Factor Va Is Inactivated by Activated Protein C in the Absence of Cleavage Sites at Arg-306, Arg-506, and Arg-679

Abstract

Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1 = 5.4 × 104m-1 s-1) and a slow phase (kx2 = 3.2 × 103m-1 s-1). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained ∼80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV. Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1 = 5.4 × 104m-1 s-1) and a slow phase (kx2 = 3.2 × 103m-1 s-1). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained ∼80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV. Human blood coagulation factor V (FV) 1The abbreviations used are: FV, factor V; APC, activated protein C; DOPS, dioleoylphosphatidylserine; DOPC, dioleoylphosphatidylcholine; FVa, activated factor V; rFV, recombinant FV, rFVΔB-GQA, recombinant factor V with a partial deletion of the B-domain containing the following mutations, R306G, R506Q, R679A; rFVa-GQA, activated rFVΔB-GQA; FXa, activated factor X; BSA, bovine serum albumin; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; FVL, FV Leiden. 1The abbreviations used are: FV, factor V; APC, activated protein C; DOPS, dioleoylphosphatidylserine; DOPC, dioleoylphosphatidylcholine; FVa, activated factor V; rFV, recombinant FV, rFVΔB-GQA, recombinant factor V with a partial deletion of the B-domain containing the following mutations, R306G, R506Q, R679A; rFVa-GQA, activated rFVΔB-GQA; FXa, activated factor X; BSA, bovine serum albumin; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; FVL, FV Leiden. is the inactive precursor of activated factor V (FVa), the non-enzymatic cofactor of the serine protease factor Xa (FXa) (1Owren P.A. Lancet. 1947; : 446-448Abstract PubMed Scopus (123) Google Scholar, 2Suzuki K. Dahlback B. Stenflo J. J. Biol. Chem. 1982; 257: 6556-6564Abstract Full Text PDF PubMed Google Scholar). FVa is formed via limited proteolysis of its single chain precursor FV (∼330 kDa) by thrombin. It consists of a heavy chain (∼105 kDa) and a light chain (71-74 kDa), which are held together via a Ca2+ ion. FVa is an essential part of the prothrombinase complex, which composes FVa, FXa, calcium ions, and a negatively charged membrane surface. In this complex FVa acts as a non-enzymatic cofactor that accelerates FXa-catalyzed prothrombin activation 103-105-fold (3Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar, 4Rosing J. Tans G. Govers-Riemslag J.W. Zwaal R.F. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 5van Rijn J.L. Govers-Riemslag J.W. Zwaal R.F. Rosing J. Biochemistry. 1984; 23: 4557-4564Crossref PubMed Scopus (69) Google Scholar). Proteolytic inactivation of FVa by activated protein C (APC) is an essential reaction in the anticoagulant protein C pathway (6Esmon C.T. Biochim. Biophys. Acta. 2000; 1477: 349-360Crossref PubMed Scopus (265) Google Scholar), which is important in the regulation of thrombin formation and in the inhibition of acute inflammation triggered by coagulation (7Esmon C.T. Thromb. Haemostasis. 2001; 86: 51-56Crossref PubMed Scopus (251) Google Scholar). Congenital deficiencies in this pathway are associated with thrombotic disease (8Griffin J.H. Evatt B. Zimmerman T.S. Kleiss A.J. Wideman C. J. Clin. Investig. 1981; 68: 1370-1373Crossref PubMed Scopus (972) Google Scholar, 9Comp P.C. Nixon R.R. Cooper M.R. Esmon C.T. J. Clin. Investig. 1984; 74: 2082-2088Crossref PubMed Scopus (478) Google Scholar). Inactivation of FVa activity by APC is associated with cleavage of three peptide bonds in the heavy chain of FVa, which have been localized at Arg-306, Arg-506, and Arg-679 (10Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). mutations have been in the APC cleavage sites of FV. The most important is FV in which been replaced by P.A. 1994; PubMed Scopus Google Scholar, J.H. Evatt B. Lancet. 1994; PubMed Scopus Google Scholar, J. J. J.W. K. Lancet. 1994; PubMed Scopus Google Scholar). in the of APC resistance and is associated with an of P.A. 1994; PubMed Scopus Google Scholar, PubMed Google Scholar, K. J. PubMed Scopus Google Scholar). The of in M. J.B. Lancet. PubMed Scopus Google this the most important factor FVL, three mutations have been in the APC cleavage sites of FV. three the Arg-306 and are FV K. C. PubMed Google Scholar, PubMed Google Scholar, B. Thromb. Haemostasis. Scholar, R.F. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). are that the mutations at Arg-306 are associated with an of PubMed Google Scholar, R.F. J. PubMed Scopus Google Scholar). the of mutations the to APC is using recombinant FV have that mutations at Arg-306 in an unexpectedly mild M. J. PubMed Scopus Google Scholar, M. Tans G. Rosing J. PubMed Scopus Google Scholar, Dahlback B. PubMed Scopus Google Scholar). have been to obtain more insight in the complex of FVa that cleavage of the heavy chain of FVa by APC is by negatively charged Esmon C.T. Biochim. Biophys. Acta. 1979; PubMed Scopus Google Scholar, K. Stenflo J. Dahlback B. