Biosynthesis of Dog Fibrinogen

Dogs were injected with 3H‐labeled L‐amino acids and the incorporation of radioactivity into fibrinogen, its component polypeptide chains and various well characterized fibrinogen fragments, was determined in newly secreted plasma fibrinogen and in nascent fibrinogen isolated from the rough endoplasmic reticulum of hepatocytes. At 15‐20 min after the administration of 3 H‐labeled l ‐amino acids, radioactive fibrinogen was secreted onto the blood and the specific radioactivities of the Aα, Bβ and y chains were approximately the same at all times of secretion up to 2 h. Newly secreted fibrinogen was almost entirely in its phosphorylated form. At 15‐18 min, a time at which maximal incorporation of radioactivity had occurred in proteins, fibrinogen was isolated from a deoxycholate‐soluble fraction of the rough endoplasmic reticulum by affinity chromatography using columns of fibrin monomer as well as those prepared from monospecific antibodies to dog fibrinogen and to the fibrinogen chains. The nascent fibrinogen thus isolated was further characterized by analyses of thrombin‐ released fibrinopeptides A and B and their tryptic peptides. Furthermore, the nascent fibrinogen was cleaved with cyanogen bromide, the NH 2 ‐terminal disulfide knot was prepared and its component chain fragments as well as the tryptic peptides derived from these fragments were isolated. The specific radioactivities of the various fibrinogen fragments were determined and it is concluded that all three fibrinogen chains are synthesized to the same extent and that, within the rough endoplasmic reticulum, nascent fibrinogen has already been fully disulfide‐bonded and exists as a dimer. In contrast to plasma fibrinogen, the Aa chain of nascent fibrinogen within the rough endoplasmic reticulum is not phosphorylated and the Bβ chain is not sulfated.