The soluble protease ADAMDEC1 released from activated platelets hydrolyzes platelet membrane pro-epidermal growth factor (EGF) to active high-molecular-weight EGF

Platelets are the sole source of EGF in circulation, yet how EGF is stored or released from stimulated cells is 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 the transmembrane domains of pro-EGF. We synthesized a fluorogenic peptide encompassing residues surrounding the putative sessile arginyl residue and found stimulated platelets released 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 (tumor necrosis factor-α-converting enzyme) 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 Arg1023 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 that activated platelets release ADAMDEC1, which hydrolyzes pro-EGF to soluble HMW-EGF, that HMW-EGF is active, that proteolytic cleavage of pro-EGF first occurs at the C-terminal arginyl residue of the EGF domain, and that proteolysis is the regulated and rate-limiting step in generating soluble EGF bioactivity from activated platelets.