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• W2110007918 abstract "We have used recombinant von Willebrand factor (vWF) fragments to investigate the properties regulating A1 domain interaction with platelet glycoprotein (GP) Ibα. One fragment,rvWF508–704, represented the main portion of domain A1 (mature subunit residues 497–716) within the Cys509-Cys695 disulfide loop. The other,rvWF445–733, included the carboxyl-terminal region of domain D3, preceding A1, and corresponded to the proteolytic fragment originally identified as the GP Ibα-binding site (residues 449–728). Conformational changes were induced by reduction and alkylation of the Cys509-Cys695 bond and/or exposure to acidic pH. The cyclic rvWF445–733fragment exhibited the function of native vWF A1 domain. When immobilized onto a surface, it tethered platelets at shear rates up to 6,300 s−1 mediating low velocity translocation but not stable attachment; in solution, it exhibited limited interaction with GP Ibα. In contrast, fragments with perturbed conformation could not tether platelets at high shear rates but promoted stable adhesion at lower shear and bound tightly to GP Ibα. Only in the presence of the exogenous modulator, botrocetin, did cyclicrvWF445–733 mediate irreversible adhesion. Thus, conformational transitions in the vWF A1 domain may influence differentially the efficiency of bond formation with GP Ibα and the stability of binding. We have used recombinant von Willebrand factor (vWF) fragments to investigate the properties regulating A1 domain interaction with platelet glycoprotein (GP) Ibα. One fragment,rvWF508–704, represented the main portion of domain A1 (mature subunit residues 497–716) within the Cys509-Cys695 disulfide loop. The other,rvWF445–733, included the carboxyl-terminal region of domain D3, preceding A1, and corresponded to the proteolytic fragment originally identified as the GP Ibα-binding site (residues 449–728). Conformational changes were induced by reduction and alkylation of the Cys509-Cys695 bond and/or exposure to acidic pH. The cyclic rvWF445–733fragment exhibited the function of native vWF A1 domain. When immobilized onto a surface, it tethered platelets at shear rates up to 6,300 s−1 mediating low velocity translocation but not stable attachment; in solution, it exhibited limited interaction with GP Ibα. In contrast, fragments with perturbed conformation could not tether platelets at high shear rates but promoted stable adhesion at lower shear and bound tightly to GP Ibα. Only in the presence of the exogenous modulator, botrocetin, did cyclicrvWF445–733 mediate irreversible adhesion. Thus, conformational transitions in the vWF A1 domain may influence differentially the efficiency of bond formation with GP Ibα and the stability of binding. The A1 domain of von Willebrand factor (vWF) 1The abbreviations used are: vWF, von Willebrand factor; GP, platelet membrane glycoprotein; PPACK, d-phenyl alanyl-l-prolyl-l-arginine chloromethyl ketone dihydrochloride. immobilized onto exposed surfaces at sites of vascular injury initiates platelet adhesion and thrombus formation by interacting with the glycoprotein (GP) Ibα receptor (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 2Ruggeri Z.M. J. Clin. Invest. 1997; 99: 559-564Crossref PubMed Scopus (192) Google Scholar, 3Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). This function is absolutely required for hemostasis in vessels such as arterioles or arterial capillaries, where rapid blood flow creates high shear rates (4Tangelder G.J. Slaaf D.W. Arts T. Reneman R.S. Am. J. Physiol. 1988; 254: H1059-H1064PubMed Google Scholar, 5Goldsmith H.L. Turitto V.T. Thromb. Haemostasis. 1986; 55: 415-435Crossref PubMed Scopus (461) Google Scholar), and may also precipitate thrombosis in larger arteries, for example the coronary arteries of the heart, particularly at sites of stenosis caused by atherosclerotic lesions (6Back C.H. Radbill J.R. Crawford D.W. J. Biomech. 