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• W2009736469 abstract "Membrane-bound thrombin-activated factor VIII (fVIIIa) functions as a cofactor for factor IXa in the factor Xase complex. We found that binding of heterotrimeric fVIIIa (A1·A2·A3-C1-C2) to synthetic vesicles with a physiologic content of 4% phosphatidylserine (PS), 76% phosphatidylcholine, and 20% phosphatidylethanolamine occurs with a 10-fold higher affinity than that of factor VIII (fVIII). The increased affinity of fVIIIa for PS-containing membranes resulted from the reduced rate of fVIIIa dissociation from the vesicles compared with that of fVIII. Similar affinities of A3-C1-C2, A1·A2·A3-C1-C2, and A3-C1-C2·heavy chain for interaction with PS-containing membranes demonstrate that removal of the light chain (LCh) acidic region by thrombin is responsible for these increased affinities of fVIIIa and its derivatives. Similar kinetic parameters of fVIII and its LCh and C2 domain for binding to PS-containing membranes and to activated platelets indicated that the C2 domain is entirely responsible for the interaction of fVIII with membranes. We conclude that the increased fVIIIa affinity for PS-containing membranes is a result of conformational change(s) within the C2 domain upon removal of the acidic region of the LCh. This conclusion is based on the finding that binding of the monoclonal antibody ESH8 to the C2 domain, which is known to prevent this conformational transition, resulted in fVIIIa binding to PS/phosphatidylcholine/phosphatidylethanolamine vesicles (4/76/20) with a lower affinity similar to that of fVIII. In addition, stabilization of the low affinity binding conformation of the C2 domain of fVIIIa by this antibody led to an inhibition of the fVIIIa activity in the factor X activation complex. Membrane-bound thrombin-activated factor VIII (fVIIIa) functions as a cofactor for factor IXa in the factor Xase complex. We found that binding of heterotrimeric fVIIIa (A1·A2·A3-C1-C2) to synthetic vesicles with a physiologic content of 4% phosphatidylserine (PS), 76% phosphatidylcholine, and 20% phosphatidylethanolamine occurs with a 10-fold higher affinity than that of factor VIII (fVIII). The increased affinity of fVIIIa for PS-containing membranes resulted from the reduced rate of fVIIIa dissociation from the vesicles compared with that of fVIII. Similar affinities of A3-C1-C2, A1·A2·A3-C1-C2, and A3-C1-C2·heavy chain for interaction with PS-containing membranes demonstrate that removal of the light chain (LCh) acidic region by thrombin is responsible for these increased affinities of fVIIIa and its derivatives. Similar kinetic parameters of fVIII and its LCh and C2 domain for binding to PS-containing membranes and to activated platelets indicated that the C2 domain is entirely responsible for the interaction of fVIII with membranes. We conclude that the increased fVIIIa affinity for PS-containing membranes is a result of conformational change(s) within the C2 domain upon removal of the acidic region of the LCh. This conclusion is based on the finding that binding of the monoclonal antibody ESH8 to the C2 domain, which is known to prevent this conformational transition, resulted in fVIIIa binding to PS/phosphatidylcholine/phosphatidylethanolamine vesicles (4/76/20) with a lower affinity similar to that of fVIII. In addition, stabilization of the low affinity binding conformation of the C2 domain of fVIIIa by this antibody led to an inhibition of the fVIIIa activity in the factor X activation complex. factor VIII thrombin-activated fVIII light chain of fVIII fVIII residues 1690–2332 heavy chain of fVIII phosphatidylserine phosphatidylethanolamine phosphatidylcholine 4-morpholineethanesulfonic acid resonance units factor X activation complex von Willebrand factor. The plasma glycoprotein factor VIII (fVIII)1 functions as a cofactor for the factor X activation complex (factor Xase) in the intrinsic pathway of blood coagulation (1van Dieijen G. Tans G. Rosing J. Hemker H.C. J. Biol. Chem. 1981; 256: 3433-3442Abstract Full Text PDF PubMed Google