Fibrinogen biosynthesis in isolated guinea pig megakaryocytes

Abstract

Fibrinogen synthesis was investigated in guinea pig megakaryocytes. Purified megakaryocytes were incubated with 35S-methionine in methionine-free incubation medium for 18 hours. Newly synthesized fibrinogen in megakaryocyte lysates enriched with purified carrier guinea pig fibrinogen was immunoprecipitated with a specific anti-guinea pig fibrinogen antiserum produced in rabbits. Proteins in the immunoprecipitates were analyzed with a 3.5% to 10.0% gradient polyacrylamide slab gel electrophoresis and auto-radiography. Radioactivity was detected in a protein band of 340,000 daltons. In order to verify fibrinogen synthesis, immunoprecipitate was analyzed by two-dimensional slab gel electrophoresis: (1) the first dimension separated unreduced fibrinogen using a 3.5% to 10.0% gradient gel; (2) following reduction by 2-beta-mercaptoethanol, fibrinogen chains were separated in the second dimension using a 10% gel. Alpha, beta, and gamma fibrinogen chains, which represented carrier guinea pig plasma fibrinogen, were visualized by Coomassie brilliant blue. Autoradiography identified the incorporation of radioactivity into the three fibrinogen chains. In control experiments, immunoprecipitates, produced by exposing megakaryocyte lysates to preimmune rabbit serum and goat anti-rabbit IgG, were also analyzed by the two-dimensional gel system. Radioactivity was not detected in sites corresponding to the migration of fibrinogen subunits. The study demonstrates that isolated guinea pig megakaryocytes can synthesize fibrinogen. The electrophoretic mobility of newly synthesized fibrinogen and subunits is similar to that of guinea pig plasma fibrinogen.