A Critical Role for N- and O-Linked Carbohydrates In Modulating Von Willebrand Factor Clearance In Vivo

Abstract 382^FN2

VWF is a multimeric plasma sialoglycoprotein essential for normal haemostasis. Although the biosynthesis, structure and functional properties of VWF have been well characterized, the molecular mechanism(s) underlying its clearance remain poorly understood. Nevertheless, enhanced VWF clearance is important in the pathophysiology of VWD. Moreover, emerging data suggest that variation in VWF glycosylation (notably ABO blood group) may constitute an important regulator of in vivo clearance rates. To define the role of VWF glycans in modulating clearance, VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF glycosylation was modified using exoglycosidases and quantified by specific lectin-binding ELISAs. Finally, the effect of altered glycosylation on VWF plasma half-life was characterized by administration of VWF glycan variants to VWF^−/− mice.

Wild type pdVWF was cleared in biphasic manner, characterized by a rapid initial phase followed by a slower secondary phase (t_1/2 = 46.9 min). Enzymatic desialylation of VWF with α2–3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced VWF clearance (t_1/2 = 3.7 min; p<0.01). Digestion of pdVWF with α2–3 neuraminidase to remove predominantly O-linked sialic acid (which constitutes less than 20% total VWF sialylation) was also sufficient to markedly enhance VWF clearance (t_1/2 = 13.1 min; p<0.05). In the presence of the asialoglycoprotein receptor (ASGPR)-antagonist ASOR, the mean residence time of Neu-VWF was identical to that of pd-VWF. Recent studies have shown that macrophages may be important in VWF clearance. Since the ASGPR is expressed on both hepatocytes and macrophages, the effect of macrophage depletion on VWF clearance was assessed. Pre-treatment with liposome-encapsulated clodronate depleted F4/80+CD11b+ murine macrophages by 75%, and significantly prolonged Neu-VWF survival. However Neu-VWF survival was not corrected to that observed in the presence of ASOR. For example, plasma Neu-VWF survival after 5 mins was corrected from 30±6% to 92±7% in the presence of ASOR, compared to 78±10% following clodronate macrophage-depletion. Cumulatively, these findings demonstrate that both N- and O-linked sialylation are critical in protecting VWF against ASGPR-mediated clearance. Moreover, ASGPR-modulated clearance is at least in part macrophage-dependent. ß-galactose residues exposed following removal of capping sialic acid are recognised by the ASGPR. To further define the role of specific sugars in regulating VWF clearance, the effect of terminal sialic acid and sub-terminal galactose removal by sequential neuraminidase and galactosidase digestions was studied. Surprisingly, VWF exposed to sequential neuraminidase and galactosidase digestions (NeuGal-VWF) was cleared rapidly from the plasma in a monophasic fashion (t_1/2 = 4.8 min). Moreover, treatment with PNGase F to completely remove N-linked carbohydrate structures also markedly decreased the plasma half-life (PNG-VWF; t_1/2 = 2.1 min). In keeping with their lack of exposed galactose residues, the enhanced clearance of NeuGal-VWF and PNG-VWF were not mediated via the ASGPR (ASOR had no significant effect). In contrast, macrophage depletion by liposomal clodronate significantly inhibited the enhanced clearance of both NeuGal-VWF and PNG-VWF respectively. These data suggest that the ASGPR is not the only macrophage receptor involved in modulating VWF clearance, which is consistent with the relatively minor prolongation in VWF survival previously reported in Asgpr1^−/− mice.

These novel data demonstrate that variation in the N- or O-linked carbohydrate structures significantly modulate VWF half-life in vivo. Moreover, VWF clearance is not mediated solely through the ASGPR, but may also require additional as yet unidentified macrophage receptors for full clearance. Therefore, qualitative and quantitative variation in VWF glycosylation represents a key regulator of VWF clearance, and as such is likely to be of direct pathophysiological significance.