Scholar). Within the factor Xase complex, thrombin-activated factor VIII (fVIIIa) associated with membranes of activated platelets (2Gilbert G.E. Sims P.J. Wiedmer T. Furie B. Furie B.C. Shattil S.J. J. Biol. Chem. 1991; 266: 17261-17268Abstract Full Text PDF PubMed Google Scholar, 3Nesheim M.E. Pittman D.D. Wang J.H. Slonosky D. Giles A.R. Kaufman R.J. J. Biol. Chem. 1988; 263: 16467-16470Abstract Full Text PDF PubMed Google Scholar) or with synthetic phospholipid vesicles (4Duffy E.J. Parder E.T. Mutucumarana V.P. Johnson A.E. Lollar P. J. Biol. Chem. 1992; 267: 17006-17011Abstract Full Text PDF PubMed Google Scholar) binds to factor X (5Lapan K. Fay P.J. J. Biol. Chem. 1997; 272: 2082-2088Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and to activated factor IX (4Duffy E.J. Parder E.T. Mutucumarana V.P. Johnson A.E. Lollar P. J. Biol. Chem. 1992; 267: 17006-17011Abstract Full Text PDF PubMed Google Scholar, 6Ahmad S.S. Rawala-Sheikh R. Walsh P.N. J. Biol. Chem. 1989; 264: 3244-3251Abstract Full Text PDF PubMed Google Scholar). Activation of factor X by the fVIIIa·activated factor IX complex assembled on a membrane is 100,000-fold more efficient than in the absence of phospholipid (7Gilbert G.E. Arena A.A. J. Biol. Chem. 1996; 271: 11120-11125Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Assembly of the factor Xase complexin vivo is localized to sites of vascular damage where the activated platelets are adherent (2Gilbert G.E. Sims P.J. Wiedmer T. Furie B. Furie B.C. Shattil S.J. J. Biol. Chem. 1991; 266: 17261-17268Abstract Full Text PDF PubMed Google Scholar, 3Nesheim M.E. Pittman D.D. Wang J.H. Slonosky D. Giles A.R. Kaufman R.J. J. Biol. Chem. 1988; 263: 16467-16470Abstract Full Text PDF PubMed Google Scholar). The fVIII protein consists of homologous A and C domains and a unique B domain, which are arranged in the order A1-A2-B-A3-C1-C2 (8Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (658) Google Scholar). It is processed to a series of Me2+-linked heterodimers produced by cleavage at the B-A3 junction (9Fay P.J. Anderson M.T. Chavin S.I. Marder V.J. Biochim. Biophys. Acta. 1986; 871: 268-278Crossref PubMed Scopus (123) Google Scholar), generating a light chain (LCh) consisting of the acidic region (AR) and A3, C1, and C2 domains and a heavy chain (HCh), which consists of the A1, A2, and B domains (Fig. 1). The site involved in fVIII binding to synthetic phospholipid vesicles or platelets was localized to the C2 domain residues 2303–2332 (10Arai M. Scandella D. Hoyer L.W. J. Clin. Invest. 1989; 83: 1978-1984Crossref PubMed Scopus (150) Google Scholar). The presence of at least 8% phosphatidylserine (PS) is required for fVIII binding to synthetic PS/phosphatidylcholine (PC) membranes (11Gilbert G.E. Drinkwater D. Biochemistry. 1993; 32: 9577-9585Crossref PubMed Scopus (88) Google Scholar). The additional presence of phosphatidylethanolamine (PE) induces high affinity binding sites for fVIII on membranes with physiologic (<8%) molar fractions of PS (12Gilbert G.E. Arena A.A. J. Biol. Chem. 1995; 270: 18500-18505Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Expression of fVIII binding sites on platelets occurs only upon their activation by thrombin or other agonists (2Gilbert G.E. Sims P.J. Wiedmer T. Furie B. Furie B.C. Shattil S.J. J. Biol. Chem. 1991; 266: 17261-17268Abstract Full Text PDF PubMed Google Scholar, 3Nesheim M.E. Pittman D.D. Wang J.H. Slonosky D. Giles A.R. Kaufman R.J. J. Biol. Chem. 1988; 263: 16467-16470Abstract Full Text PDF PubMed Google Scholar). This leads to the reorientation of PS and PE from the inner to the outer layer of the plasma membrane (13Zwaal R.F.A. Comfurius P. Bevers E.M. Biochem. Soc. Trans. 1993; 21: 248-253Crossref PubMed Scopus (106) Google Scholar, 14Bevers E.M. Comfurius P. van Rijn J.L.M.L. Hemker H.C. Zwaal R.F.A. Eur. J. Biochem. 