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• W2028983608 abstract "We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ibα. Recombinant VWF A1 domain (residues Glu497-Pro705 of VWF) bound stoichiometrically to a GPIbα-calmodulin fusion protein (residues His1-Val289 of GPIbα; GPIbα-CaM) immobilized on W-7-agarose with a K d of 3.3 μm. The variant VWF A1(R545A) bound to GPIbα-CaM 20-fold more tightly, mainly because the association rate constantk on increased from 1,100 to 8,800m−1 s−1. The GPIbα mutations G233V and M239V cause platelet-type pseudo-von Willebrand disease, and VWF A1 bound to GPIbα(G233V)-CaM and GPIbα(M239V)-CaM with aK d of 1.0 and 0.63 μm, respectively. The increased affinity of VWF A1 for GPIbα(M239V)-CaM was explained by an increase in k on to 4,500m−1 s−1. GPIbα-CaM bound with similar affinity to recombinant VWF A1, to multimeric plasma VWF, and to a fragment of dispase-digested plasma VWF (residues Leu480/Val481-Gly718). VWF A1 and A1(R545A) bound to platelets with affinities and rate constants similar to those for binding to GPIbα-CaM, and botrocetin had the expected positively cooperative effect on the binding of VWF A1 to GPIbα-CaM. Therefore, allosteric regulation by botrocetin of VWF A1 binding to GPIbα, and the increased binding affinity caused by mutations in VWF or GPIbα, are reproduced by isolated structural domains. The substantial increase in k on caused by mutations in either A1 or GPIbα suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The exceptionally slow k on andk off provide important new constraints on models for rapid platelet tethering at high wall shear rates. We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ibα. Recombinant VWF A1 domain (residues Glu497-Pro705 of VWF) bound stoichiometrically to a GPIbα-calmodulin fusion protein (residues His1-Val289 of GPIbα; GPIbα-CaM) immobilized on W-7-agarose with a K d of 3.3 μm. The variant VWF A1(R545A) bound to GPIbα-CaM 20-fold more tightly, mainly because the association rate constantk on increased from 1,100 to 8,800m−1 s−1. The GPIbα mutations G233V and M239V cause platelet-type pseudo-von Willebrand disease, and VWF A1 bound to GPIbα(G233V)-CaM and GPIbα(M239V)-CaM with aK d of 1.0 and 0.63 μm, respectively. The increased affinity of VWF A1 for GPIbα(M239V)-CaM was explained by an increase in k on to 4,500m−1 s−1. GPIbα-CaM bound with similar affinity to recombinant VWF A1, to multimeric plasma VWF, and to a fragment of dispase-digested plasma VWF (residues Leu480/Val481-Gly718). VWF A1 and A1(R545A) bound to platelets with affinities and rate constants similar to those for binding to GPIbα-CaM, and botrocetin had the expected positively cooperative effect on the binding of VWF A1 to GPIbα-CaM. Therefore, allosteric regulation by botrocetin of VWF A1 binding to GPIbα, and the increased binding affinity caused by mutations in VWF or GPIbα, are reproduced by isolated structural domains. The substantial increase in k on caused by mutations in either A1 or GPIbα suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The exceptionally slow k on andk off provide important new constraints on models for rapid platelet tethering at high wall shear rates. von Willebrand factor glycoprotein von Willebrand disease calmodulin Tris-buffered saline bovine serum albumin polyacrylamide gel electrophoresis high pressure liquid chromatography N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide polymerase chain reaction Von Willebrand factor (VWF)1 is a multimeric plasma glycoprotein that plays a crucial role in primary hemostasis by enabling platelets to adhere at sites of vascular injury (1.Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1149) Google Scholar). Platelet adhesion requires the binding of VWF to the platelet membrane glycoprotein (GP) Ib-IX complex. This interaction is essential for normal hemostasis (2.Ruggeri Z.M. Zimmerman T.S. Blood. 