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.
INSERM U311, Centre Régional de Transfusion Sanguine, Strasbourg, France.
C
C
Ravanat
C
C
Gachet
J M
JM
Herbert
S
S
Schuhler
J C
JC
Guillemot
F
F
Uzabiaga
C
C
Picard
P
P
Ferrara
M
M
Freund
J P
JP
Cazenave
D000595:Amino Acid Sequence; D000818:Animals; D001769:Blood; D001792:Blood Platelets / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism; D007120:Immunochemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010978:Platelet Factor 4 / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats
D010978:Platelet Factor 4; D006493:Heparin