An Assay for High-Sensitivity Detection of Thrombin Activity and Determination of Proteases Activating or Inactivating Protease-Activated Receptors

✍ Scribed by Ludger M. Altrogge; Denis Monard

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 336 KB
• Volume: 277
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4356

✦ Synopsis

This paper describes the development of galactosidase protease-activated receptor (GPAR) as a recombinant protein obtained by fusion of ␤-galactosidase, the extracellular domains of protease-activated receptors (PARs), and a biotin acceptor domain. Used as an immobilized substrate, this protein allows the detection of thrombin in the sub-picomolar range. A comparative analysis for proteolytic cleavage of murine PAR1, PAR2, and PAR3 and human PAR4 was performed, involving mutated and nonmutated GPAR fusion proteins. Thrombin cleaved GPAR1 (2.6 mol ␤-galactosidase /(mol thrombin * min)), GPAR3 (410 mmol ␤-galactosidase /(mol thrombin * min)), and GPAR4 (4.3 mmol ␤-galactosidase /(mol thrombin * min)) specifically at the proteolytic activation site. A second possible cleavage site for thrombin is present in murine PAR1 and PAR3. Trypsin and plasmin cleaved all receptor fusion proteins with little specificity for the activation site, except for a marked preference of trypsin for cleavage at the activation site of GPAR2. Chymotrypsin cleaves GPAR1 at a rate (58 mmol ␤-galactosidase /(mol thrombin * min)) that suggests the possibility of chymotryptic inactivation of PAR1. Elastase may inactivate PAR1 and PAR3, but probably not PAR2 and PAR4. Neither activated protein C nor the plasminogen activators cleave any GPAR fusion protein at considerable rates.