Terminal α2-6 Linked Sialic Acid Expression On VWF Specifically Enhances Proteolysis by ADAMTS13.

Abstract 30

VWF is a large plasma sialoglycoprotein that mediates platelet tethering at sites of vascular injury. VWF function is dependent upon VWF multimeric composition, which is regulated in plasma by ADAMTS13. ABO(H) blood group determinants expressed on VWF N-linked glycans significantly influence susceptibility to ADAMTS13 proteolysis. In this study, we investigated whether terminal sialic acid residues expressed on the N- and O-linked glycans of VWF may also regulate proteolysis by ADAMTS13. VWF was initially purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF sialylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. The rate of glycosidase-treated VWF proteolysis by ADAMTS13 was determined by incubation with recombinant ADAMTS13 and subsequent measurement of residual VWF collagen binding activity.

Complete VWF deglycosylation has been shown to enhance the rate of proteolysis by ADAMTS13. In contrast, enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired the rate of ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to only 50±14% by ADAMTS13, compared to 11±7% for untreated VWF (p<0.01) at the same time point. Despite this, Neu-VWF exhibited increased susceptibility to proteases other than ADAMTS13 (trypsin, chymotrypsin and cathepsin B; all p<0.05). VWF sialylation is therefore a specific enhancer of ADAMTS13-mediated proteolysis. Consequently, quantification and molecular distribution of VWF sialylation was examined by sequential digestion and HPLC analysis. Total sialic acid expression on pdVWF was 167nmol/mg, of which the majority (133.4nmol/mg or 80.1%) was present on its N-linked glycan chains. Interestingly, despite the resistance to ADAMTS13 proteolysis observed upon complete desialylation, digestion of pdVWF with α2-3 neuraminidase to remove predominantly O-linked sialic acid did not influence the rate of ADAMTS13 proteolysis.

Previous studies have demonstrated that VWF expressing different blood groups exhibit altered rates of proteolysis by ADAMTS13 (O ≥ B > A ≥ AB). Since α2-6 linked sialic acid and ABO(H) determinants are both expressed as terminal antigens on VWF N-linked glycans, the effect of desialylation upon blood group-specific VWF proteolysis by ADAMTS13 was determined. As expected, untreated group O VWF was cleaved significantly faster than group AB-VWF (p<0.05). However, the ability of ABO blood group to regulate ADAMTS13 proteolysis was completely ablated upon VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. This indicates that VWF sialylation constitutes a more important determinant of susceptibility to ADAMTS13 proteolysis than ABO(H) expression.

Sialic acid can mediate protein-protein interactions through either conformational and/or charge-mediated mechanisms. Despite this, sodium metaperiodate treatment of pdVWF to remove sialic acid anionic charge did not influence the rate of proteolysis by ADAMTS13. In contrast, the ability of sialic acid to specifically enhance ADAMTS13 proteolysis of VWF was significantly attenuated at high urea concentrations (≥2M), supporting the hypothesis that VWF sialylation enhances proteolysis by ADAMTS13 by promoting a ADAMTS13-specific permissive conformation.

These novel data demonstrate that although sialic acid protects VWF against proteolysis by serine and cysteine proteases, it also specifically enhances susceptibility to proteolysis by ADAMTS13. Moreover, the magnitude of this sialic acid-specific effect on VWF proteolysis by ADAMTS13 is more marked than that attributable to N-linked ABO(H) blood group antigen expression. Therefore, quantitative variation in VWF sialylation represents a key regulator of VWF multimeric composition, and as such, is likely to be of clear patho-physiological significance.