Conversion of factor X to factor Xa results in release of a heavily glycosylated activation peptide. Analysis of protease-digested glycopeptides derived from the activation peptides of bovine and human blood coagulation factor X allowed the identification of sites of the 0-linked oligosaccharide chains in these peptides. Glycopeptides were prepared from the activation peptides by digestion with chymotrypsin or StaphyZococcus aureus V8 protease. By combined analysis of amino acid sequence and sialic acid content, we found that bovine factor X had an 0-linked oligosaccharide chain linked to Thr26, and human factor X had four carbohydrate-attachment sites, namely, 0-glycosidic linkages to Thrl7 and Thr29, respectively, and N-glycosidic linkages to Am39 and Asn49, respectively, in their activation peptides. The 0-linked carbohydrate-attachment sites were identified since the yields of phenylthiohydantoin derivatives of amino acids that corresponded to their residues were increased during amino acid sequencing after deglycosylation of the glycopeptides with sialidase and 0-glycanase. The effect of deglycosylation of bovine factor XI was investigated with factor-X-activating enzyme from Russell's viper venom or extrinsic Xase (factor VIId tissue factor/phosholipid) by examining the activation rates of derivatives of factor X prepared using 0-glycanase, sialidase, and/or N-glycanase. The removal of 0-linked carbohydrate resulted in a decrease in the rate of activation. It appears that carbohydrate residues in factor X play an important role in the activation of the zymogen.Blood coagulation factor X is a vitamin-K-dependent protein that circulates in blood as the precursor of a serine protease. Factor X is a glycoprotein composed of a heavy chain and a light chain which are held together by a single disulfide bond. The complete amino acid sequence [l, 21 and the cDNA sequence [3] of bovine factor X and the cDNA sequence [4] of human factor X have been reported. The glycoprotein is synthesized in hepatocytes and undergoes specific post-translational modifications prior to secretion. The modifications include vitamin-K-dependent y-carboxylation of glutamic acid residues in the amino-terminal region of the light chain [5-71, /?-hydroxylation of an aspartic acid residue located in the first epidermal-growth-factor-like domain [7], and glycosylation. It was previously reported that oligosaccharide chains are linked to Asn36 and Thr3OO of Correspondence to T. Morita, Department of Biochemistry, Meiji College of Pharmacy, Tanashi, Tokyo, Japan 188Abbreviations. AP, activation peptide; AP, and AP,, the activation peptides (residues 1-51 of the heavy chain) of bovine factor X, and X,, respectively ; Pth, phenylthiohydantoin; RVV-XCP, the factor-X-activating enzyme from Russell's viper venom; Boc, tertbutoxycarbonyl ; NH-Np, p-nitroanilide.Enzymes. Coagulation factor Xa (EC 3.4.21.6) ; peptide N-glycosidase F (EC 3.5.1.52) ; endo-a-N-acetylgalactosaminidase (EC 3.2.1.97); sialidase (EC 3.2.1.18); chymotrypsin (EC 3.4.21.1); Stap...