The epidermal growth factor-like domain from tissue plasminogen activator. Cloning in E. coli, purification and ESR studies of its interaction with human blood platelets
Pietrucha, T.; Stec, W.J.; Okruszek, A.; Uznański, B.; Koziołkiewicz, M.; Wilk, A.; Płucienniczak, A.; Swiatkowska, M.; Cierniewski, C.S.
Acta Biochimica Polonica 41(1): 25-34
1994
ISSN/ISBN: 0001-527X PMID: 8030371 Document Number: 438396
To examine whether the epidermal growth factor (EGF)-like domain Pro-47-Asp-87 is involved in the interaction of tissue plasminogen activator (t-PA) with platelets, we have expressed this domain in E. coli. The peptide fragment was produced from a plasmid expression vector as a fusion protein with beta-galactosidase Met-1-Val-444 at high yield in eight clones of E. coli. The fusion protein was purified and subjected to mild acid hydrolysis with formic acid, then the peptide Pro-47-Asp-87, identified by immunoblotting using specific antibodies to t-PA, was isolated by HPLC. After incubation with blood platelets spin labelled with 16-doxylstearic acid or 5-doxylstearic acid, the Pro-47-Asp-87 peptide fragment reduced fluidity of the membranae lipid bilayer to the same extent as did intact t-PA as indicated by ESR measurements. Our data suggest that the EGF-like domain of t-PA can directly interact with blood platelets and thus it seems to contain those sites of the t-PA molecule that bind the platelet membrane components.