1977; 10: 339-353Crossref PubMed Scopus (110) Google Scholar, 7Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Crossref PubMed Scopus (2923) Google Scholar, 8Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 310-318Crossref PubMed Scopus (1414) Google Scholar). The efficient interaction between GP Ibα and immobilized vWF is in apparent contrast to the lack of measurable binding of soluble plasma vWF (9Ruggeri Z.M. Ware J. FASEB J. 1993; 7: 308-316Crossref PubMed Scopus (273) Google Scholar). This observation has led to the generally accepted concept that conformational changes induced by surface adsorption regulate A1 domain function. Such an effect is thought to be caused in vivo by binding to collagen (10Baumgartner H.R. Tschopp T.B. Weiss H.J. Thromb. Haemostasis. 1977; 37: 17-28Crossref PubMed Google Scholar, 11Pareti F.I. Niiya K. McPherson J.M. Ruggeri Z.M. J. Biol. Chem. 1987; 262: 13835-13841Abstract Full Text PDF PubMed Google Scholar, 12Cruz 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 structures (13de 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, 14Katayama 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), whereas in vitro it may be mimicked by interaction with modulators such as ristocetin (15Howard M.A. Firkin B.G. Thromb. Haemostasis. 1971; 26: 362-369Crossref Scopus (386) Google Scholar, 16Scott J.P. Montgomery R.R. Retzinger G.S. J. Biol. Chem. 1991; 266: 8149-8155Abstract Full Text PDF PubMed Google Scholar) or botrocetin (17Read M.S. Shermer R.W. Brinkhous K.M. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4514-4518Crossref PubMed Scopus (125) Google Scholar, 18Read M.S. Smith S.V. Lamb M.A. Brinkhous K.M. Blood. 1989; 74: 1031-1035Crossref PubMed Google Scholar, 19Sugimoto M. Mohri H. McClintock R.A. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 18172-18178Abstract Full Text PDF PubMed Google Scholar). Indeed, surface-bound vWF may change shape under the influence of high shear stress, appearing as an elongated filament (20Siediecki C.A. Lestini B.J. Kottke-Marchant K. Eppell S.J. Wilson D.L. Marchant R.E. Blood. 1996; 88: 2939-2950Crossref PubMed Google Scholar) rather than the loosely coiled structure predominantly seen under static conditions (21Fowler W.E. Fretto L.J. Hamilton K.K. Erickson H.P. McKee P.A. J. Clin. Invest. 1985; 76: 1491-1500Crossref PubMed Scopus (149) Google Scholar). Extended multimers expose repeating functional sites, thus supporting multiple and more efficient adhesive interactions. Whether the change in molecular shape parallels specific conformational transitions in the A1 domain is unknown at present. Nevertheless, physicochemical modifications of the isolated A1 domain in solution can result in heightened interaction with GP Ibα (22Miyata S. Goto S. Federici A.B. Ware J. Ruggeri Z.M. J. Biol. Chem. 1996; 271: 9046-9053Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), in agreement with the notion that the native conformation is functionally unfavorable for receptor recognition but can be positively modulated. Consequently, it is generally assumed that the mechanisms leading to soluble vWF binding to GP Ibα reflect conditions that endow the A1 domain with the ability to initiate platelet adhesion. The physiologic characteristics of the interaction between surface-immobilized vWF A1 domain and GP Ibα have been well established under relevant flow conditions, with the demonstration that the process supports efficient tethering even at high shear rates. Platelets kept in contact with the surface by this ligand receptor pairing, however, are not irreversibly adherent; rather, they translocate constantly in the direction of flow albeit at a markedly reduced velocity relative to freely flowing platelets (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). This is sufficient to allow the formation of additional bonds, mediated by receptors other than GP Ibα, resulting in irreversible attachment and subsequent thrombus formation (3Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). We have now studied the GP Ibα-binding function of isolated recombinant A1 domain fragments of distinct conformation to evaluate how adhesive properties under flow correlate with the ability to bind to the receptor in solution. We found that