1987; 70: 895-904Crossref PubMed Google Scholar) and can contribute to the development of arterial thrombosis (3.Nichols T.C. Samama C.M. Bellinger D.A. Roussi J. Reddick R.L. Bonneau M. Read M.S. Bailliart O. Koch G.G. Vaiman M. Sigman J.L. Pignaud G.A. Brinkhous K.M. Griggs T.R. Drouet L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2455-2459Crossref PubMed Scopus (79) Google Scholar). The binding site for platelet GPIb-IX is within the A1 domain of the mature VWF subunit (approximately Glu497-Gly716) (4.Fujimura Y. Titani K. Holland L.Z. Russell S.R. Roberts J.R. Elder J.H. Ruggeri Z.M. Zimmerman T.S. J. Biol. Chem. 1986; 261: 381-385Abstract Full Text PDF PubMed Google Scholar, 5.Andrews R.K. Gorman J.J. Booth W.J. Corino G.L. Castaldi P.A. Berndt M.C. Biochemistry. 1989; 28: 8326-8336Crossref PubMed Scopus (103) Google Scholar). The GPIb-IX complex consists of up to four gene products as follows: GPIbα, GPIbβ, GPIX, and possibly GPV (6.Du X. Beutler L. Ruan C. Castaldi P.A. Berndt M.C. Blood. 1987; 69: 1524-1527Crossref PubMed Google Scholar, 7.Modderman P.W. Admiraal L.G. Sonnenberg A. von dem Borne A.E.G.K. J. Biol. Chem. 1992; 267: 364-369Abstract Full Text PDF PubMed Google Scholar). The binding site for VWF is in the N-terminal 293 residues of GPIbα, and optimal interaction requires sulfation of tyrosine residues at positions 276, 278, and 279 (8.Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Multimeric normal VWF does not bind appreciably to platelets in the circulation. Once VWF is immobilized in the extracellular matrix, however, platelets bind and adhere tightly to it. This apparent induction of GPIbα binding in vivo is associated with VWF binding to collagen (9.Pareti F.I. Niiya K. McPherson J.M. Ruggeri Z.M. J. Biol. Chem. 1987; 262: 13835-13841Abstract Full Text PDF PubMed Google Scholar, 10.Cruz M.A. Yuan H. Lee J.R. Wise R.J. Handin R.I. J. Biol. Chem. 1995; 270: 10822-10827Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) or other subendothelial components (11.de Groot P.G. Ottenhof-Rovers M. van Mourik J.A. Sixma J.J. J. Clin. Invest. 1988; 82: 65-73Crossref PubMed Scopus (59) Google Scholar,12.Katayama M. Nagata S. Hirai S. Miura S. Fujimura Y. Matusi T. Kato I. Titani K. J. Biochem. (Tokyo). 1995; 117: 331-338Crossref PubMed Scopus (8) Google Scholar). High affinity binding of VWF to GPIbα is induced in vitro by certain exogenous modulators, including the antibiotic ristocetin (13.Howard M.A. Firkin B.G. Thromb. Diath. Haemorrh. 1971; 26: 362-369PubMed Google Scholar, 14.Scott J.P. Montgomery R.R. Retzinger G.S. J. Biol. Chem. 1991; 266: 8149-8155Abstract Full Text PDF PubMed Google Scholar) and the snake venom protein botrocetin (15.Read M.S. Smith S.V. Lamb M.A. Brinkhous K.M. Blood. 1989; 74: 1031-1035Crossref PubMed Google Scholar). These phenomena suggest that induced conformational changes modulate the affinity of VWF-GPIbα binding. A role for conformational change is supported indirectly by the effects of mutations on the binding affinity and structure of VWF A domains. Gain-of-function mutations in the VWF A1 domain promote spontaneous binding to GPIbα and cause a bleeding disorder, von Willebrand disease (VWD) type 2B (reviewed in Ref. 16.Sadler J.E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. 7th Ed. McGraw-Hill Book Co., New York1995: 3269-3287Google Scholar). The VWD type 2B mutation I546V was shown to cause a significant change in the crystal structure of recombinant VWF domain A1, altering the positions of residues proposed to bind GPIbα (17.Ruggeri, Z. M., Celikel, R., Ware, J., and Varughese, K. I. (1999) Thromb. Haemostasis (suppl.) 682Google Scholar). The relevance of conformational changes within domain A1 to the normal hemostatic function of VWF remains controversial (1.Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1149) Google Scholar, 18.Tsuji S. Sugimoto M. Kuwahara M. Nishio K. Takahashi Y. Fujimura Y. Ikeda Y. Yoshioka A. Blood. 1996; 88: 3854-3861Crossref PubMed Google Scholar). The kinetic parameters of VWF-GPIb interactions are central to the mechanism by which VWF mediates platelet adhesion under conditions of high wall shear stress (19.Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 20.Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). Binding of platelet GPIbα to VWF facilitates the initial transient tethering of platelets to the vessel wall that is visualized as rolling or slow translocation by video microscopy (19.Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 20.Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). Subsequently, integrin-ligand interactions can mediate stable platelet adhesion. The high efficiency of platelet tethering has suggested that the kinetics of VWF-GPIb binding must be fast and that rapid tethering facilitates the formation of much tighter integrin-dependent bonds that require more time to form (19.Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 21.Ruggeri Z.M. J. Clin. Invest. 1997; 99: 559-564Crossref PubMed Scopus (192) Google Scholar). To date, however, direct measurements of the kinetics of VWF A1-GPIbα binding have not been reported. To investigate the mechanism of VWF-platelet binding, we have studied the interactions between recombinant VWF domain A1 and the N-terminal domain of platelet GPIbα. The behavior of these isolated domains was similar to that of plasma VWF and the GPIb-IX complex in situ on platelets. In the absence of exogenous modulators of binding, the association rate constant for VWF A1-GPIbα-(1–289) binding was only 1,100 m−1 s−1but was increased substantially by gain-of-function mutations in either the VWF A1 domain or GPIbα-(1–289). The exceptionally slow rate of binding and the acceleration caused by mutations are consistent with a model in which rate-limiting conformational changes in both proteins are necessary for productive VWF-GPIbα binding. The slow rate constants for binding provide new constraints on models for how VWF mediates rapid platelet tethering under conditions of high wall shear stress in vivo. Plasmid pGEM-4ZNae contains an 1140-base pair NgoMI-KpnI fragment of human VWF cDNA that encodes amino acid residues 442–821 of the mature VWF subunit (22.Dong Z. Thoma R.S. Crimmins D.L. McCourt D.W. Tuley E.A. Sadler J.E. J. Biol. Chem. 1994; 269: 6753-6758Abstract Full Text PDF PubMed Google Scholar). Plasmid pGEM-4ZNaeR545A is similar but encodes the substitution R545A (23.Matsushita T. Sadler J.E. J. Biol. Chem. 1995; 270: 13406-13414Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Fragments of these plasmids were amplified by polymerase chain reaction (PCR) with Pfu polymerase (Stratagene, La Jolla, CA) and primers 5′-acc act ctg catatg gag gac atc-3′ (sense, NdeI site underlined) and 5′-gtc ggg gggatccta aga agg agg-3′ (antisense, BamHI site underlined). PCR products were digested with NdeI and BamHI and cloned into pET-11b (Stratagene), and their sequences were confirmed by DNA sequencing. Plasmid pET-VWF497–705 or pET-VWF497–705(R545A) encode a methionine followed by amino acid residues Glu497-Pro705 of mature VWF without or with the substitution R545A, respectively. Escherichia coli BL21 (DE3) cells (Stratagene) containing pET plasmids were grown in 1 liter of LB/ampicillin medium at 37 °C to an absorbance OD (600 nm) of 0.7. 