The Conversion of Prothrombin to Thrombin: II. DIFFERENTIATION BETWEEN THROMBIN- AND FACTOR Xa-CATALYZED PROTEOLYSES

Abstract

Abstract Prothrombin is activated by activated Factor X ± (Factor V and phospholipid) in the presence of diisopropylfluorophosphate which preferentially inhibits thrombin. Under these conditions a single activation fragment, designated Fragment 1·2, is released which represents the complete nonthrombin portion of the prothrombin molecule. The same fragment is seen as a transient species in the absence of diisopropylfluorophosphate when the activation reaction is monitored by disc electrophoresis in 8 m urea. The NH2terminal residue of both prothrombin and Fragment 1·2 is alanine, suggesting that Fragment 1·2 is derived from the NH2-terminal end of the prothrombin molecule. Fragment 1·2 is not cleaved by activated Factor X ± (Factor V and phospholipid), but is cleaved by thrombin to form two fragments which are identical by electrophoresis, ion exchange chromatography, and amino acid composition to Fragment 1, the fragment released from prothrombin on incubation with thrombin, and Fragment 2, the fragment released by activated Factor X when Intermediate 1 is converted to Intermediate 2 (Paper I; Owen, W. G., Esmon, C. T., and Jackson, C. M. (1974) J. Biol. Chem. 249, 594–605). It is proposed that the formation of Intermediate 1 during prothrombin activation is solely the result of thrombin-catalyzed proteolysis of prothrombin.