American Society of Hematology

Figure 2.

$Figure 2. Purification of Bombay VWF and susceptibility to ADAMTS13 proteolysis. To investigate further the mechanism underlying the low plasma VWF:Ag levels associated with the Bombay phenotype, we purified HMW-VWF from 2 individual Bombay subjects and from normal pooled group AB and group O plasmas. (A) Serial analysis of eluate fractions from the Sepharose CL-2B gel filtration column demonstrated that VWF began to elute in fraction B6. OD indicates optic density. (B) Analysis of the early fractions (B6-B9) by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions followed by silver staining demonstrated a single major protein band at 250 kDa consistent with monomeric VWF. Multimer analysis showed that fractions also contained predominantly HMW-VWF. (C) In order to investigate susceptibility of HMW-VWF to ADAMTS13 proteolysis, comparable eluate fractions (range, B7-B9) were chosen for group AB, group O, and Bombay VWF purifications, containing comparable HMW-VWF. These were incubated with recombinant human ADAMTS13 (5-20 nM) at 1.5-M urea concentration, and rate of VWF cleavage was assessed by determining the rate of fall in VWF:CB. Results (mean of 6 experiments ± SEM) are expressed as a percentage residual collagen-binding activity. In some cases, the SEM cannot be seen due to its small size. After 90 minutes of incubation, the rates of proteolysis between groups AB, O, and Bombay were significantly different (O versus AB, P < .01; Bombay versus O, P < .01; and Bombay versus AB, P < .001). (D) The effect of ABO on susceptibility of VWF to ADAMTS13 proteolysis was investigated at different concentrations of urea (0.5 to 4 M). As urea concentration was raised from 0.5 M to 2 M, the rate of VWF proteolysis increased for each blood group. However, the difference between AB, O, and Bombay HMW-VWF, which was most marked at 1.5 M urea, became progressively less apparent as urea concentration was increased. In the presence of 4 M urea, proteolysis of HMW-VWF by ADAMTS13 was inhibited (data not shown).$

Purification of Bombay VWF and susceptibility to ADAMTS13 proteolysis. To investigate further the mechanism underlying the low plasma VWF:Ag levels associated with the Bombay phenotype, we purified HMW-VWF from 2 individual Bombay subjects and from normal pooled group AB and group O plasmas. (A) Serial analysis of eluate fractions from the Sepharose CL-2B gel filtration column demonstrated that VWF began to elute in fraction B6. OD indicates optic density. (B) Analysis of the early fractions (B6-B9) by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions followed by silver staining demonstrated a single major protein band at 250 kDa consistent with monomeric VWF. Multimer analysis showed that fractions also contained predominantly HMW-VWF. (C) In order to investigate susceptibility of HMW-VWF to ADAMTS13 proteolysis, comparable eluate fractions (range, B7-B9) were chosen for group AB, group O, and Bombay VWF purifications, containing comparable HMW-VWF. These were incubated with recombinant human ADAMTS13 (5-20 nM) at 1.5-M urea concentration, and rate of VWF cleavage was assessed by determining the rate of fall in VWF:CB. Results (mean of 6 experiments ± SEM) are expressed as a percentage residual collagen-binding activity. In some cases, the SEM cannot be seen due to its small size. After 90 minutes of incubation, the rates of proteolysis between groups AB, O, and Bombay were significantly different (O versus AB, P < .01; Bombay versus O, P < .01; and Bombay versus AB, P < .001). (D) The effect of ABO on susceptibility of VWF to ADAMTS13 proteolysis was investigated at different concentrations of urea (0.5 to 4 M). As urea concentration was raised from 0.5 M to 2 M, the rate of VWF proteolysis increased for each blood group. However, the difference between AB, O, and Bombay HMW-VWF, which was most marked at 1.5 M urea, became progressively less apparent as urea concentration was increased. In the presence of 4 M urea, proteolysis of HMW-VWF by ADAMTS13 was inhibited (data not shown).

This Feature Is Available To Subscribers Only