2 mmisopropyl-β-d-thiogalactopyranoside (Sigma) was added, and incubation was continued for 5 h. The cell pellet was resuspended in 60 ml of 50 mm Tris-HCl, pH 8.0, 2 mm EDTA-Na, 0.1% Triton X-100 and sonicated on ice. Inclusion bodies were collected by centrifugation at 12,000 ×g for 20 min at 4 °C, then washed three times by resuspension, and centrifuged in 60 ml of 50 mm Tris-HCl, pH 8.0, 2 mm EDTA, containing 2.5, 0.5, and 0% Triton X-100, respectively. Inclusion bodies were solubilized in 10 ml of 100 mmTris-HCl, pH 8.6, 1 mm EDTA-Na, 6 m guanidine HCl, and 150 mm dithiothreitol and incubated for 4 h at room temperature. VWF A1 proteins were dialyzed and refolded as described previously for recombinant tissue plasminogen activator (24.Kohnert U. Rudolph R. Verheijen J.H. Weening-Verhoeff E.J.D. Stern A. Opitz U. Martin U. Lill H. Prinz H. Lechner M. Kresse G.-B. Buckel P. Fischer S. Protein Eng. 1992; 5: 93-100Crossref PubMed Scopus (134) Google Scholar) with modifications (25.Lu D. Fütterer K. Korolev S. Zheng X. Tan K. Waksman G. Sadler J.D. J. Mol. Biol. 1999; 292: 361-373Crossref PubMed Scopus (93) Google Scholar). The final solution in refolding buffer was stirred for 24 h at room temperature followed by 24 h at 4 °C and then dialyzed against 10 mm Tris-HCl, pH 7.4, 150 mm NaCl (TBS). Refolded VWF A1 domains were purified by adsorption onto a 5-ml HiTrap-heparin column (Amersham Pharmacia Biotech) in TBS, followed by elution with a linear gradient of 150–500 mm NaCl in TBS (60 ml, 2 ml/min). Fractions containing VWF were pooled and applied to a 5-ml column of Thiopropyl-Sepharose 6B (Amersham Pharmacia Biotech) to remove any remaining reduced species. Fractions containing purified VWF fragments were dialyzed against TBS, concentrated by ultrafiltration (Centriprep-10 and Centricon-10; Amicon, Beverly, MA), and stored at −70 °C. Plasmid pDXα containing the full-length GPIbα cDNA sequence (26.Lopez J.A. Leung B. Reynolds C.C. Li C.Q. Fox J.E. J. Biol. Chem. 1992; 267: 12851-12859Abstract Full Text PDF PubMed Google Scholar) was used as a template for PCR amplification with primers 5′-ta ctcgagatg cct ctc ctc ctc ttg ctg ctc ct-3′ (sense XhoI site underlined) and 5′-ta gcggccgccacc tta tcg ccc tca gtg tcc tct tct-3′ (antisense, NotI site underlined). The PCR product was digested with XhoI andNotI and ligated into pDN162, which contains the calmodulin (CaM) gene (kindly provided by Dr. Dario Neri). A fragment containing the GPIbα sequence and the CaM gene was obtained by digestion withXhoI and EcoRI and ligated into pAcSG2 (PharMingen, San Diego, CA), a baculovirus vector, to give plasmid pWIbαwt. The sequence of the insert was confirmed by DNA sequencing. Plasmid pWIbαwt encodes a chimeric protein (GPIbα-CaM) consisting of amino acid residues His1-Val289 of GPIbα, two Ala residues, and calmodulin. Mutations G233V, M239V, or both were introduced into plasmid pWIbαwt using the QuickChange site-directed mutagenesis method (Stratagene) and suitable primers. Plasmids pWIbα233V, pWIbα239V, and pWIbα233V239V encode proteins similar to GPIbα-CaM but with the corresponding mutations (GPIbαG233V-CaM, GPIbαM239V-CaM, and GPIbα233V239V-CaM). Constructs pWIbαwt, pWIbα233V, pWIbα239V, and pWIbα233V239V were cotransfected with BacVector-3000 (Novagen, Madison, WI) into Sf9 cells (Invitrogen, Carlsland, CA), and high titer recombinant baculovirus stocks were prepared by repeated infection. For large scale expression, a 1-liter shaker flask was seeded with 500 ml of High Five cells (Invitrogen) in Insect Express Media (BioWhittaker, Walkersville, MD) at a density of 2 × 106 cells/ml, infected with 10% volume of high titer virus stock, and incubated 4 days at 27 °C with constant stirring at 100 rpm. The medium was centrifuged at 500 × g for 10 min at 4 °C to gently pellet the cells and prevent the release of proteases. The supernatant was further centrifuged at 10,000 ×g for 10 min at 4 °C to remove cell debris. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7; Sigma) coupled to agarose was prepared as described previously (27.Endo T. Tanaka T. Isobe T. Kasai H. Okuyama T. Hidaka H. J. Biol. Chem. 1981; 256: 12485-12489Abstract Full Text PDF PubMed Google Scholar). Medium (400 ml) containing GPIbα-CaM (or variant) was adjusted to 20 mm CaCl2 and applied to a column of W-7-agarose (50 ml) equilibrated with 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm CaCl2. The column was washed with 50 mm Tris, 150 mm NaCl, 1 mm CaCl2, pH 7.4, and GPIbα-CaM was eluted with 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 20 mm EGTA. After dialysis against TBS, the GPIbα-CaM was concentrated by ultrafiltration (Centriprep-30, Amicon, Beverly, MA) and stored at −80 °C. Purified human plasma VWF was kindly provided by Dr. Claudine Mazurier (CRTS, Lillie, France). Residual human serum albumin was removed by gel filtration chromatography on a column (1 × 30 cm) of Superose 12 HR (Amersham Pharmacia Biotech). Botrocetin (28.Fujimura Y. Titani K. Usami Y. Suzuki M. Oyama R. Matsui T. Fukui H. Sugimoto M. Ruggeri Z.M. Biochemistry. 1991; 30: 1957-1964Crossref PubMed Scopus (117) Google Scholar) was purified as described previously. The p39/34 fragment of dispase-digested plasma VWF was purified as described previously (5.Andrews R.K. Gorman J.J. Booth W.J. Corino G.L. Castaldi P.A. Berndt M.C. Biochemistry. 1989; 28: 8326-8336Crossref PubMed Scopus (103) Google Scholar) and corresponds to amino acid residues Leu480/Val481-Gly718. W-7- agarose was resuspended in 1 volume of TBS. W-7-agarose suspension (50-μl total volume, 25-μl beads) was added to 7.8 μg (0.14 nmol) of GPIbα-CaM in 100 μl of TBS containing 20 mm CaCl2 and incubated for 2 h at room temperature. The beads were washed once with TBS, blocked with 50 μl of TBS containing 3% bovine serum albumin (BSA) (Sigma) for 30 min at room temperature, and washed again with TBS. Unbound GPIbα-CaM was quantitated by protein assay (Micro BCA, Pierce) and indicated that 5.5 μg (0.1 nmol) was bound. The beads were resuspended in 330 μl of TBS, giving a concentration of 300 nm GPIbα-CaM in the bead suspension. Immobilized variants of GPIbα-CaM were prepared similarly. Botrocetin (1 mg/ml, 500 μl) was dialyzed against 0.1 m sodium bicarbonate, mixed on ice with 500 μl of 1 mg/ml EZ-link sulfo-NHS-LC-Biotin (Pierce) in water, incubated on ice for 2 h, and dialyzed against TBS. Biotinylated botrocetin (0.5 μg) in 100 μl of TBS was incubated with 25 μl of TBS-washed streptavidin-agarose beads (Pierce) for 30 min at room temperature. The beads were washed with TBS, blocked with 50 μl of TBS containing 3% BSA for 30 min at room temperature, washed again with TBS, and resuspended in a total volume of 330 μl of TBS. All of the biotinylated botrocetin was bound, and the concentration of botrocetin in the bead suspension was 56 nm. VWF fragments were radiolabeled using IODO-GEN (29.Fraker P.J. Speck Jr., J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3626) Google Scholar) (Pierce) to a specific radioactivity of 3–5 × 109cpm/μmol. 125I-Labeled A1 or A1(R545A) at varying concentrations was incubated at room temperature with wild-type or mutant GPIbα-CaM-agarose (final concentration 20 nmGPIbα-CaM for assays in the absence of botrocetin or 3 nmfor assays in the presence of botrocetin) and 1.5% BSA in a total volume 100 μl of TBS with or without 1 μm botrocetin. After 1 h, the reaction mixture was layered over 300 μl of 20% sucrose in a 500-μl microcentrifuge tube and centrifuged for 5 min at 12,000 rpm to separate bound and free ligand. Radioactivity associated with the bead pellet was quantitated by γ radiation counting. Nonspecific binding was estimated in a mixture containing the same reagents and either 400 μm unlabeled VWF A1 or 60 μm unlabeled VWF A1(R545A). Specific binding was calculated by subtracting nonspecific binding from total binding. Dissociation constants and stoichiometry of binding were determined by Scatchard analysis. The time course of binding was investigated with an identical assay design except that the time of incubation was varied. To obtain rate constants for the reaction, the time course data were fitted to a bimolecular binding mechanism (A + B =AB) with the program KINSIM (30.Barshop B.A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (670) Google Scholar), and the dissociation constants were determined by equilibrium binding and Scatchard analysis as described above. 