Activation of Fluorescein-labeled Prothrombin

Abstract Bovine prothrombin can be labeled with fluorescein isothiocyanate to produce a fluorescent proenzyme which maintains up to 85% of native prothrombin-specific activity. Other proteins labeled similarly were analyzed by sodium dodecyl sulfate gel electrophoresis, and it was found that the chromophores did not affect the molecular weights as determined by the electrophoretic system. Sodium citrate activation of both labeled and unlabeled prothrombin produced molecular weight species consistent with our previously proposed model (Mann, K. G., Heldebrant, C. M., and Fass, D. N. (1971) J. Biol. Chem. 246, 6106–6114): prothrombin (72,000), Intermediate 1 (65,000), Intermediate 2 (39,000), and Intermediate 3 (25,000). The thrombins are produced from Intermediate 2 and consist of multichain disulfide-linked structures; α-thrombin (33,000 + 6,000), β-thrombin (18,000 + 10,000), and γ-thrombin (14,000 + 10,000 + 4,000). An analog of the physiological coagulation system was constructed using serum and a phospholipid source. Labeled prothrombin when activated in this mixture yielded products which, by virtue of their fluorescence, could be distinguished from the serum proteins. All precursors of thrombin which are produced during citrate activation are also produced during serum activation. The large thrombin, α-thrombin, is composed of 33,000- and 6,000-dalton chains in both activating systems. These results indicate that prothrombin activation in biological systems produces intermediates and products identical with those formed in highly purified synthetic activation systems. Our studies also indicate that the kinetics of the activation process in serum is demonstrably different from that previously described in highly purified systems.