The Activation of Prothrombin

Abstract

Abstract An unambiguous assignment of the product-precursor relationship is made for each step in the activation of prothrombin with Factor Xa. Prothrombin and each of its activation intermediates were reacted with trypsin-produced Factor Xa and with thrombin. [3H]Diisopropylphosphorofluoridate incorporation studies were performed to identify the active thrombin produced from its precursors. The results obtained allow us to order the events of the activation process in terms of the production of the intermediates of the activation process previously described by this laboratory (Mann, K. G., Heldebrant, C. M., and Fass, D. N. (1971) J. Biol. Chem. 246, 6106–6114). The initial activation step, the conversion of prothrombin to Intermediate 1 and Intermediate 3 may be catalyzed by either Factor Xa or by thrombin, however, later activation steps require Factor Xa as the catalytic agent. Treatment of prothrombin with thrombin yields two fragments which are indistinguishable from those produced by Factor Xa. Intermediate 4, a previously unreported activation fragment, is produced concomitant with Intermediate 2 during cleavage of Intermediate 1 with Factor Xa. Intermediate 2 and Intermediate 4 are readily resolved by DEAE-cellulose chromatography. The intermediates derived from prothrombin by citrate-Factor Xa activation (Interemdiates 1, 2, 3, and 4) or by the action of thrombin (Intermediates 1t and 3t) have also been characterized with respect to their molecular weights, amino acid compositions, and NH2-terminal amino acid residues. The NH2-terminal amino acids and the molecular weights are: prothrombin-alanine, 70,000; Intermediate 1-serine, 51,000; Intermediate 1t-serine, 51,000; Intermediate 2-threonine, 41,000; Intermediate 3-alanine, 23,000; Intermediate 3t-alanine, 23,000; Intermediate 4, 13,000. The sum of the molecular weights and amino acid compositions of the products are equivalent to the molecular weight and amino acid composition of the proposed precursor (within experimental error) in every case. Intermediates 1 and 1t, and Intermediates 3 and 3t are indistinguishable in every analysis, and appear to be identical. Furthermore, the intermediates produced by other activators appear to be identical to those produced by citrate-Factor Xa activation. The NH2-terminal amino acid residues of prothrombin and the intermediates permit the formulation of a structural model of prothrombin and the activation process which indicates that the activation proceeds by the sequential cleavage of portions of the NH2-terminal regions of prothrombin and the intermediates. These results support a mechanism in which prothrombin is first cleaved, either by Factor Xa or thrombin, to yield Intermediate 1 from its COOH-terminal region and Intermediate 3 from its NH2-terminal region. Intermediate 1 is then cleaved by Factor Xa to yield Intermediate 2 from its COOH-terminal region and Intermediate 4 from its NH2-terminal region. Intermediate 2, the immediate thrombin precursor, which contains the A chain of α-thrombin as its NH2-terminal segment, and the B chain of α-thrombin as its COOH-terminal segment, is then cleaved by Factor Xa to generate the active, two-chain α-thrombin molecule.