Platelets are the sole source of EGF in the circulation, yet how EGF is stored or released from stimulated cells are undefined. In fact, we found platelets did not store EGF, synthesized as a single 6 kDa domain in pro-EGF, but rather expressed intact pro-EGF precursor on granular and plasma membranes. Activated platelets released high-molecular-weight (HMW)-EGF, produced by a single cleavage between the EGF and transmembrane domains of pro-EGF. We synthesized a fluorogenic peptide encompassing residues surrounding the putative sessile arginyl residue and found stimulated platelets released a soluble activity that cleaved this pro-EGF1020-1027 peptide. High throughput screening identified chymostatins, bacterial peptides with a central cyclic arginyl structure, as inhibitors of this activity. In contrast, the matrix metalloproteinase/TACE inhibitor GM6001 was ineffective. Stimulated platelets released the soluble protease ADAMDEC1, recombinant ADAMDEC1 hydrolyzed pro-EGF1020-1027, and this activity was inhibited by chymostatin and not GM6001. Biotinylating platelet surface proteins showed ADAMDEC1 hydrolyzed surface pro-EGF to HMW-EGF that stimulated HeLa EGF receptor (EGFR) reporter cells, and EGFR-dependent tumor cell migration. This proteolysis was inhibited by chymostatin and not GM6001. Metabolizing pro-EGF R1023 to citrulline with recombinant polypeptide arginine deiminase 4 (PAD4) abolished ADAMDEC1-catalyzed pro-EGF1020-1027 peptidolysis, while pretreating intact platelets with PAD4 suppressed ADAMDEC1-, thrombin-, or collagen-induced release of HMW-EGF. We conclude activated platelets release ADAMDEC1 that hydrolyzes pro-EGF to soluble HMW-EGF, HMW-EGF is active, proteolytic cleavage of pro-EGF first occurs at the carboxyl terminal arginyl residue of the EGF domain, and proteolysis is the regulated and rate-limiting step in generating soluble EGF bioactivity.
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Vol.48 No.2 from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/1S2Z7n2 via IFTTT
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