1982; 122: 429-436Crossref PubMed Scopus (399) Google Scholar) to provide sufficient concentrations of PE and PS for the formation of fVIII binding sites. Maintenance of a normal fVIII level in the circulation is dependent on its complex formation with vWf, which prevents fVIII from binding to PS-containing membranes (15Gilbert G.E. Drinkwater D. Barter S. Clouse S.B. J. Biol. Chem. 1992; 267: 15861-15868Abstract Full Text PDF PubMed Google Scholar, 16Fay P.J. Coumans J.-V. Walker F.J. J. Biol. Chem. 1991; 266: 2172-2177Abstract Full Text PDF PubMed Google Scholar) and to activated platelets (17Nesheim M. Pittman D.D. Giles A.R. Fass D.N. Wang J.H. Slonosky D. Kaufman R.J. J. Biol. Chem. 1991; 266: 17815-17826Abstract Full Text PDF PubMed Google Scholar). Cleavage of the LCh at Arg1689 releases the acidic region residues 1649–1689 and leads to the dissociation of fVIIIa from vWf (18Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar, 19Ahmad S.S. Rawala-Sheikh R. Ashby B. Walsh P.N. J. Clin. Invest. 1989; 84: 824-828Crossref PubMed Scopus (51) Google Scholar, 20Hill-Eubanks D.C. Parker C.G. Lollar P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6508-6512Crossref PubMed Scopus (71) Google Scholar). Activation of fVIII by thrombin cleavage at Arg372, Arg740, and Arg1689 (21Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (397) Google Scholar) results in at least a 100-fold increase of cofactor activity. The product, fVIIIa, is a A1·A2·A3-C1-C2 heterotrimer (22Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar) in which domains A1 and A3 retain the metal ion linkage (Fig. 1) and the stable dimer A1·A3-C1-C2 (23Lollar P. Parker C.G. J. Biol. Chem. 1990; 265: 1688-1692Abstract Full Text PDF PubMed Google Scholar) is weakly associated with the A2 subunit mainly through electrostatic forces (22Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar). Spontaneous dissociation of the A2 subunit from the dimer results in nonproteolytic inactivation of fVIIIa. A conformational change(s) occurs within the C2 domain upon removal of the acidic region of the LCh (residues 1649–1689), leading to the loss of the optimal binding conformation of the vWf site within the C2 domain of thrombin-cleaved LCh (A3-C1-C2) (24Saenko E.L. Scandella D. J. Biol. Chem. 1997; 272: 18007-18014Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). We hypothesized that this change(s) could also affect fVIIIa affinity for phospholipid. In this report, we determined the effect of activation of fVIII by thrombin on its binding to synthetic phospholipid PS/PC surfaces with a high content of PS, as well as its binding to phospholipid vesicles containing physiological mole fractions of PS, PE, and PC and to membranes of activated platelets. To elucidate the role of the conformational changes occurring in the C2 domain upon removal of the acidic region of the LCh (24Saenko E.L. Scandella D. J. Biol. Chem. 1997; 272: 18007-18014Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), we used fragments of fVIII and fVIIIa for measurement of their binding to phospholipids. IgG of monoclonal antibody ESH8 (epitope, C2 residues 2248–2285) was obtained from American Diagnostica. Plasma fVIII was purified from therapeutic concentrates of Method M (American Red Cross) (25Saenko E.L. Shima M. Gilbert G.E. Scandella D. J. Biol. Chem. 1996; 271: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). HCh, LCh, A3-C1-C2 and fVIII HCh·A3-C1-C2 heterodimer were prepared as described previously (24Saenko E.L. Scandella D. J. Biol. Chem. 1997; 272: 18007-18014Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The recombinant C2 domain was expressed and purified as described(26). vWf was purified from cryoprecipitate (Cutter Biological) (26Saenko E.L. Scandella D. J. Biol. Chem. 1995; 270: 13826-13833Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Concentrations of antibody and vWf were determined by the method of Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar) and those of purified A2 and recombinant C2 by the Quantigold assay (Diversified Biotech). The concentrations of plasma-derived fVIII, LCh, and HCh were determined by absorbance at 280 nm, using extinction coefficients of 1.2 (28Lollar P. Parker C.G. Tracy R.P. Blood. 