125I-Labeled VWF A1 or125I-labeled VWF(R545A) at varying concentrations was added to 28 nm biotinylated botrocetin-agarose in 100 μl of TBS containing 1.5% BSA. After 1 h at room temperature, the mixture was centrifuged through 20% sucrose as described above, and the radioactivity bound to streptavidin-agarose was determined by γ radiation counting. Nonspecific binding was estimated in similar reactions containing 1 μm unlabeled VWF A1 or VWF A1(R545A). 125I-Labeled VWF A1 at varying concentrations was incubated at room temperature with formalin-fixed platelets (1 × 108/ml) and 3% BSA in a total volume of 100 μl of TBS. After 1 h, the reaction mixture was layered over 300 μl of 20% sucrose in a 500-μl microcentrifuge tube and centrifuged for 5 min at 12,000 rpm. Radioactivity associated with the platelet pellet was quantitated by γ radiation counting. Nonspecific binding was determined similarly but in the presence of 400 μm unlabeled VWF A1. Specific binding was calculated by subtracting nonspecific binding from total binding. For competitive inhibition assays of the VWF-GPIbα interaction, a fixed concentration of 2.5 μm or 10 nm125I-labeled VWF A1 was used in the absence or presence, respectively, of 1 μm botrocetin. For competitive inhibition assays of VWF-botrocetin interaction, a fixed concentration of 25 nm125I-labeled VWF A1 was used. Nonspecific binding was estimated with the addition of a 100-fold excess of unlabeled A1 fragment and was always <15% of total binding; the corresponding values were subtracted from all data points. Binding was expressed as a percentage of maximal specific binding (100%) and was used to calculate the concentration of VWF fragments required to inhibit 50% of specific 125I-labeled A1 binding to recombinant GPIbα-CaM fusion protein (EC50) by fitting to the equation B = EC50/(EC50 + [I]). Microtiter plates were coated overnight at 4 °C with 10 μg/ml VWF in TBS (50 μl/well), blocked with 3% BSA in TBS for 1 h at room temperature, and washed three times with phosphate-buffered saline, pH 7.4, containing 0.1% Tween 20 (PBST). NMC-4 (0.2 μg/ml) and varying concentrations (0.6–300 nm) of VWF A1, VWF A1(R545A), dispase-digested fragment, or VWF were added to each well. After incubation for 1 h at room temperature, plates were washed three times with PBST and incubated with anti-mouse IgG horseradish peroxidase-conjugated sheep F(ab′)2 fragment (Amersham Pharmacia Biotech) for 1 h. After washing three times with PBST, 50 μl of 0.8 mg/mlo-phenylenediamine was added to each well. The reaction was stopped after 3 min with 3 m H2SO4, and absorbance was measured at 490 nm. Nonspecific binding was estimated in the presence of 0.5 mg/ml VWF and was 5% of total binding. The corresponding values were subtracted from all data points. NMC-4 binding was expressed as a percentage of maximal specific binding, and the concentration of each competitive ligand required to inhibit 50% of specific NMC-4 binding (EC50) was determined. Amino acid sequencing was performed by the Protein Chemistry Laboratory of the University of Texas Medical Branch (Galveston), with an Applied Biosystems 494/HT PROCISE Sequencing System and model 477A pulsed-liquid sequencer, with a model 120A phenylthiohydantoin amino acid analyzer controlled by a 610 Data Analysis System. Electrospray ionization mass spectrometry was performed with a Finnegan LCQ spectrometer (Finnegan, San Jose, CA). SDS-15% polyacrylamide gel electrophoresis (PAGE) was performed as described (31.