cDNA cloning of thrombin-receptor on platelet membrane glycoprotein Ib.

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GPIb was not detected on the surface of HEL-cells with flow-cytometrical studies using a monoclonal anti-GPIb antibody. TM60. However, when HEL-cells were cultured for 24hrs in a medium containing 25ng/ml TPA and then for 44 hrs in a TPA-free medium, the cells were found to express GPIb on the surfaces. MRNA was extracted from the cells which had been stimulated by TPA as above and harvested after 24 hr-culture in a TPA-free medium. A phage lambda gt10 cDNA library was prepared and screened using probes synthesized based on Lopez's report. Several milloin recombinant phage from HEL-cell lambda gt10 library were screened. Cloning of GPIb -chain was unsuccessful.In order to determine a thrombin-binding site on GPIb -chain more accurately than previously estimated, 11 different, partially overlapping peptides consisting of 11-28 amino acid residues analogous to GPIb -chain were synthesized and their effects on 0.02U/ml thrombin-induced aggregation of human washed platelets were observed. A peptide, FT25(#216-240) showed the strongest inhibition (IC_<50>=12mu M) and two other peptides close to FT25 in the region (#216-274) also had inhibitory effects (IC_<50> of less than 100 mu M). In contrast, peptides out of the region (#216-274), for examples, DN24(#18-41), EP26(#181-206) and ETP26(#281-306) did not inhibit thrombin-induced aggregations (IC_<50> of more than 150mu M). Peptides with shorter than 18 amino acid residues in the region (#216-274) close to FT25 had no effect on the aggregation. These results suggest that thrombinbinding site exists on the region #216-274 with a core portion on FT25.