1988; 71: 137-143Crossref PubMed Google Scholar), 1.34 (18Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar), and 1.0 (18Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar), respectively. The molar concentrations of fVIII, HCh, LCh, A3-C1-C2, and A2, were calculated using molecular masses of 300 kDa (8Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (658) Google Scholar), 136 kDa (29Fay P.J. Arch. Biochem. Biophys. 1988; 262: 525-531Crossref PubMed Scopus (61) Google Scholar), 80 kDa (29Fay P.J. Arch. Biochem. Biophys. 1988; 262: 525-531Crossref PubMed Scopus (61) Google Scholar), 73 kDa (30Fay P.J. Biochim. Biophys. Acta. 1987; 952: 181-190Crossref Scopus (49) Google Scholar) and 43 kDa (30Fay P.J. Biochim. Biophys. Acta. 1987; 952: 181-190Crossref Scopus (49) Google Scholar), respectively. Molecular masses of HCh·A3-C1-C2 and A1·A2·A3-C1-C2 (Fig. 1) were calculated as the sums of the masses of their components to be 209 and 167 kDa, respectively. Purified fVIIIa, generously provided by Baxter Biotech Group (Duarte, CA), was prepared from recombinant fVIII (31Curtis J.E. Helgerson S.L. Parker E.T. Lollar P. J. Biol. Chem. 1994; 269: 6246-6251Abstract Full Text PDF PubMed Google Scholar). Activated plasma-derived fVIII was prepared by activation of fVIII (2.6 μm) by thrombin (0.1 μm) for 10 min at 37 °C in 20 mm HEPES, pH 7.4, 0.15m NaCl (HEPES-buffered saline) containing 5 mmCaCl2. Activation was stopped by hirudin (0.15 μm), and the pH was adjusted to 6.0 by the addition of 0.2 m MES. Activity of fVIIIa was measured using a chromogenic factor Xase assay (25Saenko E.L. Shima M. Gilbert G.E. Scandella D. J. Biol. Chem. 1996; 271: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In the assay, the limiting concentration of fVIIIa (0.2 nm) (25Saenko E.L. Shima M. Gilbert G.E. Scandella D. J. Biol. Chem. 1996; 271: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) was added to a solution containing PS/PC vesicles (20 μm), factor IXa (2 nm), and factor X (300 nm), and the reaction was initiated by the addition of 5 mm CaCl2. Aliquots were withdrawn after 15, 30, 45, and 60 s, and factor X activation was stopped with 0.05 m EDTA. Factor Xa generation was measured by cleavage of 0.3 mm synthetic substrate S-2765 (Amersham Pharmacia Biotech) using aV max microplate reader (Molecular Devices). A purified factor Xa standard (Enzyme Research Laboratories) was used to convert absorbance (410 nm) into factor Xa concentration. FVIII was iodinated as described (24Saenko E.L. Scandella D. J. Biol. Chem. 1997; 272: 18007-18014Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The specific radioactivity of fVIII was 4 μCi/μg of protein. The activity of 125I-fVIII determined in the one-stage clotting assay (3800 units/μg) was similar to that of unlabeled fVIII. Phospholipids PS, PC, and PE were purchased from Sigma, and biotin-LC-dipalmitoyl-PE was purchased from Pierce. Phospholipid vesicles with various PS, PC, and/or PE content and vesicles containing biotin-LC-dipalmitoyl-PE (0.5%) were prepared as described (32Barenholz Y. Gibbes D. Litman B.J. Goll J. Thompson T.E. Carlson F.D. Biochemistry. 1977; 16: 2806-2810Crossref PubMed Scopus (729) Google Scholar). The kinetics of protein-phospholipid interaction were determined by surface plasmon resonance using the Biacore biosensor instrument (Amersham Pharmacia Biotech, Sweden) or IAsys biosensor (FISONS, United Kingdom), which measure protein binding and subsequent dissociation in real time (33Johnsson B. Lofas S. Lindquist G. Anal. Biochem. 