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Tans G. Hemker H.C. J.H. Rosing J. J. PubMed Scopus Google and protein Tans G. Hemker H.C. J.H. Rosing J. J. PubMed Scopus Google Scholar, J. Biol. Chem. 1980; 255: Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar), which is a non-enzymatic cofactor of APC. In the of negatively charged the inactivation of FVa by APC via a rapid and a slow which are associated with at Arg-506 and Arg-306, (10Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). at Arg-506 a reaction intermediate with FVa activity, which is completely inactivated by the cleavage at Arg-306. Inactivation of activated is and associated with cleavage at Arg-306, which was by protein J. Hemker H.C. K. Tans G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the of negatively charged the of the cleavage at Arg-679 to the inactivation of FVa to and is to Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The original of the study was to the functional and structural implications of each individual APC cleavage in more factor V mutants were constructed in which a part of the B-domain was and two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. rFVa-GQA in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, was still inactivated by APC. In this we the of inactivation of were from and were from were from fragments were from using the and were from serum was from and were from and were from was from and were from was from was from APC, protein S, and thrombin were from The APC was from APC and activity APC was by using of protein C whereas APC activity was with using the by 1984; PubMed Google Scholar). Human factor Xa and monoclonal and were from antibody against the heavy chain of FV was a from B. with was from was from Human was from to Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). FV light chain was by using two monoclonal against the light chain of FV J. PubMed Scopus Google Scholar). In this ELISA, was used as antibody and as this study expression were used containing the of FV in which two or three APC cleavage sites were M. Tans G. Rosing J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In mutants the arginines at positions 306, 506, or 679 were replaced by glycine, glutamine, and alanine, The were used to FV expression a part of the B-domain from as M. Thromb. Haemostasis. PubMed Scopus Google Scholar). were by using the from V used in this study were the at the APC cleavage sites at positions 306, 506, and 679 using the The that a part of the B-domain is positions are and from R506Q, APC cleavage sites in a and of recombinant FV mutants were in COS-1 using from were with and with was at and at FV expression was by functional FV and mutants were in a as M. Thromb. Haemostasis. PubMed Scopus Google Scholar). was with and an which was with the was was from the with containing FVa activity were with against a containing and at a of to an of The was with and with were FV and by and FV containing were with against and at activity was in a as M. Thromb. Haemostasis. PubMed Scopus Google Scholar). was using of with FV. Inactivation of FVa by was with APC in BSA, were to of prothrombinase FXa, and of FXa, and After of at prothrombin activation was by in of of this were to a and of was which the of thrombin formed was a from the formation of thrombin was the FVa in the prothrombinase The FVa activity the of APC = was at of of FVa of FVa inactivation were by of FVa activity as a of in this that inactivation of the activated by APC via two and is to the inactivation of FVa via at Arg-506 and Arg-306 Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and are cleavage at the as cleavage sites and at which the inactivation is to the factor and APC the of FVa cofactor activity is by Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this is the FVa activity at is the FVa activity at is the activity of the FVa intermediate generated cleavage at and are the observed from which the were as = = and = in which and are the of inactivation of the FVa intermediate and FVa, and Immunoblot was to PubMed Scopus Google Scholar). to was a from To FV, monoclonal against the heavy and light chain and were with in with the was used to the were with in the FV and were with was to degradation fragments from the heavy chain to the of this of heavy chains still M. Dahlback B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and of V mutants were constructed with mutations in two or in three of the APC cleavage sites Arg-506, and In to the FV HongKong and FV mutations, Arg-306 was replaced by and Arg-506 by glutamine, whereas Arg-679 was replaced by After of COS-1 cells, containing the were and at V expression and of and its mutants were and the between and After and of were by and The mutants were activated with thrombin and by by or showed two with a of and which to the of the heavy and light chain of factor from two of fragment were which was most likely the of thrombin cleavage at M. M. Mann K.G. Thromb. Biol. PubMed Scopus Google Scholar). The of the rFV mutants were in of Activated were activated with thrombin at and in the or of The of the activated mutants was by following the of FVa activity as a of in a reaction that APC. The inactivation of activated and the activated mutants were × which to a of FVa activity of The of the of the activated rFV Inactivation of Activated showed that from the activated mutants the activated rFVa-GQA was inactivated by APC and that this inactivation was the of negatively charged In the of inactivation of rFVa-GQA by APC was the of of APC with a monoclonal antibody against protein C which the of APC, or with of serine the activation of rFVa-GQA by APC, whereas with a monoclonal antibody against protein the of inactivation that the of FVa activity