Sialic acids (SA) are negatively charged monosaccharides present on the terminus of N- and O-linked glycans. They carry out diverse functions such as protection from proteolytic degradation and modulation of protein clearance. Both the N- and O-linked glycans of VWF are decorated with SA residues, which have been shown to block recognition and clearance of VWF via the asialoglycoprotein receptor (ASGPR). It has been previously shown that VWF devoid of SA residues exhibits increased platelet binding and decreased susceptibility to ADAMTS13 proteolysis, however this has only been shown under static conditions. In this study we have investigated the role of VWF SA under the physiological conditions of shear stress.
Firstly, using a plate-based ELISA assay we confirmed that desialylated (ds)VWF demonstrated enhanced sensitivity to ristocetin-mediated binding to GPIbα Interestingly, this effect was attributed to α 2–3 linked SA which are present primarily on O-linked glycans. For analysis under flow conditions, a recombinant VWF fragment spanning the D'-A3 domains was immobilised on Ibidi flow slides and perfused with washed, labelled platelets and red blood cells at a range of shear rates. This fragment contains the majority of the O-linked glycans on VWF which are clustered either side of the A1 domain. At low shear rates (400–600s−1), dsD'A3 captured more platelets than wild type (wt) D'A3; whilst a similar number of platelets was captured to both proteins at high shear rates (1000–2000s−1). Platelet translocation velocity over dsD'A3 was decreased at all shear rates (400–2000s−1) when compared to wtD'A3: however the dissociation rate constant was unaltered. Interestingly, when perfused over a collagen type III coated surface in reconstituted blood, dsVWF and VWF devoid of only α 2–3 linked sialic acid residues (α 2–3dsVWF), both mediated platelet capture to a similar extent as wtVWF. In the absence of VWF, no platelet capture was detected, confirming that platelet capture under the shear rate applied (1500s−1) was VWF dependent. Subsequently, to assess susceptibility of ds- and α 2–3ds VWF to ADAMTS13 proteolysis under shear, recombinant ADAMTS13 was added to plasma free blood supplemented with VWF and perfused over collagen. In control experiments, the presence of ADAMTS13, but not the inactive ADAMTS13 variant (M225Q), diminished platelet capture demonstrating the assay was sensitive to ADAMTS13 proteolysis of VWF. Interestingly, the decrease in platelet capture in the presence of ADAMTS13 was significantly less with dsVWF and α 2–3dsVWF than with control VWF, demonstrating that dsVWF and α 2–3dsVWF are proteolysed slower by ADAMTS13 under shear stress and confirming the relevance of the results from static assays. Furthermore, these data suggest that it is the α 2–3 linked sialic acid residues capping mainly O-linked oligosaccharides which modulate VWF sensitivity to ADAMTS13 cleavage under high shear stress.
In summary, these data demonstrate that sialylation of VWF increases its susceptibility to ADAMTS13 cleavage under flow but does not otherwise affect VWF mediated platelet capture to collagen. ADAMTS13 is crucial for regulating haemostasis, as it controls the size of newly secreted ultra large VWF multimers and can cleave VWF at sites of haemostatic plug formation. It has been previously shown that sialyltransferase expression levels vary between different tissues, and sialic acid expression can change in different pathological conditions, it is therefore important to characterise how the differences in VWF sialylation status affect its key properties such as platelets binding and susceptibility to proteolysis.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.