1991; 198: 268-277Crossref PubMed Scopus (1203) Google Scholar). Binding and subsequent dissociation was measured in HEPES-buffered saline, pH 7.4, 5 mmCaCl2 at 22 °C for all ligands. A supported PS/PC or PC monolayer was formed on the surface of an HPA hydrophobic chip (Amersham Pharmacia Biotech) by incubation of unilamellar PS/PC (25/75) or 100% PC vesicles at 400 μg/ml in HEPES-buffered saline for 20 min at 22 °C, which produced a signal of 1100 resonance units (RU). The phospholipid-coated chip was blocked by 0.1 mg/ml bovine serum albumin for 20 min. Binding of fVIII and its derivatives to a supported PS/PC or PC monolayer was measured using the Biacore biosensor instrument, where 1 ng of protein bound per mm2 of the biosensor chip produces a resonance signal of 1000 RU. To regenerate the cuvette, complete dissociation of bound ligands was achieved by the addition of 10 mm NaOH for 30 s. Binding to intact vesicles was measured using the IAsys biosensor, where 1 ng of protein bound per mm2 of the biosensor chip produces a signal of 600 RU. Biotin-coated cuvettes (Affinity Sensors) were incubated with 20 μg/ml streptavidin (Sigma) for 10 min, followed by the addition of biotinylated PS/PC/PE (4/76/20) or PC/PE (80/20) vesicles at 200 μg/ml for 10 min. The resonance response due to binding of biotinylated phospholipid vesicles was 1000 Arc seconds. To regenerate the cuvette, complete dissociation of the biotin-streptavidin complex was achieved by the addition of 5m NaOH for 2 min. The values of the rate constants for the dissociation (k off) of fVIII and its derivatives from monolayers or immobilized intact vesicles were determined by fitting the dissociation kinetics data to the following equation describing a single phase dissociation process,dR/dt=−koffR(Eq. 1) where the surface plasmon resonance signal observed,R, is proportional to the formation of a complex between immobilized component and added ligand. Ref. 34O'Shannessy D.J. Brigham-Burke M. Sonenson K.K. Hensley P. Brooks I. Anal. Biochem. 1993; 212: 457-468Crossref PubMed Scopus (520) Google Scholar showed the following,dR/dt=konCRmax−konC+koffR(Eq. 2) where R max is the maximal binding capacity of the immobilized ligand surface expressed in resonance units (Arc seconds) and C is the concentration of polypeptide in solution. In Figs. Figure 2, Figure 3, Figure 4, Figure 5, the values of k on were determined from individual association kinetics data using the integrated form of the rate equation (Equation 2),R=CkonRmax1−e−(Ckon+koff)tCkon+koff(Eq. 3) The values of k on and R max were derived from nonlinear regression analysis fitting R versus t to Equation 3. The value of the k off constant used in Equation 3 was derived from the dissociation kinetics data fitted to Equation 1. The values of equilibrium dissociation constants (K d) were calculated ask off/k on.Figure 3Determination of the kinetic parameters for C2 domain binding to phospholipid monolayers and intact vesicles.The association (A) of C2 with PS/PC (25/75) monolayer and corresponding dissociation (B) kinetic data were obtained at 6 (■), 24 (○), and 60 nm (▵) of C2. The original kinetic data and fitted curves (solid lines) were obtained as in Fig. 2. In the control experiment (▿), binding of C2 (60 nm) to a control 100% PC monolayer was measured. InC and D, C2 association with and dissociation from PS/PC/PE (4/76/20) vesicles was measured at 6 (■), 24 (○), and 60 nm (▵) of C2 as described in Fig. 2 C. Thesolid lines show the fitted curves obtained as above. In the control experiment (▿), binding of C2 (60 nm) to PCPE (80/20) vesicles was measured.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4The kinetics of A3-C1-C2 and HCh·A3-C1-C2 interaction with phospholipid monolayers and intact vesicles.Association of 24 nm each of A3-C1-C2 (○) or HCh·A3-C1-C2 (■) with an immobilized PS/PC (25/75) monolayer (A) and dissociation from it (B) was measured as in Fig. 2 A. A, binding of 24 nmHCh·A3-C1-C2 (▿) to a control biosensor cuvette with 100% PC monolayer was measured as above. C and D, association of 24 nm of each A3-C1-C2 (○) or HCh·A3-C1-C2 (■) with PS/PC/PE (4/76/20) vesicles and dissociation from vesicles was measured as in Fig. 2 B. C, binding of HCh·A3-C1-C2 (▿) to the control PCPE (80/20) vesicles was measured as above. The solid lines in A and B show the fitted curves obtained as in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Determination of the kinetic parameters for fVIIIa interaction with phospholipid vesicles from association and dissociation kinetics. Association of a 16 nmconcentration of fVIIIa (○) or fVIII (■) with PS/PC/PE (4/76/20) vesicles (A) and