FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2001165687> ?p ?o ?g. }

• W2001165687 endingPage "16787" @default.
• W2001165687 startingPage "16779" @default.
• W2001165687 abstract "In platelets, αIIbβ3 exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert αIIbβ3 to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute αIIbβ3 activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which αIIbβ3 activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected αIIbβ3 but also endogenous αvβ3. Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIbα that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to αIIbβ3 activation and suggest that the signaling pathways by which GPIb-IX induces αIIbβ3 activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings. In platelets, αIIbβ3 exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert αIIbβ3 to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute αIIbβ3 activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which αIIbβ3 activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected αIIbβ3 but also endogenous αvβ3. Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIbα that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to αIIbβ3 activation and suggest that the signaling pathways by which GPIb-IX induces αIIbβ3 activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings. von Willebrand factor glycoproteins Chinese hamster ovary Arg-Gly-Asp-Ser tetramethylrhodamine isothiocyanate fluorescence-activated cell sorter monoclonal antibody actin-binding protein Integrins are adhesion receptors that regulate cell-cell and cell-matrix interactions (1.Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 2.Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar). Often an integrin cannot bind ligand until a signal from inside the cell causes a conformational change that increases its affinity for ligand. In other cases, signals cause an integrin to cluster, thus increasing its avidity for ligand (3.Hughes P.E. Pfaff M. Trends Cell Biol. 1998; 8: 359-364Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Changes in the functional activity of integrins can be induced by agonists that interact with heterotrimeric G-protein-coupled receptors. For example, in platelets thrombin, ADP, or thromboxane A2can all induce signals leading to activation of the integrin αIIbβ3 (4.Shattil S.J. Brass L.F. J. Biol. Chem. 1987; 262: 992-1000Abstract Full Text PDF PubMed Google Scholar, 5.Bennett J.S. Vilaire G. J. Clin. Invest. 1979; 64: 1393-1401Crossref PubMed Scopus (532) Google Scholar); interaction of αIIbβ3 with vWf1 in the blood vessel wall leads to irreversible adhesion and cell spreading (6.Phillips D. Charo I. Scarborough R. Cell. 1991; 65: 359-362Abstract Full Text PDF PubMed Scopus (479) Google Scholar), whereas interaction with fibrinogen in plasma leads to platelet aggregate formation at the site of injury. Investigation of the signaling molecules involved in G-protein receptor-induced αIIbβ3 activation has implicated roles for protein kinase C and the actin filament-based cytoskeleton (7.Qi W. Loh E. Vilaire G. Bennett J.S. J. Biol. Chem. 1998; 273: 15271-15278Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 8.Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar). Because platelet aggregation can lead to thrombotic events, there is considerable interest in elucidating the mechanisms involved in αIIbβ3 activation. To this end, efforts have been made to reconstitute αIIbβ3activation in several transfected cell systems. However, attempts to activate αIIbβ3 by induction of signaling pathways involved in thrombin-induced activation have been unsuccessful (9.O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (573) Google Scholar, 10.Kieffer N. Fitzgerald L. Wolf D. Cheresh D. Phillips D. J. Cell Biol. 1991; 113: 451-461Crossref PubMed Scopus (102) Google Scholar). An exception is an Epstein-Barr virus-transformed B-lymphocyte system in which αIIbβ3 can be activated by phorbol 12-myristate 13-acetate and by formyl-Met-Leu-Phe, an agonist that acts on a G-protein-coupled receptor (7.Qi W. Loh E. Vilaire G. Bennett J.S. J. Biol. Chem. 1998; 273: 15271-15278Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 11.Loh E. Beaverson K. Vilaire G. Qi W. Poncz M. Bennett J.S. J. Biol. Chem. 1995; 270: 18631-18636Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Thus, the suggestion has been made that the pathway involved in αIIbβ3 activation is specific for hematopoietic cells (11.Loh E. Beaverson K. Vilaire G. Qi W. Poncz M. Bennett J.S. J. Biol. Chem. 1995; 270: 18631-18636Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Although integrin activation has been studied in much detail following G-protein-coupled receptor signaling, it is becoming apparent that other receptor families may activate integrins. For example, the presence of serum accelerates integrin-induced adhesion in cultured cells, suggesting that growth factor receptor signaling activates pathways that influence integrin activity (12.Gullberg D. Tingstrom A. Thuresson A.C. Olsson L. Terracio L. Borg T.K. Rubin K. Exp. Cell Res. 1990; 186: 264-272Crossref PubMed Scopus (236) Google Scholar, 13.Kinashi T. Escobedo J.A. Williams L.T. Takatsu K. Springer T.A. Blood. 