Figure 1
Glycosylation of platelet-VWF and susceptibility to ADAMTS13 proteolysis. (A) Platelet (PT)-VWF was purified from lysed (Lys) human platelets by immunoaffinity chromatography and analyzed by SDS-polyacrylamide gel electrophoresis and subsequent silver staining. (B) ABO(H) blood group antigen expression on plasma (PL)-VWF and PT-VWF were quantified using lectin plate-binding assays. All experiments were performed in triplicate, and the results shown represent the mean ± SEM (***P < .001). (C) Reverse-phase HPLC analysis was used to quantify sialic acid expression on purified PL-VWF and PT-VWF. Total sialic acid expression on PT-VWF was significantly reduced compared with PL-VWF (76 vs 167 nmol/mg; ***P < .001). To determine relative quantitative sialic acid expression levels on the N-linked glycans of VWF, HPLC analysis of residual VWF-bound sialic acid was performed following digestion with O-glycosidase. To determine quantitative sialic acid expression on the O-linked glycans of VWF, HPLC analysis of residual VWF-bound sialic acid was performed following digestion with PNGase F. (ns, P value is nonsignificant). (D) To investigate whether altered glycosylation on platelet-VWF influences susceptibility to ADAMTS13 proteolysis, PT-VWF and PL-VWF were incubated with 3 nM recombinant human ADAMTS13 in the presence of 1.5 M urea. Rate of cleavage was assessed by determining the reduction in VWF:CB over time. Results (mean of 5 experiments ± SEM) are expressed as the percentage residual collagen-binding activity. In some cases, SEM cannot be seen due to its small size. (E) The susceptibility of PT-VWF and PL-VWF multimers to digestion with recombinant human ADAMTS13 in the presence of 1.5 M urea was further assessed by performing standard nonreducing SDS agarose gel electrophoresis at baseline (T0) and following a 30-minute incubation (T30). (F) To establish whether the altered glycosylation profile of PT-VWF influenced susceptibility to other nonspecific proteases, PL-VWF and PT-VWF were also treated with chymotrypsin (30 U/mg VWF) and carboxypeptidase Y (19 U/mg VWF) at 37°C for 90 minutes. Results are shown as residual VWF:CB at 90 minutes ± SEM.

Glycosylation of platelet-VWF and susceptibility to ADAMTS13 proteolysis. (A) Platelet (PT)-VWF was purified from lysed (Lys) human platelets by immunoaffinity chromatography and analyzed by SDS-polyacrylamide gel electrophoresis and subsequent silver staining. (B) ABO(H) blood group antigen expression on plasma (PL)-VWF and PT-VWF were quantified using lectin plate-binding assays. All experiments were performed in triplicate, and the results shown represent the mean ± SEM (***P < .001). (C) Reverse-phase HPLC analysis was used to quantify sialic acid expression on purified PL-VWF and PT-VWF. Total sialic acid expression on PT-VWF was significantly reduced compared with PL-VWF (76 vs 167 nmol/mg; ***P < .001). To determine relative quantitative sialic acid expression levels on the N-linked glycans of VWF, HPLC analysis of residual VWF-bound sialic acid was performed following digestion with O-glycosidase. To determine quantitative sialic acid expression on the O-linked glycans of VWF, HPLC analysis of residual VWF-bound sialic acid was performed following digestion with PNGase F. (ns, P value is nonsignificant). (D) To investigate whether altered glycosylation on platelet-VWF influences susceptibility to ADAMTS13 proteolysis, PT-VWF and PL-VWF were incubated with 3 nM recombinant human ADAMTS13 in the presence of 1.5 M urea. Rate of cleavage was assessed by determining the reduction in VWF:CB over time. Results (mean of 5 experiments ± SEM) are expressed as the percentage residual collagen-binding activity. In some cases, SEM cannot be seen due to its small size. (E) The susceptibility of PT-VWF and PL-VWF multimers to digestion with recombinant human ADAMTS13 in the presence of 1.5 M urea was further assessed by performing standard nonreducing SDS agarose gel electrophoresis at baseline (T0) and following a 30-minute incubation (T30). (F) To establish whether the altered glycosylation profile of PT-VWF influenced susceptibility to other nonspecific proteases, PL-VWF and PT-VWF were also treated with chymotrypsin (30 U/mg VWF) and carboxypeptidase Y (19 U/mg VWF) at 37°C for 90 minutes. Results are shown as residual VWF:CB at 90 minutes ± SEM.

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