dissociation from vesicles (B) was studied in the presence of exogenous A2 (200 nm) in 0.02 m MES, pH 6.0, 0.1 m NaCl, and 5 mm CaCl2. In the control experiment (▿), binding of 16 nm fVIIIa to PCPE (80/20) vesicles was measured as above. The solid lines in A and B show the fitted curves obtained as in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In Figs. 6 and 7, the values of k on and K d were determined from multiple association curves as follows. A linear fit of dR/dt versus R yields an apparent first order association rate constant corresponding to each concentration of fVIIIa or ESH8·fVIIIa,ks=konC+koff(Eq. 4) The value of k on was determined from the linear fit of k s versus C(concentration of fVIIIa or ESH8·fVIIIa) to Equation 4. TheK d was derived from the best fit of the resonance response corresponding to an equilibrium ligand binding (R e) achieved at different ligand concentrationsversus F, the concentration of unbound ligand, to Equation 5,R0=RmaxF/Kd+F(Eq. 5) Under the experimental conditions, the concentration of bound ligand was much less than the concentration of added ligand, indicating that the concentration of unbound ligand (F) is similar to the concentration of added ligand. The k offvalue was calculated as K d ×k on using the values of K dand k on determined as above. All the fitting procedures were performed using Sigmaplot 1.02 (Jandel Scientific).Figure 7Determination of the kinetic parameters for ESH8·fVIIIa interaction with phospholipid vesicles. A, the ESH8·fVIIIa complex was prepared by incubation of 500 nm fVIII with monoclonal antibody ESH8 (1000 nm) for 30 min at room temperature prior to activation by thrombin (125 nm) for 1 min at 37 °C followed by inhibition of thrombin by hirudin (200 nm). Association of ESH8·fVIIIa with PS/PC/PE (4/76/20) vesicles was measured in the presence of 200 nm of exogenous A2 as in Fig. 5 A. The concentrations of ESH8·fVIIIa corresponding tocurves 1–7 were 1, 2, 4, 8, 16, 32, and 64 nm, respectively. In experiment 8, binding of ESH8·fVIIIa (64 nm) to PC/PE (80/20) vesicles was measured as above.B, determination of the K d value for ESH8·fVIIIa binding to PS/PC/PE vesicles. The open symbols are the values of equilibrium binding determined from curves 1–7. The solid line shows the best fit of the B e values to Equation 5.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The binding affinities of fVIII or its fragments for activated platelets were determined by homologous or heterologous ligand displacement assays. Platelets were isolated from platelet-rich plasma and activated by thrombin as described(3).125I-fVIII (0.25 nm) and increasing concentrations of unlabeled fVIII, C2, LCh, or A3-C1-C2 were incubated with activated platelets in Tyrode's solution (35Greco N.J. Yamamoto N. Jackson B.W. Tandon N.N. Moos Jr., M. Jamieson G.A. J. Biol. Chem. 1991; 266: 13627-13633Abstract Full Text PDF PubMed Google Scholar) at 22 °C for 30 min. Aliquots (75 μl) from the samples were loaded onto 20% sucrose (350 μl) and centrifuged for 1 min at 10,000 × g. Negative controls contained unactivated platelets and all the other components. The negative control values (≤5% of the maximal binding values) were subtracted from average values of triplicates of all other samples. For heterologous ligand displacement studies, the data were analyzed assuming two distinct equilibria, R +L 1 ⇄ RL 1 and R + L 2 ⇄RL 2, where R is the concentration of fVIII binding sites on activated platelets, L 1is 125I-fVIII, and L 2 is C2, LCh, or A3-C1-C2. These equilibria are described by the equilibrium constantsK d and K i, respectively. The data of homologous and heterologous displacements were fitted to a model assuming a single class of fVIII binding sites on activated platelets using the LIGAND (36Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7771) Google Scholar) program. Since in most previous fVIII binding studies, the synthetic phospholipid membranes were composed of 15–25% PS and PC (4Duffy E.J. Parder E.T. Mutucumarana V.P. Johnson A.E. Lollar P. J. Biol. Chem. 1992; 267: 17006-17011Abstract Full Text PDF PubMed Google Scholar, 11Gilbert G.E. Drinkwater D. Biochemistry. 