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

Summary

Vascular 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.

Keywords

Extracellular haemoglobin - von Willebrand factor - ADAMTS-13 activity - sickle cell disease

• References

• 1 Reiter CD, Wang X, Tanus-Santos JE. et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat Med 2002; 8: 1383-1389.
• 2 Savitsky JP, Doczi J, Black J. et al. A clinical safety trial of stroma-free hemoglobin. Clin Pharmacol Ther 1978; 23: 73-80.
• 3 Gladwin MT, Sachdev V, Jison ML. et al. Pulmonary hypertension as a risk factor for death in patients with sickle cell disease. N Engl J Med 2004; 350: 886-895.
• 4 Olsen SB, Tang DB, Jackson MR. et al. Enhancement of platelet deposition by cross-linked hemoglobin in a rat carotid endarterectomy model. Circulation 1996; 93: 327-332.
• 5 Schnog JJ, Hovinga JA, Krieg S. et al. ADAMTS13 activity in sickle cell disease. Am J Hematol 2006; 81: 492-498.
• 6 Krishnan S, Siegel J, Pullen Jr G. et al. Increased von Willebrand factor antigen and high molecular weight multimers in sickle cell disease associated with nocturnal hypoxemia. Thromb Res 2008; 122: 455-458.
• 7 Roberts DD, Rao CN, Liotta LA. et al. Comparison of the specificities of laminin, thrombospondin, and von Willebrand factor for binding to sulfated glycolipids. J Biol Chem 1986; 261: 6872-6877.
• 8 Wick TM, Moake JL, Udden MM. et al. Unusually large von Willebrand factor multimers increase adhesion of sickle erythrocytes to human endothelial cells under controlled flow. J Clin Invest 1987; 80: 905-910.
• 9 Kaul DK, Nagel RL, Chen D. et al. Sickle erythrocyte-endothelial interactions in microcirculation: the role of von Willebrand factor and implications for vasoocclusion. Blood 1993; 81: 2429-2438.
• 10 Bernardo A, Ball C, Nolasco L. et al. Effects of inflammatory cytokines on the release and cleavage of the endothelial cell-derived ultra large von Willebrand factor multimers under flow. Blood 2004; 104: 100-106.
• 11 Studt JD, Hovinga JA, Antoine G. et al. Fatal congenital thrombotic thrombocytopenic purpura with apparent ADAMTS13 inhibitor: in vitro inhibition of ADAMTS13 activity by hemoglobin. Blood 2005; 105: 542-544.
• 12 Rother RP, Bell L, Hillmen P. et al. The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease. J Am Med Assoc 2005; 293: 1653-1662.
• 13 Cruz MA, Whitelock J, Dong JF. Evaluation of ADAMTS-13 activity in plasma using recombinant von Willebrand Factor A2 domain polypeptide as substrate. Thromb Haemost 2003; 90: 1204-1209.
• 14 Whitelock JL, Nolasco L, Bernardo A. et al. ADAMTS-13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF-A2 domain as substrate. J Thromb Haemost 2004; 2: 485-491.
• 15 Tao Z, Peng Y, Nolasco L. et al. Recombinant CUB-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions. Blood 2005; 106: 4139-4145.
• 16 Choi H, Aboulfatova K, Pownall HJ. et al. Shear-induced disulfide bond formation regulates adhesion activity of von Willebrand factor. J Biol Chem 2007; 282: 35604-35611.
• 17 Li Y, Choi H, Zhou Z. et al. Covalent regulation of ULVWF string formation and elongation on endothelial cells under flow conditions. J Thromb Haemost 2008; 6: 1135-1143.
• 18 Wick TM, Moake JL, Udden MM. et al. Unusually large von Willebrand factor multimers preferentially promote young sickle and nonsickle erythrocyte adhesion to endothelial cells. Am J Hematol 1993; 42: 284-292.
• 19 Sadrzadeh SM, Graf E, Panter SS. et al. Hemoglobin. A biologic fenton reagent. J Biol Chem 1985; 259: 14354-14356.
• 20 Furlan M, Robles R, Lamie B. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 1996; 87: 4223-4234.
• 21 Vischer UM, Jornot L, Wollheim CB. et al. Reactive oxygen intermediates induce regulated secretion of von Willebrand factor from cultured human vascular endothelial cells. Blood 1995; 85: 3164-3172.
• 22 Wagener FA, Eggert A, Boerman OC. et al. Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase. Blood 2001; 98: 1802-1811.
• 23 Joneckis CC, Ackley RL, Orringer EP. et al. Inte-grin α4β1 and glycoprotein IV (CD36) are expressed on circulating reticulocytes in sickle cell anemia. Blood 1993; 82: 3548-3555.
• 24 Mohandas N, Evans E. Sickle erythrocyte adherence to vascular endothelium. Morphologic correlates and the requirement for divalent cations and collagen-binding plasma proteins. J Clin Invest 1985; 76: 1605-1612.
• 25 Moake JL, Rudy CK, Troll JH. et al. Unusually large plasma factor VIII:von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med 1982; 307: 1432-1435.
• 26 Hebbel RP, Boogaerts MA, Eaton JW. et al. Erythrocyte adherence to endothelium in sickle-cell anemia: A possible determinant of disease severity. N Engl J Med 1980; 302: 992-995.