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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 α2-3,6,8,9 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.

Abstract 2234 Von Willebrand Factor (VWF) is extensively glycosylated with both N- and O-linked carbohydrates. Moreover, these complex glycan structures influence VWF functional properties, including susceptibility to ADAMTS13 proteolysis, and plasma clearance. The molecular mechanisms through which VWF glycosylation (including ABO blood group antigens) act to influence VWF physiology remains unexplained. However, recent data suggest that VWF circulates in normal plasma bound to various carbohydrate-binding proteins, including specific members of the galectin family. In addition, galectin-3 binding has been reported to influence VWF cleavage by ADAMTS13. In this context, we sought to elucidate the role of specific VWF glycan determinants in modulating galectin interaction. VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. VWF glycosylation was then modified using exoglycosidases and quantified by specific lectin ELISAs. Blood group specific VWF was also purified from pooled group AB, O, or Bombay plasmas. Galectins-1 and -3 were transiently expressed in competent E-coli cells with an N-terminal histidine tag, and purified by nickel chromatography. Finally, binding interactions were characterized via modified immunosorbant assay. In keeping with the previous report of Lenting et al, human pdVWF bound to both galectin-1 and galectin-3 in a dose-dependent manner. Enzymatic desialylation of pdVWF with α2-3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced binding to galectin-1 (231±6%, p ABO(H) blood group antigens are expressed on both the N-linked and O-linked glycans of human VWF. Moreover, ABO(H) determinants influence VWF susceptibility to ADAMTS13 proteolysis and plasma VWF half-life, through unknown mechanisms. Purified VWF from normal group AB individuals bound to both galectin-1 and galectin-3 significantly better than group O VWF (146±8% and 483±19%; p These novel data demonstrate that both the N- and O-linked oligosaccharide structures of VWF are involved in mediating galectin binding. In particular, expression of terminal AB blood group antigens, and expression of sub-terminal galactose moieties following loss of capping sialic acid, both markedly enhance galectin binding affinity. Further studies will be required to define how galectin binding is involved in mediating the functional consequences of variation in VWF glycans. Disclosures: No relevant conflicts of interest to declare.

Abstract 382FN2 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 (t1/2 = 46.9 min). Enzymatic desialylation of VWF with α2–3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced VWF clearance (t1/2 = 3.7 min; p 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. Disclosures: No relevant conflicts of interest to declare.

Abstract 2119 Poster Board II-96 ABO(H) blood group antigen expression on platelets varies widely among normal blood donors. An ABO ‚High Expresser' phenotype (HXP) that exhibits significantly increased A and/or B antigen expression on platelets has been identified in ∼7% of normal donors. HXP has been implicated in both platelet-refractoriness and neonatal alloimmune thrombocytopenic purpura, however, the underlying molecular and genetic elements that mediate this phenomenon are not well-defined. To investigate the mechanisms underlying HXP, blood samples were collected from 231 group A (180 A1and 51 A2) and 310 group O individual apheresis platelet donors. Quantitative expression of platelet A and H antigen were then assessed by flow cytometry of platelet-rich plasma. In total, 10 A1 donors (5.6%) exhibited HXP. Analysis of the platelet A antigen expression in these individuals identified 8 HXP donors who exhibited ‚type I' HXP (normal bimodal population of platelets, but with predominant A antigen expression) whereas 2 individuals exhibited ‚type II' HXP (a single uniform population of platelets, strongly positive for blood group A expression). Both types of HXP were found to be a stable donor characteristic. ABO(H) determinants have also been identified on the N-linked glycans of the plasma von Willebrand factor (VWF), and influence plasma VWF levels and susceptibility to proteolysis by ADAMTS13. To determine whether HXP was platelet-specific, blood group A antigen expression on plasma VWF from group A donors was determined. Interestingly, blood group A antigen expression on plasma VWF was concordantly increased in donors with type I and type II HXP, indicative of increased glycosyltransferase expression in HXP individuals. To ascertain whether increased glycosyltransferase expression contributes to HXP, ABO genotype was determined for all 231 group A donors by PCR-RFLP analysis. Genotype at the ABO locus on 9q34 exerts a dosage effect on glycosyltransferase expression. 80% HXP (all type I) donors were genotyped A1A1. suggesting increased A transferase activity contributes to type I HXP. Despite this, the majority of A1A1individuals (67%) did not exhibit HXP, and 2 HXP donors were found to possess the A2 allele, which expresses limited A transferase enzymatic activity. Collectively, this data clearly demonstrates the contribution of additional factors to ABO genotype that contribute to HXP. To identify additional HXP modifiers, potential enhancer repeat elements upstream of the ABO gene were examined in group A donors, including those with HXP. Typically, A1alleles contain a single 43-base pair repeat within a minisatellite positive regulatory region upstream of the ABO gene. In contrast, A2and O1alleles contain four 43bp repeats, which are associated with a 100-fold enhancement of transcriptional activity. Analysis of this enhancer region demonstrated two HXP donors with A1alleles containing four copies of the 43-base pair repeat. Consequently, this allele would be predicted to modulate A transferase expression via enhanced ABO gene transcription. In conclusion, we have demonstrated the multi-factorial nature of the regulatory elements mediating platelet type I and type II HXP. A1alleles containing novel upstream enhancer repeats identified in donor individuals may represent a novel genetic mediator of HXP, and contribute to the pathophysiology associated with this phenomenon independently of ABO genotype. Disclosures: No relevant conflicts of interest to declare.

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 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 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. Disclosures: No relevant conflicts of interest to declare.