Expression of terminal α2-6–linked sialic acid on von Willebrand factor specifically enhances proteolysis by ADAMTS13

Abstract

von Willebrand factor (VWF) multimeric composition is regulated in plasma by ADAMTS13. VWF deglycosylation enhances proteolysis by ADAMTS13. In this study, the role of terminal sialic acid residues on VWF glycans in mediating proteolysis by ADAMTS13 was investigated. Quantification and distribution of VWF sialylation was examined by sequential digestion and high-performance liquid chromatography analysis. Total sialic acid expression on VWF was 167nmol/ mg, of which the majority (80.1%) was present on N-linked glycan chains. Enzymatic desialylation of VWF by ␣2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to 50% (؎ 14%) by ADAMTS13, compared with 11% (؎ 7%) for untreated VWF. Despite this, Neu-VWF exhibited increased susceptibility to other proteases, including trypsin, chymotrypsin, and cathepsin B. VWF expressing different blood groups exhibit altered ADAMTS13 proteolysis rates (O > B > A > AB). However, ABO blood group regulation of ADAMTS13 proteolysis was ablated on VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. These novel data show that sialic acid protects VWF against proteolysis by serine and cysteine proteases but specifically enhances susceptibility to ADAMTS13 proteolysis. Quantitative variation in VWF sialylation therefore represents a key determinant of VWF multimeric composition and, as such, may be of pathophysiologic significance. (Blood. 2010;115(13): 2666-2673) Introduction von Willebrand factor (VWF) is a large multimeric plasma sialoglycoprotein that plays 2 essential roles in normal hemostasis. 1 First, it mediates platelet adhesion to exposed subendothelial tissues at sites of vascular injury. 2 Second, VWF acts as a carrier molecule for procoagulant factor VIII (FVIII), thereby protecting it from premature proteolytic degradation and clearance. 3 In vivo expression of VWF is restricted to endothelial cells (ECs) and megakaryocytes only. VWF synthesized within ECs is either secreted constitutively into the plasma or alternatively stored in specific intracellular organelles known as Weibel-Palade bodies. 4 In contrast, VWF synthesized within megakaryocytes is subsequently stored within the ␣ granules of their platelet progeny. Consequently, plasma VWF is derived chiefly from ECs. 5 Within ECs, VWF undergoes complex posttranslational modification before secretion. 6 In the endoplasmic reticulum, individual VWF monomers are assembled into dimers through the formation of C-terminal disulphide bonds. 7 Subsequently, VWF dimers form high-molecular-weight multimers in the Golgi after another round of N-terminal disulphide bond formation. 8 The multimeric composition of plasma VWF is a critical determinant of its functional activity, because larger multimers bind both collagen and platelets with significantly higher affinities and are thus more efficient in inducing platelet aggregation under high-shear conditions. 9,10 In normal plasma, VWF multimer distributio...

Altered glycosylation of platelet-derived von Willebrand factor confers resistance to ADAMTS13 proteolysis

“…11,14 In recent studies, we and others have clearly demonstrated the key role played by VWF glycans in regulating proteolysis by ADAMTS13. [15][16][17][18] Importantly, expression of both ABO blood group antigens and terminal sialic acid on VWF were shown to be of critical importance. 15,16,18 In this study, we have characterized terminal carbohydrate expression on platelet-VWF.…”

“…[15][16][17][18] Importantly, expression of both ABO blood group antigens and terminal sialic acid on VWF were shown to be of critical importance. 15,16,18 In this study, we have characterized terminal carbohydrate expression on platelet-VWF. Our findings demonstrate that platelet-VWF exists as a distinct natural glycoform, with a particular marked reduction in N-linked sialic acid expression compared with plasma-VWF.…”

“…Finally, platelet-VWF was purified by immunoaffinity chromatography employing the monoclonal antibody CLB-RAg20 as previously described in detail. 20 Plasma-derived VWF was purified by cryoprecipitation and 2BCL gel filtration as before, 18 followed by CLB-RAg20 immunoaffinity chromatography.…”

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

“…6,7,[9][10][11] Although the O-linked glycans of VWF also demonstrate marked heterogeneity, disialyl core 1 structures account for ;70% of the total population. 8,12 Importantly, although the majority of its glycans are capped by negatively charged sialic acid, 7,13 VWF is unusual in that a minority of both N-linked and O-linked carbohydrate chains express terminal ABO(H) blood group determinants. 7,8,11 Although substantial progress has been achieved in understanding the biosynthesis, structure, and functions of VWF, the biological mechanism(s) responsible for modulating VWF clearance from the plasma remain poorly understood.…”

Molecular Basis of Thrombophilia / MOLEKULARNE OSNOVE TROMBOFILIJE

“…Recent studies have clarified this association and verified that B and A1 blood groups are at higher risk than O and A2 blood groups, with the relative risk of approximately 2 (38,39). It is assumed that an ABO blood group could contribute to thrombosis risk through modifications of von Willebrand factor (VWF) and factor VIII (FVIII) levels in plasma (40,41). On the other hand, several studies have revealed that ABO blood groups remain significantly associated with elevated prothrombotic risk, even after adjustment for FVIII or VWF levels (41,42).…”