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A CALCIUM-DEPENDENT PROTEASE CLEAVES VWF73 AT THE PEPTIDE BOND CORRESPONDING TO V1607-T1608 IN THE A2 DOMAIN
Abstract number: P-W-185
Raife1 T.J., Bedell1 B., Zheng2 X., Montgomery3 R.R., Johnson1 G.
11Department of Pathology, University of Iowa, Iowa City, IA 22Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 33Blood Research Institute, The Blood Center of Wisconsin and Children's Hospital of Wisconsin, Milwaukee, WI, United States
How-to-cite Raife TJ, Bedell B, Zheng X, Montgomery RR, Johnson G. A CALCIUM-DEPENDENT PROTEASE CLEAVES VWF73 AT THE PEPTIDE BOND CORRESPONDING TO V1607-T1608 IN THE A2 DOMAIN. J Thromb Haemost 2007; 5 Supplement 2: P-W-185
Abstract
Introduction: Mucocutaneous bleeding is a common manifestation of von Willebrand disease (VWD). Saliva contains a number of enzymes whose role in oropharyngeal hemostasis is poorly understood. We previously reported a salivary protease with activity against von Willebrand factor (VWF) with functional similarities to ADAMTS13.
Methods: We further characterized the salivary protease using the 73 amino acid VWF substrates GST-VWF73 and FRET-VWF73.
Results: Immunoblotting demonstrated that protease in saliva cleaved GST-VWF73 yielding a cleavage fragment slightly smaller than the fragment generated by plasma. Purified IgG from a TTP patient abolished proteolytic activity in plasma but did not affect activity in saliva. The proteolytic activity determined by FRET-VWF73 in 23 healthy volunteer saliva samples ranged from undetectable to more than 20-fold greater than normal pooled plasma when normalized to protein concentration. Mass spectrometry analysis of plasma-cleaved FRET-VWF73 yielded a 7027 kD peak consistent with cleavage at Y1605-M1606. Saliva yielded a 6796 kD peak, consistent with cleavage at V1607-T1608. Saliva dialyzed against 10 mM EDTA was inactive; addition of CaCl2, but not NaCl, restored activity. Among protease inhibitors, only EDTA inhibited plasma activity. By constrast, saliva activity was completely abolished by phenylmethylsulfonyl fluoride (PMSF), and substantially inhibited by several other serine protease inhibitors.
Conclusions: Saliva contains a potent calcium-dependent protease that cleaves VWF two peptide bonds from the ADAMTS13 cleavage site and is markedly variable between individuals. Salivary proteases may be modifying factors and targets for therapy in mucocutaneous bleeding.
To cite this abstract use the following format:
Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number
Session Details
| Date: |
01/08/2007
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| Time: |
00:00-00:00
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| Session name: |
XXIst ISTH Congress |
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| Location: |
Oxford, UK |
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