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• W2085809902 abstract "SummaryThe binding of factor (FVa) to phosphatidylserine (PS) membranes regulates assembly of the prothrombinase complex. Two pairs of solvent‐exposed amino acids, Tyr1956/Leu1957 in the C1 domain and Trp2063/Trp2064 in the C2 domain, each make significant contributions to the affinity of FVa for PS membranes, but individually neither pair of amino acids is required for prothrombinase assembly on 25% PS membranes. In this study we characterize a FVa mutant with alanine substitutions in both the C1 and C2 domains: (Y1956,L1957,W2063,W2064)A. We conclude that: (i) prothrombinase assembly on PS membranes requires Trp2063/Trp2064 and/or Tyr1956/Leu1957; (ii) combined mutation of Trp2063/Trp2064 and Tyr1956/Leu1957 results in only a modest 4‐fold decrease in the rate of thrombin generation in the absence of membranes; (iii) the present data provide experimental support for the joint participation of the C1 and C2 domains in the binding of FVa to phospholipid membranes as suggested by the recently solved structure for FVai (A1/A3‐C1‐C2). The binding of factor (FVa) to phosphatidylserine (PS) membranes regulates assembly of the prothrombinase complex. Two pairs of solvent‐exposed amino acids, Tyr1956/Leu1957 in the C1 domain and Trp2063/Trp2064 in the C2 domain, each make significant contributions to the affinity of FVa for PS membranes, but individually neither pair of amino acids is required for prothrombinase assembly on 25% PS membranes. In this study we characterize a FVa mutant with alanine substitutions in both the C1 and C2 domains: (Y1956,L1957,W2063,W2064)A. We conclude that: (i) prothrombinase assembly on PS membranes requires Trp2063/Trp2064 and/or Tyr1956/Leu1957; (ii) combined mutation of Trp2063/Trp2064 and Tyr1956/Leu1957 results in only a modest 4‐fold decrease in the rate of thrombin generation in the absence of membranes; (iii) the present data provide experimental support for the joint participation of the C1 and C2 domains in the binding of FVa to phospholipid membranes as suggested by the recently solved structure for FVai (A1/A3‐C1‐C2). Activated coagulation factor V (FVa) is a protein cofactor that binds to membranes containing phosphatidylserine (PS) and regulates the production of thrombin by the prothrombinase complex [1Kane W.H. Davie E.W. Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders.Blood. 1988; 71: 539-55Crossref PubMed Google Scholar, 2Mann K.G. Kalafatis M. Factor V: a combination of Dr Jekyll and Mr Hyde.Blood. 2003; 101: 20-30Crossref PubMed Scopus (173) Google Scholar]. The prothrombinase complex consists of FVa, the serine protease factor Xa (FXa), calcium and a negatively charged phospholipid membrane. Formation of the prothrombinase complex increases the catalytic activity of FXa for prothrombin approximately 100 000‐fold [2Mann K.G. Kalafatis M. Factor V: a combination of Dr Jekyll and Mr Hyde.Blood. 2003; 101: 20-30Crossref PubMed Scopus (173) Google Scholar]. FV is synthesized as a single‐chain glycoprotein with the domain structure of A1‐A2‐B‐A3‐C1‐C2 [1Kane W.H. Davie E.W. Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders.Blood. 1988; 71: 539-55Crossref PubMed Google Scholar]. Activation of FV by thrombin releases the B‐domain peptides producing an active heterodimer A1‐A2/A3‐C1‐C2 comprised of the amino terminal heavy chain and carboxyl terminal light chain [3Esmon C.T. The subunit structure of thrombin‐activated factor V. Isolation of activated factor V, separation of subunits, and reconstitution of biological activity.J Biol Chem. 1979; 254: 964-73Abstract Full Text PDF PubMed Google Scholar]. The heavy and light chains of FVa are homologous to factor VIIIa (FVIIIa), which serves as the protein cofactor for the intrinsic FX‐ase complex which consists of FVIIIa, factor IXa, calcium and a phospholipid membrane surface [1Kane W.H. Davie E.W. Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders.Blood. 1988; 71: 539-55Crossref PubMed Google Scholar]. Membrane binding sites in FVa have been localized within the A3 [4Kalafatis M. Rand M.D. Mann K.G. Factor Va–membrane interaction is mediated by two regions located on the light chain of the cofactor.Biochemistry. 1994; 33: 486-93Crossref PubMed Google Scholar], C1 [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar] and C2 [6Ortel T.L. Devore‐Carter D. Quinn‐Allen M. Kane W.H. Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C‐type domain.J Biol Chem. 1992; 267: 4189-98Abstract Full Text PDF PubMed Google Scholar, 7Kim S.W. Quinn‐Allen M.A. Camp T. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. Identification of functionally important amino acid residues within the C2 domain of human factor V using alanine scanning mutagenesis.Biochemistry. 2000; 39: 1951-8Crossref PubMed Scopus (0) Google Scholar, 8Nicolaes G.A. Villoutreix B.O. Dahlback B. Mutations in a potential phospholipid binding loop in the C2 domain of factor V affecting the assembly of the prothrombinase complex.Blood Coagul Fibrinolysis. 2000; 11: 89-100Crossref PubMed Google Scholar, 9Srivastava A. Quinn‐Allen M.A. Kim S.W. Kane W.H. Lentz B.R. Soluble phosphatidylserine binds to a single identified site in the C2 domain of human factor Va.Biochemistry. 2001; 40: 8246-55Crossref PubMed Scopus (0) Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar] domains. In contrast, the membrane binding sites for FVIIIa appear to be contained solely within the C2 domain [11Saenko E.L. Scandella D. Yakhyaev A.V. Greco N.J. Activation of factor VIII by thrombin increases its affinity for binding to synthetic phospholipid membranes and activated platelets.J Biol Chem. 1998; 273: 27918-26Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 12Gilbert G.E. Kaufman R.J. Arena A.A. Miao H. Pipe S.W. Four hydrophobic amino acids of the factor VIII, C2 domain are constituents of both the membrane‐binding and von Willebrand factor‐binding motifs.J Biol Chem. 2002; 277: 6374-81Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. The structures of FVa‐C2 [13Macedo‐Ribeiro S. Bode W. Huber R. Quinn‐Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes‐Prior P. Crystal structures of the membrane‐binding C2 domain of human coagulation factor V.Nature. 1999; 402: 434-9Crossref PubMed Scopus (0) Google Scholar] and FVIIIa‐C2 [14Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.L. Structure of the C2 domain of human factor VIII at 1.5 A resolution.Nature. 1999; 402: 439-42Crossref PubMed Scopus (285) Google Scholar] have been solved, revealing that each is composed of a common distorted jelly roll β‐barrel structure with three variable loops or ‘spikes’ located at the membrane binding surface. In both structures spike 1 and spike 3 consist of a β‐hairpin structure with one or two hydrophobic amino‐acid side chains located at the apex. (In this manuscript, the C‐domain variable loops or ‘spikes’ are identified by number as described by Macedo‐Ribeiro et al. [13Macedo‐Ribeiro S. Bode W. Huber R. Quinn‐Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes‐Prior P. Crystal structures of the membrane‐binding C2 domain of human coagulation factor V.Nature. 1999; 402: 434-9Crossref PubMed Scopus (0) Google Scholar].) In FVa, Trp2063/Trp2064 located at the apex of spike 1 contribute to the interaction of FVa with PS membranes [7Kim S.W. Quinn‐Allen M.A. Camp T. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. Identification of functionally important amino acid residues within the C2 domain of human factor V using alanine scanning mutagenesis.Biochemistry. 2000; 39: 1951-8Crossref PubMed Scopus (0) Google Scholar, 8Nicolaes G.A. Villoutreix B.O. Dahlback B. Mutations in a potential phospholipid binding loop in the C2 domain of factor V affecting the assembly of the prothrombinase complex.Blood Coagul Fibrinolysis. 