Evidence That Calpains and Elastase Do Not Produce the von Willebrand Factor Fragments Present in Normal Plasma and IIA von Willebrand Disease

• Published in: Blood 1988-08, Vol.72 (2), p.721-727
• Main Authors: Berkowitz, Scott D., Nozaki, Hiroyuki, Titani, Koiti, Murachi, Takashi, Plow, Edward F., Zimmerman, Theodore S.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biological and medical sciences; Calpain - pharmacology; Hematologic and hematopoietic diseases; Humans; Medical sciences; Pancreatic Elastase - pharmacology; Peptide Fragments - analysis; Peptide Fragments - blood; Platelet diseases and coagulopathies; von Willebrand Diseases - blood; von Willebrand Factor - metabolism
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V72.2.721.721

Recent evidence suggests that proteolysis plays an important role in some forms of inherited and acquired von Willebrand disease (vWD). Because calpains and one or more enzymes released from polymorphonuclear leukocytes are known to proteolyze von Willebrand factor (vWF) in vitro with resultant loss of large multimers similar to that seen in IIA vWD, they have been suggested as being responsible for the proteolysis in vivo. Using monoclonal epitope mapping, we have examined the proteolysis of the vWF subunit by porcine calcium-activated neutral proteases (calpains) and human leukocyte elastase to determine whether they produce the vWF proteolytic cleavage products seen in normal individuals and IIA vWD. Purified vWF was digested with porcine calpains I and II. We found no difference in the size, location, and quantity of the fragments produced by calpain I v calpain II. New fragments were detected of approximately 200,170,150, and 125 Kd. There was no evidence for generation of the native 140 and 176 Kd fragments. Some loss of the native fragments was seen, which suggests that they were further cleaved. Epitope mapping of the 170- and 150-Kd calpain-cleaved fragments revealed them to be from different parts of the molecule than the regions from which the native 176- and 140-Kd fragments derived. This was further supported by determination of the amino-terminal sequence of the calpain-cleaved 170- and 150-Kd fragments. Digestion of vWF with human leukocyte elastase produced new fragments at 210/205, 190, 170/165, 145/140, and 130/125 Kd. No generation of native fragments was detected. Monoclonal epitope mapping of the 145/140-Kd elastase-cleaved band proved that it derived from the carboxyl-terminal portion of the vWF molecule, whereas the native 140-Kd fragment is derived from the amino-terminal end. Neither calpains nor human leukocyte elastase produced the proteolyzed fragments present in normal and IIA vWD and, therefore, probably do not cause the loss of large multimers that is seen in that disorder. © 1988 by Grune & Stratton, Inc.