Rat platelets contain glycosylated and non-glycosylated forms of platelet factor 4. Identification and characterization by mass spectrometry.

Platelet factor 4 is a heparin-binding protein released from the alpha granules of activated platelets. This study describes the purification and identification of two forms of rat platelet factor 4, the previously characterized non-glycosylated form of 7 kDa and an additional glycosylated form of molecular mass 9 kDa. The two proteins both neutralized the antithrombin-III-dependent inhibitory activity of heparin. Although their amino acid composition was found to be the same, in the N-terminal sequence of the 9-kDa protein, the second threonine residue could not be detected and a difference of 976Da was determined by mass spectrometry. After digestion with O-glycanase and sialidase, the two proteins showed the same molecular mass. Overall consideration of these data led to identification of the higher-molecular-mass protein as a glycosylated form of rat platelet factor 4 with O-glycosylation at the second N-terminal amino acid, while the structure of the oligosaccharide core was established by mass spectrometry and sugar differentiation with lectins. The two forms of platelet factor 4 are both present in platelets and secreted after platelet activation.