1993; 32: 9577-9585Crossref PubMed Scopus (88) Google Scholar, 15Gilbert G.E. Drinkwater D. Barter S. Clouse S.B. J. Biol. Chem. 1992; 267: 15861-15868Abstract Full Text PDF PubMed Google Scholar, 37Bardelle C. Furie B. Furie B.C. Gilbert G.E. J. Biol. Chem. 1993; 268: 8815-8824Abstract Full Text PDF PubMed Google Scholar), in the present studies we also determined parameters for binding to immobilized PS/PC (25/75) monolayers formed on a hydrophobic surface, which represents a stable and structurally defined lipid environment resembling cell membranes (38Plant A.L. Brigham-Burke M. Petrella E.C. O'Shannessy D.J. Anal. Biochem. 1995; 226: 342-348Crossref PubMed Scopus (176) Google Scholar). To determine whether LCh is entirely responsible for the high affinity fVIII interaction with phospholipid membranes, we compared the kinetics of fVIII and LCh binding to and dissociation from immobilized phospholipid monolayers or intact phospholipid vesicles. Values of second order association rate constants (k on) (Table I) were determined from the representative curves (Fig. 2 A) showing the resonance response of fVIII or LCh association with immobilized PS/PC monolayer over time. The half-lives for the dissociation of fVIII or LCh from phospholipid monolayers were approximately 8 min and 7 min, respectively (Fig. 2 B). The k offvalues calculated for fVIII and LCh using the equationk off = ln2/half-life were 1.44 × 10−3 s−1 and 1.65 × 10−3s−1, correspondingly, similar to the respective values calculated for the best fits of the dissociation curves to Equation 1(Table I).Table IKinetic parameters for binding of fVIII and its derivatives to immobilized PS/PC (25/75) monolayerSoluble ligandk onk offK dm−1s−1s−1nmfVIII(6.3 ± 0.6) × 105(1.36 ± 0.04) × 10−32.2 ± 0.21LCh(6.8 ± 0.8) × 105(1.54 ± 0.02) × 10−32.3 ± 0.26C2(7.2 ± 0.04) × 105(2.3 ± 0.09) × 10−33.2 ± 0.25A3-C1-C2(8.8 ± 0.15) × 105(3.5 ± 0.05) × 10−40.4 ± 0.07HCh·A3-C1-C2(7.8 ± 0.12) × 105(2.8 ± 0.04) × 10−40.36 ± 0.06The k on and k off values were derived from individual association (A) and dissociation (B) kinetic curves shown in Figs. Figure 2, Figure 3, Figure 4 as described under “Experimental Procedures.” The K d values were calculated as k off/k on. Open table in a new tab The k on and k off values were derived from individual association (A) and dissociation (B) kinetic curves shown in Figs. Figure 2, Figure 3, Figure 4 as described under “Experimental Procedures.” The K d values were calculated as k off/k on. In a control experiment, we examined whether binding characteristics of PS/PC (25/75) monolayers are similar to those of intact vesicles of identical composition. We determined fVIII and LCh parameters for binding to immobilized small unilamellar PS/PC vesicles, containing 0.5% PE-biotin for immobilization to an SA-coated chip via biotin (kinetic data not shown). The k on and k off values for fVIII binding to such vesicles were 6.0 × 105m−1s−1 and 1.22 × 10−3 s−1, while for LCh binding the values were 6.2 × 105m−1 s−1 and 1.38 × 10−3 s−1 and are similar to the corresponding values for fVIII and LCh interaction with the PS/PC monolayer (Table I). To elucidate if LCh is also entirely responsible for fVIII binding to membranes containing PS, PC, and PE at molar fractions similar to those in the membranes of activated platelets (12Gilbert G.E. Arena A.A. J. Biol. Chem. 1995; 270: 18500-18505Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), we compared kinetic parameters for fVIII and LCh interaction with PS/PC/PE (4/76/20) membranes. In preliminary experiments, we found that a planar phospholipid monolayer containing physiologic ratios of PS, PC, and PE (4/76/20) formed on the hydrophobic biosensor" @default.
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• W2009736469 title "Activation of Factor VIII by Thrombin Increases Its Affinity for Binding to Synthetic Phospholipid Membranes and Activated Platelets" @default.
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