2000; 11: 89-100Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar]. In contrast, two pairs of hydrophobic amino acids, Met2199/Phe2200 located at the apex of spike 1 and Leu2251/Leu2252 located at the apex of spike 3, contribute to the interactions of FVIIIa with the membrane surface [12Gilbert G.E. Kaufman R.J. Arena A.A. Miao H. Pipe S.W. Four hydrophobic amino acids of the factor VIII, C2 domain are constituents of both the membrane‐binding and von Willebrand factor‐binding motifs.J Biol Chem. 2002; 277: 6374-81Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Recently, we used site‐directed mutagenesis to demonstrate that a similar pair of amino acids, Tyr1956/Leu1957, located at the apex of spike 3 in the FVa C1 domain, also contribute to the binding of FVa to PS membranes [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar]. The localization of Trp2063/Trp2064 and Tyr1956/Leu1957 to the putative membrane binding interface is further supported by the structure of inactivated FVa (FVai, A1/A3‐C1‐C2) recently solved by Adams et al. [15Adams T.E. Hockin M.F. Mann K.G. Everse S.J. The crystal structure of activated protein C‐inactivated bovine factor Va: implications for cofactor function.Proc Natl Acad Sci USA. 2004; 101: 8918-23Crossref PubMed Scopus (0) Google Scholar] in which the C1 and C2 domains are aligned in an ‘edge‐to‐edge’ orientation. The contribution of Trp2063/Trp2064 and Tyr1956/Leu1957 to assembly of the prothrombinase complex has been questioned because alanine substitutions for either Trp2063/Trp2064 or Tyr1956/Leu1957 had little effect on prothrombinase assembly on membranes containing 25% PS but did result in a 4‐fold decrease in the apparent binding affinity for FXa [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar]. We hypothesized that combined mutations in both the C1 and C2 domains would completely block assembly of the prothrombinase complex even at high concentrations of membrane PS. In order to test this hypothesis, we have characterized a FVa mutant containing mutations in both the C1 and C2 domains: (Y1956,L1957,W2063,W2064)A. Human prothrombin, human thrombin, human FXa and monoclonal antibodies AHV‐5146 were obtained from Hematologic Technologies Inc. (Essex Junction, VT, USA). Hen egg L‐α‐phosphatidylcholine (PC), brain L‐α‐phosphatidylserine (PS) and 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(5‐dimethylamino‐1‐naphthalenesulfonyl) (D‐PE) were from Avanti Polar Lipids (Alabaster, AL, USA). Cell culture media were obtained from Invitrogen (Carlsbad, CA, USA). All other reagents were from Sigma (St Louis, MO, USA). The FV mutants (W2063,W2064)A and (Y1956,L1957)A have been described previously [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 7Kim S.W. Quinn‐Allen M.A. Camp T. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. Identification of functionally important amino acid residues within the C2 domain of human factor V using alanine scanning mutagenesis.Biochemistry. 2000; 39: 1951-8Crossref PubMed Scopus (0) Google Scholar]. Mutant (Y1956,L1957,W2063,W2064)A was generated by inserting the Xba1‐Sal1 fragment encoding the C2 domain from pDX rHFV des B (W2063,W2064)A into pDX rHFV des B (Y1956,L1957)A. Native and mutant recombinant FVa were expressed in COS cells and purified as previously described [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar, 16Kane W.H. Devore‐Carter D. Ortel T.L. Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region.Biochemistry. 1990; 29: 6762-8Crossref PubMed Google Scholar, 17Kim S.W. Ortel T.L. Quinn‐Allen M.A. Yoo L. Worfolk L. Zhai X. Lentz B.R. Kane W.H. Partial glycosylation at asparagine‐2181 of the second C‐type domain of human factor V modulates assembly of the prothrombinase complex.Biochemistry. 