The Activation of Prothrombin

Abstract The intermediates formed during the activation of prothrombin in 25% sodium citrate solutions have been isolated and partially characterized. Their properties are in agreement with those predicted by a previously proposed model for prothrombin activation in 25% sodium citrate: prothrombin (72,000 daltons) → intermediate 1 (65,000 daltons) → Intermediate 2 (39,000 daltons) and Intermediate 3 (25,000 daltons); Intermediate 2 (39,000 daltons) → thrombin (α-thrombin, 39,000 daltons) (Mann, K. G., Heldebrant, C. M., and Fass, D. N. (1971) J. Biol Chem. 246, 6106–6114). Intermediates 1 and 2, as predicted, can be activated to thrombin, while Intermediate 3 cannot. The chromatographic behavior of Intermediate 2 is nearly identical with that of thrombin. Molecular weight and amino acid composition data strongly suggest that α-thrombin is formed from Intermediate 2 by the loss of little, if any, of the Intermediate 2 sequence. Intermediates 2 and 3 are shown to be substantially independent portions of the prothrombin sequence. The tryptic peptides derived from Intermediates 2 and 3, while very dissimilar account for greater than 85% of the tryptic peptides derived from prothrombin. Furthermore, Intermediate 3 contains nearly twice as much half-cystine and more than twice as much neutral sugar as Intermediate 2. A product of prothrombin activation, apparently derived from Intermediate 3, has been shown to alter the activity of thrombin toward N-α-tosyl-l-arginine methyl ester and fibrinogen. The possibility that a thrombin of altered specificity may participate in the control and regulation of coagulation is discussed.