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The Conversion of Prothrombin to Thrombin

IV. THE FUNCTION OF THE FRAGMENT 2 REGION DURING ACTIVATION IN THE PRESENCE OF FACTOR V

From the ¹ From the Division of Biology and Biomedical Sciences, Department of Biological Chemistry, Washington University, St. Louis, Missouri 63110

During activation, the prothrombin molecule is divided into a COOH-terminal half which gives rise to thrombin and an NH₂-terminal activation fragment half (Fragment 1·2) (Esmon, C. T., Owen, W. G., and Jackson, C. M. (1974) J. Biol. Chem. 249, 606–611). Thrombin-catalyzed proteolysis further divides the activation fragment into 2 parts, Fragment 1 (from the NH₂-terminal end) and Fragment 2. The Fragment 1 region of prothrombin has been shown to be responsible for the binding of prothrombin to phospholipid (Gitel, S. N., Owen, W. G., Esmon, C. T., and Jackson, C. M. (1973) Proc. Nat. Acad. Sci. U. S. A. 70, 1344–1348). It is shown here that the ability of blood clotting Factor V to accelerate thrombin precursor activation is specifically dependent upon the interaction between the Fragment 2 region of the substrate and Factor V. The evidence supporting the conclusion that the Fragment 2 region is required for Factor V function is as follows: Intermediate 2, a thrombin precursor without a Fragment 2 region, is activated more rapidly by activated Factor X and Ca²⁺ than either prothrombin or Intermediate 1, whereas when Factor V is present, the situation is reversed and the Fragment 2 region containing substrates, i.e. prothrombin or Intermediate 1, are activated more rapidly than Intermediate 2. Addition of either Fragment 2 or Fragment 1·2 to Intermediate 2 results in the rapid activation of Intermediate 2 by Factor V, activated Factor X, and Ca²⁺, and Intermediate 2 and Fragment 2 associate to form a product of 1:1 stoichiometry which is electrophoretically indistinguishable from Intermediate 1.

In contrast to the situation with Fragment 2, Fragment 1 is completely without effect on the Factor V enhancement of Intermediate 1 and Intermediate 2 plus Fragment 2 activation indicating that Fragment 1 and Fragment 2 are independent in their function in the prothrombin activation process. The course of Intermediate 1 activation by activated Factor X, Factor V, and Ca²⁺ was investigated by determining the product distributions which can be monitored by sodium dodecyl sulfate gel electrophoresis as a function of time. The results on the activation pathway obtained from these experiments are consistent with the previously demonstrated prothrombin activation pathway (Esmon, C. T., and Jackson, C. M. (1974) J. Biol. Chem. 249, 7782–7790), i.e.

[see PDF for equation]

with the added requirement for interaction between Intermediate 2 and Fragment 1·2 or Fragment 2 for Factor V to function.

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