Cleavage of human von Willebrand factor by porcine pancreatic elastase.
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.
Howard
M A
MA
Department of Medicine, Monash University, Prahran, Australia.
Greco
T
T
Coghlan
M
M
D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D003435:Crotalid Venoms / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007519:Isoantigens / Q000302:isolation & purification; D007700:Kinetics; D010196:Pancreatic Elastase / Q000378:metabolism; D013552:Swine; D014841:von Willebrand Factor / Q000276:immunology / Q000378:metabolism*
D003435:Crotalid Venoms; D007519:Isoantigens; D014841:von Willebrand Factor; C030961:botrocetin; D010196:Pancreatic Elastase