patients. Thus, this article provides a new insight into the molecular pathophysiology of SCD.

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