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 receptor (GPAR) as a recombinant protein obtained by fusion of beta-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(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 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(beta-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.
Altrogge
L M
LM
Friedrich Miescher-Institut, Postfach 2543, Basel, CH-4002, Switzerland.
Monard
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D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D011956:Receptors, Cell Surface / Q000032:analysis / Q000737:chemistry; D018179:Receptors, Thrombin / Q000032:analysis* / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D004800:Enzymes, Immobilized; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011993:Recombinant Fusion Proteins; C105295:protease-activated receptor 3; D001616:beta-Galactosidase; D002918:Chymotrypsin; D014357:Trypsin; D013917:Thrombin; D005341:Fibrinolysin; C112908:protease-activated receptor 4