an immobilized fragment with refolded conformation supported platelet tethering at high shear rates as efficiently as native vWF but had the lowest GP Ibα binding capacity in solution. In contrast, disruption of the tertiary structure (22Miyata S. Goto S. Federici A.B. Ware J. Ruggeri Z.M. J. Biol. Chem. 1996; 271: 9046-9053Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) resulted in a fragment that exhibited markedly enhanced binding to the receptor in solution, as judged by the ability to block its function, but defective support of platelet adhesion when immobilized onto a surface, particularly at high shear rates. Moreover, platelets became irreversibly attached to the fragment with disrupted conformation, rather than translocating through transient interactions as seen with native refolded fragments and multimeric vWF. After forming a complex with botrocetin, however, even A1 domain fragments with native conformation supported irreversible adhesion. Our findings indicate that the affinity regulation of vWF A1 domain binding to GP Ibα is a complex event since distinct and, in most instances, mutually exclusive structural characteristics control the ability to establish stable bonds or to initiate platelet tethering opposing elevated shear forces. Blood was collected from healthy and medication-free donors into polypropylene syringes containing as anticoagulant the α-thrombin inhibitord-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone dihydrochloride (PPACK) at the final concentration of 50 μm. All human subjects participating in these studies were aware of the experimental nature of the research and gave their informed consent in accordance with the Declaration of Helsinki. In order to eliminate potential effects of vWF and/or other plasma proteins in the experiments to be performed, washed blood cells suspended in perfusion buffer were prepared as follows. After adding the ADP scavenger apyrase and prostaglandin E1 to prevent platelet activation (10 units/ml and 10 μm, respectively, final concentration), blood was centrifuged at 2200 ×g for 15 min at room temperature (22–25 °C), and the resultant supernatant plasma was removed from the sedimented cells, including platelets and leukocytes on top of the erythrocyte cushion. After adding an equivalent volume of divalent cation-free Hepes/Tyrode buffer (10 mm Hepes, 140 mm NaCl, 2.7 mm KCl, 0.4 mm NaH2PO4, 10 mm NaHCO3, and 5 mm dextrose), pH 6.5, and mixing gently, the cell suspension was centrifuged again and the supernatant fluid was removed. This procedure was repeated three additional times, and after the final centrifugation the cells were suspended in divalent cation-free Hepes/Tyrode buffer, pH 7.4, containing 50 mg/ml bovine serum albumin. Final cell counts were within normal blood limits. In some experiments, platelet-depleted reconstituted blood was prepared by centrifuging the final cell suspension at 150 × g for 15 min, removing the resulting platelet-rich supernatant fluid, and replacing it with an equivalent volume of Hepes/Tyrode buffer, pH 7.4, containing 50 mg/ml bovine serum albumin. After counting the platelet number in both the whole cell suspension and the platelet-rich suspension, appropriate volumes of the latter were added into the former to obtain the target platelet count in the reconstituted blood, at the same time maintaining a normal hematocrit. In some experiments, whole blood containing PPACK as anticoagulant and prostaglandin E1 to prevent platelet activation (see above) was used instead of reconstituted blood. Native multimeric vWF was purified as previously reported (23Ruggeri Z.M. De Marco L. Gatti L. Bader R. Montgomery R.R. J. Clin. Invest. 1983; 72: 1-12Crossref PubMed Scopus (287) Google Scholar). Two recombinant polypeptides coding for residues 508–704 and 445–733 of the mature vWF subunit, designatedrvWF508–704 andrvWF445–733, respectively, were expressed in host Escherichia coli BL21-DE3 using plasmids containing the T7 RNA polymerase promoter (24Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4842) Google Scholar, 25Rosenberg A.H. Lade B.N. Chui D.S. Lin S.W. Dunn J.J. Studier F.W. Gene (Amst.). 