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.