Cleavage and activation of human factor IX by serine proteases.
Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.
D000906:Antibodies; D002918:Chymotrypsin / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D005164:Factor IX / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007610:Kallikreins / Q000494:pharmacology; D010196:Pancreatic Elastase / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D012697:Serine Endopeptidases; D013917:Thrombin / Q000494:pharmacology; D013925:Thromboplastin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology; D014757:Viper Venoms / Q000378:metabolism
D000906:Antibodies; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D014757:Viper Venoms; D005164:Factor IX; D013925:Thromboplastin; D010450:Endopeptidases; D007610:Kallikreins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D013917:Thrombin