1987; 56: 125-135Crossref PubMed Scopus (1044) Google Scholar) and induction with isopropyl-β-d-thiogalactopyranoside, as described previously in detail (22Miyata S. Goto S. Federici A.B. Ware J. Ruggeri Z.M. J. Biol. Chem. 1996; 271: 9046-9053Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 26Sugimoto M. Dent J. McClintock R.S. Ware J. Ruggeri Z.M. J. Biol. Chem. 1993; 268: 12185-12192Abstract Full Text PDF PubMed Google Scholar, 27Prior C.P. Chu V. Cambou B. Dent J.A. Ebert B. Gore R. Holt J. Irish T. Lee J. Mitschelen J. McClintock R.A. Searfoss G. Ricca G.A. Tarr C. Weber D. Ware J.L. Ruggeri Z.M. Hrinda M. BioTechnology. 1993; 11: 709-713Crossref PubMed Scopus (14) Google Scholar). To prevent formation of random aggregates during purification, five codons for Cys residues at positions 459, 462, 464, 471, and 474 in thervWF445–733 construct were replaced with Gly codons by site-directed mutagenesis. Expressed fragments were purified by reverse-phase high pressure liquid chromatography and subjected either to reduction and alkylation (S-carboxyamidomethylation) or oxidization of the two Cys residues at positions 509 and 695, according to previously described procedures (22Miyata S. Goto S. Federici A.B. Ware J. Ruggeri Z.M. J. Biol. Chem. 1996; 271: 9046-9053Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Oxidization created the intramolecular disulfide bond that exists in native plasma vWF (28Marti T. Roesselet S. Titani K. Walsh K.A. Biochemistry. 1987; 26: 8099-8109Crossref PubMed Scopus (204) Google Scholar). Purified recombinant fragments were dialyzed against 2 mm acetic acid titrated to pH 3.5 with HCl and stored at −70 °C. As reported in detail elsewhere (22Miyata S. Goto S. Federici A.B. Ware J. Ruggeri Z.M. J. Biol. Chem. 1996; 271: 9046-9053Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), refolding of the oxidized recombinant fragments from the denatured state following exposure to acidic pH was achieved by slow dialysis with incremental pH increase in steps of 0.5 units each up to a final value of 7.0 for rvWF508–704 or 5.0 forrvWF445–733. This was obtained by dialyzing the samples at 4 °C against 2 mm acetic acid to which an appropriate amount of ammonium hydroxide was added every 8 h. Limiting the pH for refoldingrvWF445–733 was necessary because this fragment tended to form aggregates at pH values above 5.0. Reduced and alkylated rvWF508–704 was not refolded slowly, rather it was rapidly returned to neutral pH just before use by direct mixing with an appropriate buffer. Protein concentration was determined with the micro BCA assay (Pierce) according to the instructions of the manufacturer. This modulator of vWF A1 domain binding to platelet GP Ibα was purified from the venom of the snake Bothrops jararaca as previously reported in detail (29Fujimura 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, 30Usami Y. Fujimura Y. Suzuki M. Ozeki Y. Nishio K. Fukui H. Titani K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 928-932Crossref PubMed Scopus (123) Google Scholar). It was stored in Hepes buffer (10 mm Hepes, 140 mm NaCl), pH 7.4, at −70 °C until used. The anti-vWF, anti-GP Ibα, and anti-αIIbβ3 (GP IIb-IIIa) monoclonal antibodies used in these studies have been characterized in previous publications. Pooled M13 monoclonal antibodies (31Zimmerman T.S. Dent J.A. Ruggeri Z.M. Nannini L.H. J. Clin. Invest. 1986; 77: 947-951Crossref PubMed Scopus (182) Google Scholar, 32Dent J.A. Berkowitz S.D. Ware J. Kasper C.K. Ruggeri Z.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6306-6310Crossref PubMed Scopus (312) Google Scholar) recognize epitopes in the M13 cyanogen bromide fragment of vWF comprising residues 631–710 of the mature subunit (33Titani K. Kumar S. Takio K. Ericsson L.H. Wade R.D. Ashida K. Walsh K.A. Chopek M.W. Sadler J.E. Fujikawa K. Biochemistry. 1986; 25: 3171-3184Crossref PubMed Scopus (329) Google Scholar). NMC-4 (34Fujimura Y. Usami Y. Titani K. Niinomi K. Nishio K. Takase T. Yoshioka A. Fukui H. Blood. 