Characterization of material with epidermal growth factor immunoreactivity in human serum and platelets

We investigated the molecular nature of the epidermal growth factor immunoreactivity (ir-EGF) in serum and platelets of normal subjects. In serum, ir-EGF appeared and increased during spontaneous blood coagulation, reaching a plateau in 2 h. The mean plateau measured by time-resolved immunofluorometric assay was 778 pg/mL [130 pmol/L; range, 465-1352 pg/mL (78-225 pmol/L)] for men (n = 66), and 774 pg/mL (129 pmoL/L; range, 521-1114 pg/mL (87-186 pmol/L)] for women (n = 33). Serum samples (n = 9), when examined under nonreducing conditions by high performance liquid chromatography, contained three mol wt components. Their approximate proportions averaged 56 +/- 6% (+/- SD) for a 140K component, 22 +/- 9% for a 67K component, and 22 +/- 6% for a component coeluting with the 6K EGF standard. The molecular size distribution of ir-EGF released from isolated platelets varied with the treatment of the platelets. After stimulation with a Ca2+ ionophore, the proportions of the 140K, 67K, and 6K components were 61 +/- 11%, 20 +/- 8%, and 17 +/- 4% (n = 5), respectively, as in serum. In addition, we found a small amount, (approximately 1%) of a 17K component. When platelets (n = 6) were ruptured by sonication and repeated freeze-thawing, the 67K component formed 53 +/- 7% of the total; the proportions of the 140K, 17K, and 6K components were 14 +/- 7%, 2.1 +/- 0.7%, and 31 +/- 4%, respectively. Under reducing conditions the 17K and 6K components remained intact, but part of the 140K component and all of the 67K component were cleaved to a 35-37K form. After protease digestion of the higher mol wt components, the ir-EGF was exclusively of the 6K form. The 67K and 6K components bound to the EGF receptor, whereas the 140K component bound inconsistently. We conclude that treatment of platelets with Ca2+ ionophore produces ir-EGF components similar to those found in incubated serum but different from those obtained by freeze-thawing. The 67K mol wt component appears to be the main storage form of EGF in platelets; at least two independent mechanisms appear to exist for EGF release.