Altered inactivation pathway of factor Va by activated protein C in the presence of heparin.

Inactivation of factor Va (FVa) by activated protein C (APC) is a predominant mechanism in the down-regulation of thrombin generation. In normal FVa, APC-mediated inactivation occurs after cleavage at Arg306 (with corresponding rate constant k'306) or after cleavage at Arg506 (k506) and subsequent cleavage at Arg306 (k306). We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inactivation. Peptide bond cleavage was identified by Western blotting using FV-specific antibodies. In normal FVa, unfractionated heparin (UFH) was found to inhibit cleavage at Arg506 in a dose-dependent manner. Maximal inhibition of k506 by UFH was 12-fold, with the secondary cleavage at Arg306 (k306) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'306) two- to threefold. Low molecular weight heparin (Fragmin) had the same effects on the rate constants of FVa inactivation as UFH, but pentasaccharide did not inhibit FVa inactivation. Analysis of these data in the context of the 3D structures of APC and FVa and of simulated APC-heparin and FVa-APC complexes suggests that the heparin-binding loops 37 and 70 in APC complement electronegative areas surrounding the Arg506 site, with additional contributions from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental data suggest that heparin, when bound to APC, prevents optimal docking of APC at Arg506 and promotes association between FVa and APC at position Arg306.

[1]  B. Dahlbäck,et al.  Congenital and acquired activated protein C resistance. , 2003, Seminars in vascular medicine.

[2]  J. A. Grant,et al.  Gaussian docking functions. , 2003, Biopolymers.

[3]  Jean-Luc Pellequer,et al.  Interdomain engineered disulfide bond permitting elucidation of mechanisms of inactivation of coagulation factor Va by activated protein C , 2002, Protein science : a publication of the Protein Society.

[4]  J. Griffin,et al.  Molecular Characterization of an Extended Binding Site for Coagulation Factor Va in the Positive Exosite of Activated Protein C* , 2002, The Journal of Biological Chemistry.

[5]  Cinque S. Soto,et al.  Evaluating conformational free energies: The colony energy and its application to the problem of loop prediction , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Rezaie,et al.  Contribution of basic residues of the 70-80-loop to heparin binding and anticoagulant function of activated protein C. , 2002, Biochemistry.

[7]  B. Dahlbäck,et al.  Factor V and Thrombotic Disease: Description of a Janus-Faced Protein , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[8]  R. Abagyan,et al.  Soft protein–protein docking in internal coordinates , 2002, Protein science : a publication of the Protein Society.

[9]  E. Goldsmith,et al.  The structure of a Michaelis serpin–protease complex , 2001, Nature Structural Biology.

[10]  B Honig,et al.  Electrostatic contributions to protein–protein interactions: Fast energetic filters for docking and their physical basis , 2001, Protein science : a publication of the Protein Society.

[11]  A. Blom,et al.  Structural and Energetic Characteristics of the Heparin-binding Site in Antithrombotic Protein C* , 2001, The Journal of Biological Chemistry.

[12]  B. Dahlbäck,et al.  Secondary Substrate-binding Exosite in the Serine Protease Domain of Activated Protein C Important for Cleavage at Arg-506 but Not at Arg-306 in Factor Va* , 2001, The Journal of Biological Chemistry.

[13]  T. Langer,et al.  Virtual screening: an effective tool for lead structure discovery? , 2001, Current pharmaceutical design.

[14]  J. Hirsh,et al.  Hypersulfated Low Molecular Weight Heparin with Reduced Affinity for Antithrombin Acts as an Anticoagulant by Inhibiting Intrinsic Tenase and Prothrombinase* , 2001, The Journal of Biological Chemistry.

[15]  E. Getzoff,et al.  Three-dimensional Model of Coagulation Factor Va Bound to Activated Protein C , 2000, Thrombosis and Haemostasis.

[16]  David F. Burke,et al.  Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin , 2000, Nature.