observed in the of APC and was by APC and a of APC of FVa activity of activated Activated was at in with APC, which been with C antibody antibody or The FVa activity is FVa activity in the of APC was at FVa activity in the of APC was at in a Inactivation of Activated with APC that the inactivation of rFVa-GQA by APC from or more proteolytic at still cleavage To more the of rFVa-GQA was with of APC the APC resulted in an of FVa inactivation The FVa inactivation curves with a single that inactivation at two a rapid reaction resulting in a FVa that is completely inactivated via a slow the the reaction was that the was to the the reaction and the activity of the FVa intermediate from The of the at APC and the at the an the FVa activity in the intermediate which was to that the the slow reaction is a of the APC which of the (kx2 = 3.2 × 103m-1 s-1) the cleavage The to a of × at = of the with the which most likely the of activity of the reaction intermediate as is close to that × s-1). 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Full Text Full Text PDF PubMed Scopus Google inactivation of activated by APC via peptide at cleavage sites and The s-1) in the of protein were from the of the of the the APC The in the of protein were at a single APC of The and were by the to that × 104m-1 s-1) and were by the of protein The and were from the as × × × × × × in a of the Inactivation of Activated have that in the of of protein the inactivation of FVa by APC is accelerated by stimulation of the cleavage at Arg-306 J. Hemker H.C. K. Tans G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 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FVa light chains were by During the inactivation of rFVa-GQA with APC, degradation of the light chain was observed Analysis of the heavy the of which associated with the of FVa activity The phase of inactivation was associated with of a fragment of whereas the slow phase was associated with the of a of kDa and fragments of and kDa that the heavy chain fragment of kDa heavy chain from to a fragment of kDa was which was the of APC. this fragment was formed during the activation with thrombin, which the heavy chain of FVa at M. M. Mann K.G. Thromb. Biol. PubMed Scopus Google Scholar). In the of protein S, the of FVa activity was associated with the of a fragment After and fragments were of APC in and the of FV were used to APC cleavage sites in the heavy chain of The rapid cleavage at in the of protein was associated with formation of a fragment a and which still ∼80% FVa a fragment may from a cleavage in a from the or the of the heavy the arginines and a whereas the between and a with arginines and J.L. A.J. J.H. Thromb. Haemostasis. 2000; PubMed Scopus Google Scholar), is likely that is in the in this in the formation of an fragment from the and heavy chains and is with the activity of the intermediate cleavage at the slow cleavage which in in the of explain the of the observed fragment in the heavy chain of kDa) and the fragment in the intermediate fragment of 80 cleavage close to explain the of a fragment in heavy chain fragment of kDa), which been M. M. Mann K.G. Thromb. Biol. PubMed Scopus Google Scholar). a of close to Arg-306 is with the stimulation of by protein of fragment in cleavage at Arg-306 is by protein J. Hemker H.C. K. Tans G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of and was from in which the activated mutants and were with APC. Inactivation of activated with APC generated an fragment of kDa which was a fragment that in the of APC cleavage is The of this fragment was accelerated in the of protein Inactivation of activated with APC generated fragments with the of and kDa which were accelerated in the of protein fragments were to APC cleavage in the which may to of the and the fragments cleavage at Arg-306 K.G. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of more by the of the which are the formed inactivation of activated and by APC The fragment generated during the inactivation of an of to fragment The fragment generated from the inactivation of activated most likely to was kDa that the of is carboxyl-terminal to residue the of very close to residue 506, the in the inactivation of a of of in Activated cleavage at in the of residue may as an a cleavage at Arg-506. To was activated and inactivated using the as the mutants and to analysis by and The showed a very cleavage as observed the activated with a heavy chain of and a was which was most likely the of cleavage at Arg-306 in the and a fragment of kDa was which was by cleavage of the fragment resulting in a fragment from The fragments were at to was observed during inactivation of activated the of of the carboxyl-terminal to Arg-306 To obtain more insight in the functional and structural implications of the individual at Arg-306, Arg-506, or Arg-679 in the heavy chain of FVa by APC, recombinant FV mutants were generated in which two or three of APC cleavage sites been mutated. the B-domain of FV in the inactivation of FVa, we used mutants a part of the B-domain in to the of rFV The expression of mutants was that of the which is in with M. Thromb. Haemostasis. PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar). inactivation with the recombinant showed that rFVa-GQA which the APC cleavage sites Arg-306, Arg-506, and Arg-679 have been was still inactivated by APC, at a The of