1999; 38: 11448-54Crossref PubMed Scopus (0) Google Scholar]. The concentration of purified rHFVa was estimated by measuring the absorbance at 280 nm using an extinction coefficient () of 1.74 [18Krishnaswamy S. Mann K.G. The binding of factor Va to phospholipid vesicles.J Biol Chem. 1988; 263: 5714-23Abstract Full Text PDF PubMed Google Scholar]. The concentration of rHFVa estimated by absorbance was in excellent agreement with the concentration determined by ELISA using monoclonal antibody AHV‐5146 (heavy chain) for capture and biotinylated monoclonal antibody 6A5 (C2 domain) for detection [7Kim S.W. Quinn‐Allen M.A. Camp T. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. Identification of functionally important amino acid residues within the C2 domain of human factor V using alanine scanning mutagenesis.Biochemistry. 2000; 39: 1951-8Crossref PubMed Scopus (0) Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar]. The kinetics of prothrombin activation were investigated by measuring the rate of thrombin generation catalyzed by prothrombinase in reaction mixtures containing FXa, 1.4 µm prothrombin, 5 µm phospholipid vesicles (PC : PS, 75 : 25 mol : mol) and FVa in 25 mm Tris, pH 7.4, 0.15 m NaCl, 2.7 mm KCl, 10 mg mL−1 bovine serum albumin (BSA) and 3 mm CaCl2 as described previously [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar]. The effect of membrane PS content on prothrombin activation was investigated in reaction mixtures containing 0.2 nm FVa, 0.2 nm FXa and 5 µm PC : PS phospholipid vesicles containing 0–40% PS. Prothrombin activation in the absence of phospholipid membranes was investigated in a similar manner using 1 nm FXa and 0–100 nm FVa incubated with 1.4 µm prothrombin for 5 min at 37 °C. The binding of FVa to phospholipid vesicles was determined using membranes labeled with 2.5 mol% dansyl‐phosphatidylethanolamine as described by Gilbert et al. [19Gilbert G.E. Furie B.C. Furie B. Binding of human factor VIII to phospholipid vesicles.J Biol Chem. 1990; 265: 815-22Abstract Full Text PDF PubMed Google Scholar] with minor modifications [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar]. Data were analyzed using a simple bimolecular model, in which each FVa binding site is independent and comprised of a discrete number of phospholipid monomers [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar, 18Krishnaswamy S. Mann K.G. The binding of factor Va to phospholipid vesicles.J Biol Chem. 1988; 263: 5714-23Abstract Full Text PDF PubMed Google Scholar, 19Gilbert G.E. Furie B.C. Furie B. Binding of human factor VIII to phospholipid vesicles.J Biol Chem. 1990; 265: 815-22Abstract Full Text PDF PubMed Google Scholar]. Mutant FVa was isolated using Mono S chromatography (Fig. 1A) under conditions similar to those previously described for isolation of rHFVa [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar, 16Kane W.H. Devore‐Carter D. Ortel T.L. Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region.Biochemistry. 1990; 29: 6762-8Crossref PubMed Google Scholar, 17Kim S.W. Ortel T.L. Quinn‐Allen M.A. Yoo L. Worfolk L. Zhai X. Lentz B.R. Kane W.H. Partial glycosylation at asparagine‐2181 of the second C‐type domain of human factor V modulates assembly of the prothrombinase complex.Biochemistry. 1999; 38: 11448-54Crossref PubMed Scopus (0) Google Scholar]. The preparations of (Y1956,L1957,W2063,W2064)A used in the present study were highly purified as judged by SDS–PAGE and silver staining (Fig. 1B, fractions 10 and 11). The binding of FVa to phospholipid vesicles (PC : PS : Dansyl‐PE 72.5 : 25 : 2.5) was determined by FRET (Fig. 2A) as described previously [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar]. The binding affinity of (Y1956,L1957,W2063,W2064)A was reduced by over 450‐fold compared with the rHFVa. The ability of (Y1956,L1957,W2063,W2064)A to form the prothrombinase complex on 25% PS membranes was also dramatically reduced in the presence of 0–20 nm