Role of glycans expression in regulating the ADAMTS13-resistant phenotype of PL-VWF. (A) PL-VWF and PT-VWF were incubated with PNGase F to remove N-linked glycans. Subsequently, the effects of N-linked deglycosylation in regulating susceptibility to ADAMTS13 proteolysis were investigated. Removal of N-linked glycans significantly increased PL-VWF cleavage by ADAMTS13 (PL-VWF [□] vs PNG-PL-VWF [▪]; P < .001 at all time points). Treatment of platelet-VWF with PNGase F was also associated with significantly enhanced proteolysis by ADAMTS13 (PT-VWF [○] vs PNG-PT-VWF [●]; P < .001). Nevertheless, PNG-treated PT-VWF remained more resistant to ADAMTS13 proteolysis compared with PNG-treated PL-VWF. All experiments were performed a minimum of 3 times and results are expressed as mean ± SEM at 120 minutes. (B) PL-VWF and PT-VWF were incubated with O-glycosidase (OGly) to remove O-linked glycans. Subsequently, the effects of O-linked deglycosylation in regulating susceptibility to ADAMTS13 proteolysis were investigated. Removal of O-linked glycans from PL-VWF had no significant effect on cleavage by ADAMTS13 (PL-VWF [□] vs OGly-PL-VWF [▪]. In contrast, treatment of PL-VWF with OGly was associated with significantly enhanced proteolysis by ADAMTS13 (PT-VWF [○] vs PNG-PT-VWF [●]; P < .01). (C) PL-VWF and PT-VWF were incubated with α2-3,6,8,9 neuraminidase to remove terminal sialic acid. Subsequently, the effects of desialylation in regulating susceptibility to ADAMTS13 proteolysis were investigated as previously described. Removal of sialic acid from PL-VWF (neuraminidase [Neu]-PL-VWF) significantly attenuated proteolysis by ADAMTS13 (***P < .001). In keeping with its markedly reduced sialic acid expression levels, Neu digestion of PT-VWF demonstrated only a small effect on susceptibility to ADAMTS13 cleavage.