Cleavage of human von Willebrand factor by porcine pancreatic elastase
MA Howard, T Greco and M Coghlan
Department of Medicine, Monash University, Prahran, Australia.
von Willebrand factor (vWF) was purified from pooled normal plasma,
radiolabeled with iodine then cleaved by porcine pancreatic elastase.
Cleavage was monitored by sodium dodecylsulphate-polyacrylamide gel
electrophoresis (SDS-PAGE), SDS-agarose electrophoresis, crossed immuno-
electrophoresis, ristocetin and botrocetin cofactor activities, and
ristocetin and botrocetin induced binding to fixed washed platelets.
Cleavage of vWF by porcine elastase was dependent on both the concentration
of porcine elastase and period of incubation. Incubation of vWF with
concentrations of porcine elastase greater than 20 U/mL for 30 minutes
resulted in loss of the 240 Kd vWF subunit and a corresponding loss of the
high molecular weight multimers and formation of fragments with molecular
weights on reduced SDS-PAGE of 150, 125, 115 Kd and minor bands at 44, 28,
and 24 Kd and a band at 20 Kd, which increased in intensity as either the
concentration of porcine elastase or the period of incubation increased.
More than 50% of the ristocetin cofactor activity was lost during a
five-minute incubation of vWF with 0.56 U/mL porcine elastase when no
detectable structural changes in the vWF molecule had occurred. Botrocetin
cofactor activity was more resistant. Similarly botrocetin induced vWF
binding to platelets was retained by a fraction produced by digestion of
vWF with porcine elastase, which contained predominantly a 20 Kd fragment
of vWF. This fragment retained insignificant amounts of ristocetin related
functions and therefore represents a useful piece of the vWF molecule for
further exploration of the site involved in botrocetin induced binding to
platelets.
Volume 74,
Issue 2,
pp. 673-681,
08/01/1989
Copyright © 1989 by The American Society of Hematology