1991; 77: 113-120Crossref PubMed Google Scholar) recognizes a defined epitope in the A1 domain of vWF, as demonstrated by atomic structure resolution (35Celikel R. Varughese K.I. Madhusudan A. Yoshioka A. Ware J. Ruggeri Z.M. Nat. Struct. Biol. 1998; 5: 189-194Crossref PubMed Scopus (94) Google Scholar). LJ-Ib1 reacts with the amino-terminal 45-kDa domain of GP Ibα (36Handa M. Titani K. Holland L.Z. Roberts J.R. Ruggeri Z.M. J. Biol. Chem. 1986; 261: 12579-12585Abstract Full Text PDF PubMed Google Scholar, 37Titani K. Takio K. Handa M. Ruggeri Z.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5610-5614Crossref PubMed Scopus (103) Google Scholar) and is a competitive inhibitor of vWF binding to this platelet receptor. LJ-CP8 is a complex specific antibody against the integrin αIIbβ3 and completely blocks binding of all ligands to this receptor (38Niiya K. Hodson E. Bader R. Byers-Ward V. Koziol J.A. Plow E.F. Ruggeri Z.M. Blood. 1987; 70: 475-483Crossref PubMed Google Scholar, 39Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar). All monoclonal antibodies were mouse IgG1 and were purified by protein A-Sepharose (Amersham Pharmacia Biotech) chromatography according to published procedures (40Ey P.L. Prowse S.J. Jenkin C.R. Immunochemistry. 1978; 15: 429-436Crossref PubMed Scopus (1999) Google Scholar). The inhibitory effect of specific monoclonal antibodies on platelet interaction with immobilized native vWF or recombinant fragments was assessed by incubating purified IgG with reconstituted blood at room temperature for 20 min before perfusion through the chamber. This assay, an indirect measurement of the binding to platelet GP Ibα of recombinant vWF fragments containing the A1 domain, has been described in detail in a previous publication (22Miyata S. Goto S. Federici A.B. Ware J. Ruggeri Z.M. J. Biol. Chem. 1996; 271: 9046-9053Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Platelet-rich plasma was prepared by centrifugation of blood containing PPACK at 150 × gfor 15 min at room temperature and then diluted in 10 mmHepes buffer, pH 7.4, to give a final platelet count of 1 × 108/ml. In other experiments, washed cells were prepared as described above, and a plasma-free suspension of platelets was obtained by centrifuging the final cell suspension at 150 × gfor 15 min yielding a platelet-rich supernatant fluid that could be separated from the sedimented red and white blood cells. A constant volume (equal to or less than 12.5 μl) of various concentrations of each recombinant vWF fragment was added to the platelet suspension, followed by 10 μg/ml 125I-labeled LJ-Ib1 (a concentration of antibody resulting in half-maximum binding to platelets) diluted in 10 mm Hepes buffer, pH 7.4. When indicated, botrocetin was also added to a final concentration of 5 μg/ml (0.17 μm); in this case, washed platelets (41Goto S. Salomon D.R. Ikeda Y. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 23352-23361Crossref PubMed Scopus (260) Google Scholar) were used rather than platelet-rich plasma. The final volume of each experimental mixture was 125 μl, and all indicated concentrations were final. After incubation at room temperature for 30 min, platelets were separated by centrifugation though a layer of 20% sucrose, and bound radioactivity was measured in a γ-scintillation counter. Nonspecific binding was estimated by adding a 100-fold excess of nonlabeled LJ-Ib1, and the corresponding value (always less than 10% of the total counts added) was subtracted from all data points. Binding was expressed as percentage of that measured in a control mixture containing, instead of a recombinant vWF fragment, 12.5 μl of 10 mm ammonium acetate with the same pH. Recombinant vWF fragments and native vWF were diluted to a final concentration of 100–130 μg/ml with 2 mmammonium acetate, pH 7.0. A glass coverslip (number 1, 24 × 50 mm; Corning) was coated evenly with 200 μl of protein solution and placed in a humid environment at room temperature for 1 h. In some experiments, larger volumes (300–600 μl) of solutions with lower protein concentration were used. Just before assembly of the flow chamber, unbound protein was removed by rinsing the surface