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Plasma VWF is multivalent, and this feature complicates the study of reactions between individual binding sites on VWF and platelet GPIbα. To address this limitation, monomeric recombinant VWF A1 proteins were expressed in E. coli and refolded by a method developed previously for the renaturation of tissue-type plasminogen activator (24.Kohnert U. Rudolph R. Verheijen J.H. Weening-Verhoeff E.J.D. Stern A. Opitz U. Martin U. Lill H. Prinz H. Lechner M. Kresse G.-B. Buckel P. Fischer S. Protein Eng. 1992; 5: 93-100Crossref PubMed Scopus (134) Google Scholar). Both wild-type VWF A1 and the high affinity variant A1(R545A) (23.Matsushita T. Sadler J.E. J. Biol. Chem. 1995; 270: 13406-13414Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) were prepared to determine the effect of the mutation on the affinity and the rate constants for binding. The products were purified by chromatography on heparin-agarose and thiol-agarose. Both proteins appeared to be homogeneous upon gel electrophoresis (Fig. 1 A), with apparent masses of 23.5 kDa that increased to 24 kDa upon reduction. The reduced and oxidized forms were distinguished easily by analytical reverse-phase HPLC (Fig. 1 C), and the final preparations were free of reduced precursor proteins. Both proteins were soluble at ≥20 mg/ml in TBS, pH 7.4, and gave single peaks upon gel filtration chromatography that are consistent with monomers (Fig.1 B). Amino acid sequencing of VWF A1 gave the expected N-terminal sequence Met-Glu-Asp-Ile-Ser-Glu-Pro-Pro-Leu-His. By electrospray ionization mass spectrometry, VWF A1 had a molecular mass of 24,004 Da compared with the calculated mass of 24,012.8 Da. The concentration of each fragment was determined by amino acid analysis after acid hydrolysis, allowing the determination of an extinction coefficient (280 nm) of 18,084m−1cm−1. This experimental value is similar to the calculated extinction coefficient of 16,180m−1cm−1 based on amino acid composition (32.Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar). To facilitate both purification and binding studies, chimeric proteins were designed containing the VWF binding domain of GPIbα fused to the N terminus of calmodulin. Calmodulin binds tightly to the immobilized phenothiazine derivative W-7 in the presence of calcium ions (33.Moore P.B. Dedman J.R. J. Biol. Chem. 1982; 257: 9663-9667Abstract Full Text PDF PubMed Google Scholar). Calmodulin retains this binding activity in the construct GPIbα-CaM and does not dissociate significantly from W-7-agarose during use. Mutations that increase the affinity of binding to VWF and cause platelet-type pseudo-VWD were introduced into GPIbα-CaM. All of the GPIbα-CaM proteins gave a single major band on gel electrophoresis, with an apparent mass of 55 kDa (Fig.2).Figure 2Gel electrophoresis of GPIbα-CaM proteins. Samples were analyzed by SDS-gel electrophoresis on 4–15% polyacrylamide gradient gels under non-reducing (NR) and reducing (R) conditions. The molecular masses in kDa of standard proteins (lane 1) are indicated at the left. Samples include GPIbα-CaM (lane 2), GPIbα(G233V)-CaM (lane 3), GPIbα(M239V)-CaM (lane 4), and GPIbα(G233V/M239V)-CaM (lane 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The binding of VWF A1 to GPIbα-CaM proteins reached equilibrium within 15 min and was reversed completely within 10 min by the addition of excess unlabeled ligand (data not shown). Radiolabeled VWF A1 fragments bound saturably to the various immobilized GPIbα-CaM proteins (Fig.3), and Scatchard analysis was consistent with binding to a single class of sites at a stoichiometry of 1:1. Introduction of the mutation R545A into VWF A1 increased the affinity of binding to GPIbα-CaM 20-fold (TableI); the value for K ddecreased from 3.3 μm to 160 nm. Mutations of GPIbα that cause platelet-type pseudo-VWD produced a smaller 3-fold (G233V) or 6-fold (M239V) increase in the affinity of VWF A1 binding. Combining the two GPIbα mutations in a single construct did not increase the affinity of binding further. The value forK d was 0.6 μm for the interaction of wild-type VWF A1 with either GPIbα(M239V)-CaM or GPIbα(G233V/M239V)-CaM (Table I). Also, A1(R545A) bound with similar affinity to wild-type GPIbα-CaM and to GPIbα(M239V)-CaM. Thus, the effects of these high affinity mutations were not additive.Table IDissociation constants for binding of recombinant VWF A1 and GPIbαVWF A1GPIbα-CaMPlatelets (2)wt (5)G233V (3)M239V (2)G233V/M239V (3)Kd (μm)wt3.3 ± 0.51.0 ± 0.10.63 ± 0.10.59 ± 0.44.7aThis value is based on a single experiment.R545A0.16 ± 0.030.18 ± 0.010.20 ± 0.060.14 ± 0.010.11 ± 0.01Binding of 125I-labeled A1 domains to GPIbα-CaM proteins immobilized on W-7-agarose or to formalin-fixed platelets was determined by Scatchard analysis as described under “Experimental Procedures.” Representative data are shown in Fig. 3. Values for dissociation constants are expressed as the mean ± S.D. for the number of independent determinations indicated in parentheses.a This value is based on a single experiment. Open table in a new tab Binding of 125I-labeled A1 domains to GPIbα-CaM proteins immobilized on W-7-agarose or to formalin-fixed platelets was determined by Scatchard analysis as described under “Experimental Procedures.” Representative data are shown in Fig. 3. Values for dissociation constants are expressed as the mean ± S.D. for the number of independent determinations indicated in parentheses. Recombinant A1 and A1(R545A) bound to formalin-fixed pla" @default.
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• W2028983608 title "Interaction of von Willebrand Factor Domain A1 with Platelet Glycoprotein Ibα-(1–289)" @default.
• W2028983608 cites W1427202838 @default.
• W2028983608 cites W1494526055 @default.
• W2028983608 cites W1503766585 @default.
• W2028983608 cites W1531681292 @default.
• W2028983608 cites W1534508763 @default.
• W2028983608 cites W1535032041 @default.
• W2028983608 cites W1538504702 @default.
• W2028983608 cites W1548784534 @default.
• W2028983608 cites W1571880088 @default.
• W2028983608 cites W1598021300 @default.
• W2028983608 cites W1644153661 @default.
• W2028983608 cites W164651796 @default.
• W2028983608 cites W1972684857 @default.
• W2028983608 cites W1989315829 @default.
• W2028983608 cites W2005261732 @default.
• W2028983608 cites W2029708892 @default.
• W2028983608 cites W2030665169 @default.
• W2028983608 cites W2037690358 @default.
• W2028983608 cites W2049504117 @default.
• W2028983608 cites W2050141829 @default.
• W2028983608 cites W2051446149 @default.
• W2028983608 cites W2051857903 @default.
• W2028983608 cites W2052192720 @default.
• W2028983608 cites W2058628357 @default.
• W2028983608 cites W2075013504 @default.
• W2028983608 cites W2075502795 @default.
• W2028983608 cites W2080580789 @default.
• W2028983608 cites W2084188731 @default.
• W2028983608 cites W2090664620 @default.
• W2028983608 cites W2100837269 @default.
• W2028983608 cites W2104017463 @default.
• W2028983608 cites W2108156759 @default.
• W2028983608 cites W2110007918 @default.
• W2028983608 cites W2112104641 @default.
• W2028983608 cites W2121859722 @default.
• W2028983608 cites W2131027010 @default.
• W2028983608 cites W2133326826 @default.
• W2028983608 cites W2139340234 @default.
• W2028983608 cites W2262897385 @default.
• W2028983608 cites W2270373849 @default.
• W2028983608 cites W2270564137 @default.
• W2028983608 cites W2277208912 @default.
• W2028983608 cites W2414530566 @default.
• W2028983608 cites W2417239351 @default.
• W2028983608 cites W24454078 @default.
• W2028983608 cites W2794411860 @default.
• W2028983608 cites W3113274326 @default.
• W2028983608 cites W4245562509 @default.
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