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Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader
Published on: May 24, 2024
Protease action and generation of beta-thromboglobulin-like protein followed by platelet activation
1Biomedical Technology Wing, Thrombosis Research Unit, Sree Chitra Tirunal Institute for Medical sciences and Technology, 695012, Trivandrum, India.
Abstract:
beta-Thromboglobulin (betaTG) is a platelet specific protein present in the alpha-granules and secreted into the surrounding medium on cell activation. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of platelet releasate after inhibition of metalloproteinases with ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetra acetic acid (EGTA) showed disappearance of an 8.0-kDa band. In the absence of the cation chelators, a 48-kDa band disappeared and concurrently, the 8.0-kDa band intensity increased suggesting that the former may be the immediate precursor of the latter. The Western blot stained using specific antibodies, isolated from single-cell clones of hybridoma, against 8.0-kDa protein recognized not only 48- and 8.0-kDa bands but few others too. The data suggest that one or more high molecular weight (HMW) protein is released from alpha-granules and is broken down into smaller fragments after release to form beta-thromboglobulin (beta-TG)-like proteins by the action of metal-dependent proteases.
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