An assay for high-sensitivity detection of thrombin activity and determination of proteases activating or inactivating protease-activated receptors

This paper describes the development of galactosidase protease-activated re ceptor (GPAR) as a recombinant protein obtained by fusion of beta-galactosi dase, the extracellular domains of protease-activated receptors (PARs), and a biotin acceptor domain. Used as an immobilized substrate, this protein a llows the detection of thrombin in the sub-picomolar range. A comparative a nalysis for proteolytic cleavage of murine PAR1, PAR2, and PAR3 and human P ARA was performed, involving mutated and nonmutated GPAR fusion proteins. T hrombin cleaved GPAR1 (2.6 mol(beta-galactosidase)/(mol(thrombin) * min)), GPAR3 (410 mmol(beta-galactosidase)/(mol(thrombin) * min)), and GPAR4 (4.3 mmol(beta-galactosidase)/(mol(thrombin) * min)) specifically at the proteol ytic activation site. A second possible cleavage site for thrombin is prese nt in murine PAR1 and PAR3, Trypsin and plasmin cleaved all receptor fusion proteins with little specificity for the activation site, except for a mar ked preference of trypsin for cleavage at the activation site of GPAR2. Chy motrypsin cleaves GPAR1 at a rate (58 mmol(beta-galactosidase)/(mol(thrombi n) * min)) that suggests the possibility of chymotryptic inactivation of PA R1. 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, (C) 2000 Academic Press.