[17]  J. Griffin,et al.  The autolysis loop of activated protein C interacts with factor Va and differentiates between the Arg506 and Arg306 cleavage sites. , 2000, Blood.

[18]  C. Esmon.  Regulation of blood coagulation. , 2000, Biochimica et biophysica acta.

[19]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[20]  José A Fernández,et al.  Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C , 1999, Thrombosis and Haemostasis.

[21]  B. Dahlbäck,et al.  Partial glycosylation of Asn2181 in human factor V as a cause of molecular and functional heterogeneity. Modulation of glycosylation efficiency by mutagenesis of the consensus sequence for N-linked glycosylation. , 1999, Biochemistry.

[22]  B. Dahlbäck,et al.  Interspecies Loop Grafting in the Protease Domain of Human Protein C Yielding Enhanced Catalytic and Anticoagulant Activity , 1999, Thrombosis and Haemostasis.

[23]  B. Dahlbäck,et al.  Involvement of Lys 62[217] and Lys 63[218] of Human Anticoagulant Protein C in Heparin Stimulation of Inhibition by the Protein C Inhibitor , 1999, Thrombosis and Haemostasis.

[24]  K. Mann,et al.  A model describing the inactivation of factor Va by APC: bond cleavage, fragment dissociation, and product inhibition. , 1999, Biochemistry.

[25]  Rebecca C. Wade,et al.  Docking of Glycosaminoglycans to Heparin-Binding Proteins: Validation for aFGF, bFGF, and Antithrombin and Application to IL-8 , 1999 .

[26]  B. Dahlbäck,et al.  Structural investigation of the A domains of human blood coagulation factor V by molecular modeling , 1998, Protein science : a publication of the Protein Society.

[27]  J. Abrahams,et al.  The anticoagulant activation of antithrombin by heparin. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[28]  J. Griffin,et al.  Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C. , 1997, The Journal of clinical investigation.

[29]  S Foundling,et al.  The 2.8 A crystal structure of Gla‐domainless activated protein C. , 1996, The EMBO journal.

[30]  D C Rees,et al.  Heparin Structure and Interactions with Basic Fibroblast Growth Factor , 1996, Science.

[31]  H. Hemker,et al.  Effects of Protein S and Factor Xa on Peptide Bond Cleavages during Inactivation of Factor Va and Factor VaR506Q by Activated Protein C (*) , 1995, The Journal of Biological Chemistry.

[32]  T. P. Jackson,et al.  Intermolecular interactions between protein C inhibitor and coagulation proteases. , 1995, Biochemistry.

[33]  H. Hemker,et al.  Peptide Bond Cleavages and Loss of Functional Activity during Inactivation of Factor Va and Factor VaR506Q by Activated Protein C (*) , 1995, The Journal of Biological Chemistry.

[34]  B. Honig,et al.  Classical electrostatics in biology and chemistry. , 1995, Science.

[35]  K. Mann,et al.  The mechanism of inactivation of human factor V and human factor Va by activated protein C. , 1994, The Journal of biological chemistry.

[36]  H. Hemker,et al.  Characterization of two forms of human factor Va with different cofactor activities. , 1993, The Journal of biological chemistry.

[37]  M. Forster,et al.  N.m.r. and molecular-modelling studies of the solution conformation of heparin. , 1993, The Biochemical journal.

[38]  J. Stenflo,et al.  Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. , 1991, Blood.

[39]  C. Esmon,et al.  The roles of protein C and thrombomodulin in the regulation of blood coagulation. , 1984, The Journal of biological chemistry.

[40]  J. Stenflo.  Structure and Function of Protein C , 1984, Seminars in thrombosis and hemostasis.

[41]  H C Hemker,et al.  The role of phospholipids and factor Va in the prothrombinase complex. , 1980, The Journal of biological chemistry.

[42]  K. Mann,et al.  The contribution of bovine Factor V and Factor Va to the activity of prothrombinase. , 1979, The Journal of biological chemistry.