of the coverslip with 0.04 m phosphate buffer, pH 7.4, containing 0.15 m NaCl (PBS, phosphate-buffered saline composed of 0.04 m monosodium phosphate and 0.04 m disodium phosphate with 0.15 m NaCl, pH 7.4). In some cases, after coating with the desired protein, the glass surface was saturated with a solution of 50 mg/ml bovine serum albumin in PBS for 1 h at room temperature before use. To calculate the amount of protein adsorbed onto the glass surface, the supernatant containing unbound protein was carefully recovered after coating as were three successive 200-μl aliquots of PBS used to rinse the coverslip (washing solution). The amount of adsorbed protein was calculated as the difference between the total amount added for coating and that recovered in the coating plus washing solutions. Platelet interaction with immobilized recombinant fragments or native vWF under flow conditions was observed in real-time by means of epifluorescence videomicroscopy in a modified Hele-Shaw flow chamber (42Usami S. Chen H.H. Zhao Y. Chien S. Skalak R. Ann. Biomed. Eng. 1993; 21: 77-83Crossref PubMed Scopus (256) Google Scholar), as described previously (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 3Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). The bottom of the chamber was formed by the protein-coated surface of a glass coverslip, and a flow path height of 254 μm was determined by a silicon rubber gasket designed with a shape that resulted in a linear variable wall shear rate from 1500 s−1 at the inlet to 50 s−1 near the outlet when the flow rate was maintained at 2 ml/min. The entire flow path of the chamber, mounted on the stage of an inverted epifluorescence microscope (Axiovert 135 m, Carl Zeiss Inc.), was kept at 37 °C with a thermostatic air bath. Platelets were visualized by adding mepacrine (quinacrine dihydrochloride; 10 μm final concentration), a fluorescent dye that becomes concentrated in the dense granules and has no effects on function at the concentration used (43Dise C.A. Burch J.W. Goodman D.B.P. J. Biol. Chem. 1982; 257: 4701-4704Abstract Full Text PDF PubMed Google Scholar). When native vWF was immobilized on the surface, reconstituted blood also contained the anti-αIIbβ3monoclonal antibody, LJ-CP8, at the final concentration of 50 μg/ml in order to eliminate any irreversible interaction between platelets and the RGD sequence in vWF (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 3Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). In some experiments, recombinant vWF fragments were mixed with the reconstituted blood to evaluate their capacity to inhibit, when in solution, the interaction of platelets with immobilized native vWF or recombinant fragments adsorbed onto the surface. Reconstituted blood, considered to have the same viscosity of 4 centipoise as native blood, was aspirated through the chamber, initially filled with PBS, by a syringe pump (Harvard Apparatus Inc.) for the desired time, and all experiments were continuously recorded on videotape using a video cassette recorder (VCR, Magnavox). The number of individual platelets interacting at any given time with immobilized native vWF or recombinant fragments was measured on images obtained at different positions in the flow path of the chamber, corresponding to selected wall shear rates. Each image corresponded to a single frame from the real time (30 frames/s) videotape recording, digitized and processed by computer analysis using a Sony 9500 VCR, a Matrox Image LC frame grabber, and the MetaMorph software package (Universal Imaging Corp.). The motion of platelets not irreversibly attached to the surface was analyzed as previously reported (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 3Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). To evaluate the number of platelets displaced from the point of initial interaction, a series of images at 1/3–1/15-s intervals from real time recording was digitized and binarized after application of a threshold to distinguish platelets from background. Movement was defined as spatial displacement on the surface greater than