THE COMPLETE PRIMARY STRUCTURE OF GLYCOSYLATED PORCINE PLATELET FACTOR-4

We have purified platelet factor 4 from porcine platelets and shown th at it is glycosylated. The purified protein migrated as a broad band a t approximately 14 000 Da, characteristic of glycoproteins. Electrospr ay mass spectroscopy of the intact protein gave a predominant mass of 11 111 Da, with a minor component of 10 804 Da. Sialidase digestion re duces both forms to a single mass of 10 497 Da. Upon Edman degradation , the amino terminus was found to be blocked by the presence of a pyro glutamate residue. We have determined the complete primary structure o f platelet factor 4 by peptide mapping and Edman degradation, thereby completing information on the amino-terminal and carboxy-terminal regi ons which is missing in the previously published partial sequence. Seq uencing of the intact and deglycosylated protein show that the glycosy lation site is at Thr8. The amino acid composition accounts for a mass of 9623 Da, and the carbohydrate moeity was found to contribute 1490 Da. The biological activity of the porcine protein has been compared t o recombinant human platelet factor 4 in an endothelial cell prolifera tion assay; both inhibit at a concentration giving half the maximal in hibition of 0.1 mu M. Removal of the 19 amino-terminal residues carryi ng the carbohydrate moiety results in no change in the biological acti vity.