ALTERNATIVELY SPLICED ISOFORM OF P-SELECTIN IS PRESENT IN-VIVO AS A SOLUBLE MOLECULE

To demonstrate the presence of a soluble isoform of P-selectin predict ed from cDNA sequencing (Johnston, G. I., Bliss, G. A., Newman, P. J., and McEver, R. P. (1990) J. Biol. Chem. 265, 21381-21885), we immunoi solated and compared structurally P-selectin from fresh frozen human p lasma with that from washed intact platelets. Plasma P-selectin was re active with rabbit antiserum to a synthesized peptide (residues 762-77 4 of mature P-selectin) but was significantly less reactive with antib ody to a peptide (residues 747-760). In contrast, platelet P-selectin reacted with both antibodies. S-Pyridylethylated plasma P-selectin was fractionated by reversed phase-high performance liquid chromatography into two major species. From platelets, two virtually identical speci es were separated. Sequential digestion with Achromobacter protease I and then Staphylococcus V8 protease produced peptides assigned to the tail region of the protein including the putative spliced site. From t he more hydrophilic species in both plasma and platelets, a peptide co mpletely lacking the sequence of the putative spliced site was identif ied. In contrast, the more hydrophobic species yielded a peptide with an intact transmembrane sequence. Hence, these results provide direct evidence that the previously predicted soluble isoform of P-selectin i s actually synthesized in vivo and is present as a circulating molecul e.