one platelet diameter. The first two consecutive frames in the series were superimposed by the logical AND function, so that the resultant image represented only overlapping areas of an individual platelet at two different times. This computed image was then superimposed to next frame in the time series, and the same logical AND function was applied. The process was repeated for a number of frames corresponding to a preselected time interval, or until the area of overlap was equal to 0. When the latter occurred, the platelet had moved by a distance greater than its diameter; if this did not occur, the platelet was considered firmly attached during the period of observation. To measure the velocity of translocation onto the surface, the centroid of individual platelets on each image was assigned a set of x and y coordinate values. Centroid displacement was then followed as a function of time, typically on a total of 60 frames for each analyzed position in the chamber corresponding to 4–20 s. The rate of frame acquisition from real time recording was selected in relation to the speed of translocation. The velocity of individual platelets was calculated as the distance traveled by the centroid divided by the time interval (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar,3Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). Preliminary experiments established that platelet adhesion to immobilized native vWF or recombinant fragments was maximal after coating glass with a solution at the concentration of 100 μg/ml for 1 h at room temperature (22–25 °C). The corresponding amount of bound protein was in the range of 8.7–15.2 μg per glass slide for all recombinant fragments as well as purified native vWF (Table I). The number of platelets interacting with the coated surface was directly correlated to the amount of adsorbed protein, but considerably less native multimeric vWF than recombinant fragment was required for maximum effect (Fig.1).Table IAmount of native multimeric vWF and recombinant vWF fragments absorbed onto the surface of glass coverslipsProteinCoating solutionSupernatantWashing solutionDensity of proteinVolumeConcentrationVolumeConcentrationVolumeConcentrationμlμg/mlμlμg/mlμlμg/mlμg/coverslip1st experimentNative vWF2001281501065101.78.8rvWF445–733cyclic200134155515706.415.2rvWF508–704cyclic200121145725300.813.5rvWF508–704R/A200123163755751.811.32nd experimentNative vWF2001281551005802.38.7rvWF445–733cyclic200134155555808.713.3rvWF508–704cyclic200121150735700.513.1rvWF508–704R/A200123162735731.911.7The amount of protein adsorbed onto the surface of glass coverslips was determined by subtracting from the total initial amount in the coating solution the sum of that recovered in the supernatant solution after coating and in all washing solutions. Open table in a new tab The amount of protein adsorbed onto the surface of glass coverslips was determined by subtracting from the total initial amount in the coating solution the sum of that recovered in the supernatant solution after coating and in all washing solutions. Platelet interaction with immobilized recombinant fragments as well as native multimeric vWF was evaluated in real time at wall shear rates between 50 and 6000 s−1. Fragments of different conformation exhibited considerable variation in their ability to support platelet attachment (Fig.2). ImmobilizedrvWF445–733, refolded after oxidation of the Cys509-Cys695 intrachain disulfide bond, interacted with platelets at all shear rates tested in a manner indistinguishable from multimeric vWF. As with the native molecule, the surface coated with this fragment became saturated with interacting platelets within second" @default.
• W2110007918 created "2016-06-24" @default.
• W2110007918 creator A5020778422 @default.
• W2110007918 creator A5084109223 @default.
• W2110007918 date "1999-03-01" @default.
• W2110007918 modified "2023-09-29" @default.
• W2110007918 title "Distinct Structural Attributes Regulating von Willebrand Factor A1 Domain Interaction with Platelet Glycoprotein Ibα under Flow" @default.
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