FVa (Fig. 2B) and 0–5 nm FXa (Fig. 2C). We next investigated the ability of FVa to promote assembly of the prothrombinase complex on membranes containing 0–40% PS (Fig. 2D). Half maximal rates of thrombin generation were observed at 8.0% PS for rHFVa. In contrast, the activity of (Y1956,L1957,W2063,W2064)A was dramatically reduced even on membranes containing 40% PS. For comparison, half‐maximal rates of thrombin generation for (Y1956,L1957)A and (W2063,W2064)A were observed at 13.7 and 20.6% PS, respectively (Fig. 2D).Figure 2Interaction of factor Va (FVa) with phospholipid vesicles. (A) Binding of FVa to phospholipid vesicles. FVa was added to 0.3 µm phospholipid vesicles (PC : PS : Dansyl‐PE, 72.5 : 25 : 2.5) in a buffer containing 150 mm NaCl, 20 mm Tris–HCl, pH 7.4 at 25 °C. Samples were irradiated at 280 nm and fluorescence emission (F) and background fluorescence (Fb) were measured at 545 nm as described in the experimental procedure [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar]. The relative fluorescence (F/Fb − 1) was fitted to a simple bimolecular binding model as described by Gilbert et al. [19Gilbert G.E. Furie B.C. Furie B. Binding of human factor VIII to phospholipid vesicles.J Biol Chem. 1990; 265: 815-22Abstract Full Text PDF PubMed Google Scholar]. rHFVa (circles); (Y1956,L1957,W2063,W2064)A (diamonds). (B) Prothrombinase activity as a function of FVa concentration. Rates of thrombin generation were determined in reaction mixtures containing 25 mm Tris, pH 7.4, 150 mm NaCl, 2.7 mm KCl, 10 mg mL−1 bovine serum albumin, 3 mm CaCl2, 0.2 nm factor Xa (FXa) and 5 µm phospholipid vesicles (75 : 25, PC : PS). Thrombin generation was measured using the chromogenic substrate S‐2238 as previously described [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 7Kim S.W. Quinn‐Allen M.A. Camp T. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. Identification of functionally important amino acid residues within the C2 domain of human factor V using alanine scanning mutagenesis.Biochemistry. 2000; 39: 1951-8Crossref PubMed Scopus (0) Google Scholar]. rHFVa (circles); (Y1956,L1957, W2063,W2064)A (diamonds). (C) Prothrombinase activity as a function of FXa concentration. Rates of thrombin formation were determined in reaction mixtures similar to (B) containing varying concentrations of FXa, 0.2 nm FVa and 5 µm phospholipid vesicles (75 : 25, PC : PS). rHFVa (circles); (Y1956,L1957,W2063,W2064)A (diamonds). (D) Prothrombinase activity as a function of membrane PS content. Rates of thrombin formation were determined in reaction mixtures similar to (B) containing 0.2 nm FXa, 0.2 nm FVa and 5 µm PC:PS phospholipid vesicles containing the indicated mole fraction of PS. rHFVa (circles); (W2063,W2064)A (triangles). (Y1956,L1957)A (squares). (Y1956,L1957,W2063,W2064)A (diamonds).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The near complete loss of cofactor activity observed for (Y1956,L1957,W2063,W2064)A on phospholipid membranes could be due solely to a defect in membrane binding or alternatively due to combined effects on membrane binding, FXa binding and/or intrinsic cofactor activity. We therefore investigated the cofactor activity of native and mutant FVa in the absence of phospholipid membranes (Fig. 3). Under these conditions, rates of prothrombin activation are essentially linear and do not saturate in the presence of 0–100 nm FVa due to the relatively low affinity of FXa for FVa in solution (Kd∼ 1 µm) [20Rosing J. Tans G. Govers‐Riemslag J.W. Zwaal R.F. Hemker H.C. The role of phospholipids and factor Va in the prothrombinase complex.J Biol Chem. 1980; 255: 274-83Abstract Full Text PDF PubMed Google Scholar, 21Pryzdial E.L.G. Mann K.G. The association of coagulation factor Xa and factor Va.J Biol Chem. 1991; 266: 8969-77Abstract Full Text PDF PubMed Google Scholar]. At the highest FVa concentration examined, the rate