this study was to more the this activated rFV mutants used in this were the to study FVa inactivation by APC the of a heavy chain fragment of kDa likely fragment M. M. Mann K.G. Thromb. Biol. PubMed Scopus Google generated during the activation with the of the activated rFV The inactivation of rFVa-GQA by APC is negatively charged and is by a monoclonal antibody against protein C that the observed inactivation is by of activated mutants in the or of inactivation were by of inactivation with a single were to FVa inactivation in the of APC FVa in BSA, at factor × × × × × × × × × × in a analysis of the inactivation of rFVa-GQA by APC that from to Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google rFVa-GQA was inactivated in a that to the inactivation of FVa the inactivation of rFVa-GQA was associated with at two and is by analysis of the reaction During inactivation of rFVa-GQA, an FVa intermediate with ∼80% of the original FVa activity was formed via rapid cleavage at intermediate was completely inactivated via a slow cleavage at The of the rapid reaction (kx1 = 5.4 × 104m-1 s-1) was that of the slow reaction (kx2 = 3.2 × 103m-1 and = × 103m-1 s-1). The of the rapid reaction is and cleavage at Arg-306 and Arg-506, Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), is the cleavage at which been at × 103m-1 Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the of cleavage sites APC, close to residue and close to residue 506, is important the of the cleavage at the individual sites Arg-306, Arg-506, or Arg-679 and to in the of analysis of cleavage at the inactivation of rFVa-GQA by APC analysis that the slow cleavage at that rFVa-GQA was by protein In this analysis we that cleavage at was by protein is by the that in the of protein a fragment at was generated by APC of the fragment observed in the of protein the of the cleavage at by protein to the inactivation to the FVa activity associated with the intermediate formed cleavage at To the of the cleavage sites and the fragments generated during the degradation of rFVa-GQA were by and using an antibody with an epitope between and the heavy chain of of the inactivation fragment was in the activated mutants and and activated from the heavy chain analysis of the of thrombin activation that this fragment was the of cleavage by thrombin most likely at M. M. Mann K.G. Thromb. Biol. PubMed Scopus Google Scholar). to the of the was to the of and the of the cleavage at the FVa activity was observed of and close to residue and 306, respectively, the via and of residue explain the of a fragment of kDa during the phase of the inactivation of rFVa-GQA by APC It explain the FVa activity of the intermediate formed cleavage at cleavage of factor at Arg-506 in the formation of an intermediate with factor activity Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Dahlback B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of residue explain the formation of of and kDa the heavy chains fragments of and and kDa the during the slow phase. of close to residue is with the that the cleavage at is by protein protein exerts its the inactivation of FVa by the cleavage at Arg-306 J. Hemker H.C. K. Tans G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). more of the of was by the fragment generated in the inactivation of with the fragment generated during the inactivation of activated by APC, cleavage at Arg-506 is by cleavage at The fragment of was kDa the fragment of that the of is localized carboxyl-terminal to residue the the of to the and the of very close to residue 506, the fragment during the inactivation of rFVa-GQA the as the fragment and the of the of at this residue is in a J.H. PubMed Scopus Google Scholar). cleavage at likely in an FVa intermediate with a FVa activity the activity the intermediate cleavage at are to of to the of and by the degradation of the activated and mutants of the structural implications of cleavage at Arg-306 or Arg-506. The fragments in the degradation of activated were and kDa which indicated that cleavage at Arg-306, was to APC by the of the and fragments from the FVa a that been as the FVa activity cleavage at Arg-306 K.G. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the fragment in the degradation of activated was which that still by APC cleavage at Arg-506. is the that APC cleavage sites are in the heavy chain of FVa from the sites at Arg-306, Arg-506, and Arg-679. During the inactivation of FVa by APC, cleavage at or the are cleavage at Arg-506 and Arg-306, in the of protein explain the sites of and have been identified in using FVa (10Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Tans G. Hemker H.C. K. Rosing J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). APC cleavage sites have been observed in using rFV in which Arg-306 and Arg-506 been M. J. PubMed Scopus Google Scholar, Dahlback B. PubMed Scopus Google Scholar, M. Dahlback B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Mann K.G. PubMed Scopus Google Scholar). the used in were APC and that at and During the study we observed that is by APC as an Arg-506 during the inactivation of activated most likely of of the cleavage at Arg-306. Inactivation of activated the that cleavage at may as an cleavage at Arg-306. cleavage at may in the inactivation of activated or and may explain the mild associated with the of FV J. the expression the APC