Formation of Factors IXa and Xa by the Extrinsic Pathway DIFFERENTIAL REGULATION BY TISSUE FACTOR PATHWAY INHIBITOR AND ANTITHROMBIN III

Genmin Lu; George J. Broze; Sriram Krishnaswamy

doi:10.1074/jbc.M312827200

Formation of Factors IXa and Xa by the Extrinsic Pathway

DIFFERENTIAL REGULATION BY TISSUE FACTOR PATHWAY INHIBITOR AND ANTITHROMBIN III*

^‡Joseph Stokes Research Institute, Children's Hospital of Philadelphia and the ^¶Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the ^§Division of Hematology, Jewish Hospital, Washington University, St. Louis, Missouri 63110

↵∥ To whom correspondence should be addressed: Joseph Stokes Research Institute, Children's Hospital of Philadelphia, 310A Abramson, 3615 Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-3346; Fax: 215-590-2320; E-mail: skrishna{at}mail.med.upenn.edu.

Abstract

The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. IX activation by VIIa/TF is considered to contribute catalyst necessary for further Xa production in the ensuing amplification phase. We have investigated Xa and IXaβ production by VIIa-TF in a system reconstituted with both X and IX and the principal physiologic inhibitors of this pathway TFPI and antithrombin III (AT). Kinetic studies without inhibitors established that IX and X functioned as competitive alternate substrates for VIIa/TF with similar kinetic constants. When both IX and X were present, TFPI significantly inhibited the extent of formation of either IXaβ or Xa. In contrast, AT rapidly depleted active Xa with a small effect on IXaβ formation. When both AT and TFPI were present, active IXaβ formation significantly exceeded the formation of active Xa regardless of the VIIa/TF concentration. These findings could be quantitatively accounted for by a model encompassing the kinetics of the individual activation and inhibition steps. Active Xa formation by this pathway is regulated in a principal way by its rapid inactivation by AT. In contrast, the Xa-dependent inhibitory reactions of TFPI play a primary role in limiting zymogen consumption and the formation of active IXaβ. These regulatory phenomena yield active IXaβ as a major rather than secondary product of VIIa/TF. Our findings raise the possibility that IXaβ produced by the extrinsic pathway, and its ability to function within the intrinsic Xase complex to activate X may play a significant role in producing Xa necessary for both the initiation and sustained phases of the procoagulant response following vascular damage.