of thrombin generation was accelerated over 400‐fold compared with the rate observed with FXa alone. In the presence of 100 nm (Y1956,L1957)A, (W2063,W2064)A and (Y1956,L1957,W2063,W2064)A the rates of thrombin generation were accelerated 159, 235 and 104‐fold, respectively (Table 1). The observed 2–4‐fold decrease in the cofactor activities of mutant FVa in solution are consistent with the approximately 4‐fold difference in FXa affinity previously noted for binding to membrane‐bound (Y1956,L1957)A and (W2063,W2064)A [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar]. The observed differences in activity shown in Fig. 3 are unlikely to be due to problems with estimating the concentration of mutant proteins since the protein preparations used in this study were highly purified as judged by SDS–PAGE (Fig. 1 and references [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar]). Furthermore, the protein concentration determined by absorbance measurements is in excellent agreement with that calculated by ELISA assay using monoclonal antibodies directed against the heavy chain (HV5146) and light chain (6A5). It is also unlikely that these amino‐acid substitutions have induced gross abnormalities in protein folding since this would be expected to cause a complete loss of cofactor activity in solution and failure to bind to light‐chain monoclonal antibodies [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 7Kim S.W. Quinn‐Allen M.A. Camp T. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. Identification of functionally important amino acid residues within the C2 domain of human factor V using alanine scanning mutagenesis.Biochemistry. 2000; 39: 1951-8Crossref PubMed Scopus (0) Google Scholar]. These observations are consistent with previous studies which have demonstrated that alanine substitutions for flexible solvent‐exposed residues contribute little to the overall folding and stability of protein domains [22Matthews B.W. Structural and genetic analysis of protein stability.Annu Rev Biochem. 1993; 62: 139-60Crossref PubMed Google Scholar]. Thus, the observed differences in activity for native and mutant FVa in solution are most likely due to changes in a FXa binding site, possibly localized within the C1 and/or C2 domains. Nevertheless, the major mechanism contributing to impaired prothrombinase assembly in the presence of (Y1956,L1957,W2063,W2064)A is the reduced affinity of the mutant FVa for phospholipid membranes leading to a > 580‐fold reduced rate of thrombin generation in the presence of 0.2 nm FXa (Fig. 2B).Table 1Prothrombin activation in the presence and absence of phosphatidylserine (PS) membranesCofactorPS : PC membranes (25 : 75)Solution (no PS : PC)§Rates of thrombin generation using 1 n m factor Xa (FXa) and 100 nm factor Va in the absence of membranes compared with FXa alone. See Fig. 3 for details. PC, Phosphatidylcholine.Turnover no. (min−1)K1/2Xa (nm)Thrombin (nm min−1)Relative rateFactor Va2576 ± 47*Mean ± SEM. Data from reference [5].0.10 ± 0.01*Mean ± SEM. Data from reference [5].8.5 ± 0.6413 ± 30(W2063,2064)A1537 ± 33†The values for the turnover number and K1/2Xa reported here are slightly lower than previously reported [10].0.50 ± 0.04†The values for the turnover number and K1/2Xa reported here are slightly lower than previously reported [10].4.8 ± 0.3235 ± 14(Y1956,L1957)A2920 ± 70*Mean ± SEM. Data from reference [5].0.37 ± 0.04*Mean ± SEM. Data from reference [5].3.2 ± 0.1159 ± 5(W2063,2064, Y1956,L1957)A‡Rate of thrombin generation too low to calculate kinetic parameters.‡Rate of thrombin generation too low to calculate kinetic parameters.2.1 ± 0.08104 ± 4NoneNDND0.03 ± 0.0061* Mean ± SEM. Data from reference [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar].† The values for the turnover number and K1/2Xa reported here are slightly lower than previously reported [10Peng W. Quinn‐Allen M.A. Kim S.W. Alexander K.A. Kane W.H. Trp2063 and Trp2064 in the factor Va C2 domain are required for high‐affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.Biochemistry. 2004; 43: 4385-93Crossref PubMed Scopus (0) Google Scholar].‡ Rate of thrombin generation too low to calculate kinetic parameters.§ Rates of thrombin generation using 1 n m factor Xa (FXa) and 100 nm factor Va in the absence of membranes compared with FXa alone. See Fig. 3 for details. PC, Phosphatidylcholine. Open table in a new tab The crystal structures of the C2 domains of FV and FVIII and the recently solved structure for FVai (A1/A3‐C1‐C2) have suggested models for the interaction of these cofactors with phospholipid membranes that involve insertion of solvent‐exposed hydrophobic amino‐acid side chains into the hydrophobic membrane core and interactions of positively charged amino acids with phosphatidylserine [13Macedo‐Ribeiro S. Bode W. Huber R. Quinn‐Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes‐Prior P. Crystal structures of the membrane‐binding C2 domain of human coagulation factor V.Nature. 1999; 402: 434-9Crossref PubMed Scopus (0) Google Scholar, 14Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.L. Structure of the C2 domain of human factor VIII at 1.5 A resolution.Nature. 1999; 402: 439-42Crossref PubMed Scopus (285) Google Scholar, 15Adams T.E. Hockin M.F. Mann K.G. Everse S.J. The crystal structure of activated protein C‐inactivated bovine factor Va: implications for cofactor function.Proc Natl Acad Sci USA. 2004; 101: 8918-23Crossref PubMed Scopus (0) Google Scholar]. The observation that combined mutations in the FVa C1 and C2 domains dramatically reduce prothrombin activation on phospholipid membranes with only modest effects on prothrombin activation in solution provides critical support for the proposed membrane binding model for FVa in which the putative membrane binding spikes in both the C1 and C2 domains are oriented towards the membrane [5Saleh M. Peng W. Quinn‐Allen M.A. Macedo‐Ribeiro S. Fuentes‐Prior P. Bode W. Kane W.H. The factor V, C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.Thromb Haemost. 2004; 91: 16-27Crossref PubMed Google Scholar, 15Adams T.E. Hockin M.F. Mann K.G. Everse S.J. The crystal structure of activated protein C‐inactivated bovine factor Va: implications for cofactor function.Proc Natl Acad Sci USA. 2004; 101: 8918-23Crossref PubMed Scopus (0) Google Scholar]. Lentz and coworkers have proposed that phospholipid binding enhances the cofactor activity of FVa by promoting the binding of FXa [23Majumder R. Weinreb G. Zhai X. Lentz B.R. Soluble phosphatidylserine triggers assembly in solution of a prothrombin‐activating complex in the absence of a membrane surface.J Biol Chem. 2002; 277: 29765-73Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. The molecular basis for this phenomenon has not yet been elucidated. Our finding that mutations in the phospholipid binding sites in FVa results in a 2–4‐fold decrease in the activity in the absence of phospholipid membranes suggests the possibility of a FXa binding site localized within the C1 and/or C2 domains near the protein membrane interface. The weak interaction between FVa and FXa is an obstacle to characterizing these interactions further in the absence of membranes. It is possible that soluble phospholipid analogs [23Majumder R. Weinreb G. Zhai X. Lentz B.R. Soluble phosphatidylserine triggers assembly in solution of a prothrombin‐activating complex in the absence of a membrane surface.J Biol Chem. 2002; 277: 29765-73Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] may prove useful in localizing and defining the phospholipid binding sites in FVa that regulate FXa binding and cofactor activity. This work was supported by grants HL43106 and HL54939 from the National Institutes of Health." @default.
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