1995; 86: 2086-2090Crossref PubMed Google Scholar). Similarly, engagement of adhesion receptors in leukocytes and platelets may regulate integrin function (14.Blanks J.E. Moll T. Eytner R. Vestweber D. Eur. J. Immunol. 1998; 28: 433-443Crossref PubMed Scopus (116) Google Scholar, 15.Giblin P.A. Hwang S.T. Katsumoto T.R. Rosen S.D. J. Immunol. 1997; 159: 3498-3507PubMed Google Scholar, 16.Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar, 17.Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 18.Goto S. Salomon D.R. Ikeda Y. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 23352-23361Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 19.Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). In platelets, the glycoprotein (GP) Ib-IX complex interacts with vWf in the blood vessel wall. This is a transient, low affinity interaction that, under conditions of flow, results in rolling of platelets; the subsequent interaction of the integrins αIIbβ3 and α2β1 with their ligands in the vessel wall causes irreversible adhesion (16.Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar, 17.Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 18.Goto S. Salomon D.R. Ikeda Y. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 23352-23361Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 19.Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar), “outside-in” signaling, and cytoskeletal reorganizations leading to platelet spreading (16.Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar,20.Weiss H.J. Turitto V.T. Baumgartner H.R. Blood. 1986; 67: 322-330Crossref PubMed Google Scholar, 21.Kroll M.H. Hellums J.D. McIntire L.V. Schafer A.I. Moake J.L. Blood. 1996; 88: 1525-1541Crossref PubMed Google Scholar, 22.Ruggeri Z.M. Thromb. Haemostasis. 1993; 70: 119-123Crossref PubMed Scopus (166) Google Scholar). In vitro, the interaction between GPIb-IX and vWf can be induced by exposing platelets to shear forces comparable to those in the vasculature (21.Kroll M.H. Hellums J.D. McIntire L.V. Schafer A.I. Moake J.L. Blood. 1996; 88: 1525-1541Crossref PubMed Google Scholar, 23.Peterson D.M. Stathopoulos N.A. Giorgio T.D. Hellums J.D. Moake J.L. Blood. 1987; 69: 625-628Crossref PubMed Google Scholar, 24.Moake J.L. Turner N.A. Stathopoulos N.A. Nolasco L.H. Hellums J.D. J. Clin. Invest. 1986; 78: 1456-1461Crossref PubMed Scopus (520) Google Scholar), or it can be induced under static conditions by the use of a modulator such as botrocetin (25.Read M.S. Smith S.V. Lamb M.A. Brinkhous K.M. Blood. 1989; 74: 1031-1035Crossref PubMed Google Scholar) or ristocetin (26.Howard M.A. Firkin B.G. Thromb. Diath. Haemorrh. 1971; 26: 362-369PubMed Google Scholar). Such studies have revealed that the GPIb-IX-vWF interaction induces intracellular signaling (27.Kroll M.H. Harris T.S. Moake J.L. Handin R.I. Schafer A.I. J. Clin. Invest. 1991; 88: 1568-1573Crossref PubMed Scopus (235) Google Scholar, 28.Chow T.W. Hellums J.D. Moake J.L. Kroll M.H. Blood. 1992; 80: 113-120Crossref PubMed Google Scholar). Although it is clear that GPIb-IX-vWf interactions lead to altered functional activity of αIIbβ3 (16.Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar, 17.Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 18.Goto S. Salomon D.R. Ikeda Y. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 23352-23361Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 19.Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar, 23.Peterson D.M. Stathopoulos N.A. Giorgio T.D. Hellums J.D. Moake J.L. Blood. 1987; 69: 625-628Crossref PubMed Google Scholar), it is not clear whether this results directly from activation of signaling pathways by GPIb-IX-vWf interactions or whether activation of additional membrane receptors, for example by ADP or by thromboxane secreted as a consequence of GPIb-IX signaling, is required. It also is not known whether interaction of αIIbβ3 with vWf results from GPIb-IX-induced αIIbβ3activation or from a GPIb-IX-induced alteration in the conformation of vWf that allows it to interact with αIIbβ3. GPIb-IX contains GPIbα disulfide-linked to GPIbβ; this dimer is non-covalently associated with GPIX (29.Berndt M.C. Gregory C. Kabral A. Zola H. Fournier D. Castaldi P.A. Eur. J. Biochem. 1985; 151: 637-649Crossref PubMed Scopus (150) Google Scholar). GPIbα contains the domain that interacts with vWf. The cytoplasmic domain of GPIbα interacts with actin-binding protein (ABP), linking the GPIb-IX complex to the skeleton that coats the intracellular surface of the lipid bilayer (30.Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 31.Andrews R.K. Fox J.E.B. J. Biol. Chem. 1991; 266: 7144-7147Abstract Full Text PDF PubMed Google Scholar, 32.Andrews R.K. Fox J.E.B. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google Scholar, 33.Fox J.E.B. J. Biol. Chem. 1985; 260: 11970-11977Abstract Full Text PDF PubMed Google Scholar, 34.Fox J.E.B. Boyles J.K. Berndt M.C. Steffen P.K. Anderson L.K. J. Cell Biol. 1988; 106: 1525-1538Crossref PubMed Scopus (118) Google Scholar). The cytoplasmic domain of GPIbα also contains a binding site for the putative signaling molecule 14-3-3 (35.Du X. Fox J.E.B. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Nothing is known about the importance of ABP or 14-3-3 in allowing GPIb-IX to transmit signals leading to altered integrin function. GPIb-IX can interact with another transmembrane protein, GPV (36.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, 37.Meyer S.C. Fox J.E.B. J. Biol. Chem. 1995; 270: 14693-14699Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The potential importance of GPV in allowing GPIb-IX to induce altered αIIbβ3 function is not known. In the present study, we have provided evidence that signaling across GPIb-IX leads to activation of αIIbβ3 in platelets. Moreover, we have established a CHO cell system in which αIIbβ3 and GPIb-IX are expressed. Signaling across GPIb-IX in this system leads to αIIbβ3 activation even in the absence of GPV and the binding sites for 14-3-3 and ABP. Taken together, these studies show that the function of integrin adhesion receptors can be regulated not only by interaction of soluble ligands with heterotrimeric G-protein-coupled receptors but also by signals transmitted following occupancy of a non-integrin adhesion receptor. Moreover, they suggest that signaling molecules used by GPIb-IX to induce αIIbβ3 activation differ from those used by the thrombin receptor. The monoclonal antibody against αIIbβ3 used for FACS analysis was purchased from Immunotech (Westbrook, ME) and that used for inhibition of cell spreading was purchased from Chemicon (Temecula, CA). The polyclonal antibodies against αvβ3 were purchased from Life Technologies, Inc. The F(ab′)2 fragment against αIIbβ3 (clone CRC64) was a kind gift of Tatiana Byzova (The Cleveland Clinic Foundation, Cleveland). Monoclonal antibodies against GPIbα (mAb Ib-4 and mAb Ib-23) were generously provided by Dr. B. Steiner (Hoffmann-La Roche). A monoclonal antibody against GPIb-IX was purchased from Immunotech. The polyclonal antibody against GPV was generously provided by Dr. D. Phillips (COR Therapeutics Inc., San Francisco). Fluorescently labeled PAC-1 monoclonal antibody was kindly provided by Dr. S. J. Shattil (The Scripps Research Institute, La Jolla, CA). The antibodies against major histocompatibility complex class I molecules (clone W6.32) were purchased from ATCC (Manassas, VA) and against VCAM-1 were purchased from Chemicon. Venous blood was drawn from healthy donors, and from patients M. M. and N. L. with Glanzmann's thrombasthenia (whose platelets lack αIIbβ3 (38.George J.N. Nurden A.T. Phillips D.R. N. Engl. J. Med. 1984; 311: 1084-1098Crossref PubMed Scopus (228) Google Scholar)). Blood from the thrombasthenic patients was kindly made available by Robert L. Abel of Christiana Hospital, Newark, DE. Platelets were isolated by centrifugation and washed as described previously (39.Fox J.E.B. Aggerbeck L.P. Berndt M.C. J. Biol. Chem. 1988; 263: 4882-4890Abstract Full Text PDF PubMed Google Scholar). Washed platelets were resuspended at a concentration of 1 × 109 platelets/ml at 37 °C in a Tyrode's buffer containing 138 mm sodium chloride, 2.9 mm potassium chloride, 12 mmsodium bicarbonate, 0.36 mm sodium phosphate, 5.5 mm glucose, 1.8 mm calcium chloride, and 0.4 mm magnesium chloride, pH 7.4. Platelet suspensions were stirred in the presence of 4 μg/ml vWf (American Bioproducts, Parsippany, NJ) and 1 mg/ml ristocetin (Sigma) for 5 min. In some experiments 200 μg/ml fibrinogen (isolated from human plasma and kindly provided by Dr. Stanley D'Souza of the Cleveland Clinic Foundation, Cleveland) was included. In others, platelets were preincubated with apyrase (2 units/ml) and aspirin (1 mm) for 30 min. Incubations were terminated by addition of an equal volume of ice-cold lysis buffer containing 2% Triton X-100, 10 mmEGTA, 100 mm Tris-HCl, 2 mg/ml leupeptin (Roche Molecular Biochemicals), 100 mm benzamidine (Sigma), 2 mmsodium orthovanadate (Fisher), and 2 mmphenylmethylsulfonyl fluoride (Sigma), pH 7.4 (40.Fox J.E.B. Lipfert L. Clark E.A. Reynolds C.C. Austin C.D. Brugge J.S. J. Biol. Chem. 1993; 268: 25973-25984Abstract Full Text PDF PubMed Google Scholar). Lysates were immediately centrifuged at 15,600 × g at 4 °C for 4 min to obtain the low speed detergent-insoluble pellet. Detergent-insoluble and -soluble fractions were solubilized by addition of an SDS-containing buffer in the presence of reducing agent (40.Fox J.E.B. Lipfert L. Clark E.A. Reynolds C.C. Austin C.D. Brugge J.S. J. Biol. Chem. 1993; 268: 25973-25984Abstract Full Text PDF PubMed Google Scholar), denatured at 95 °C, and electrophoresed through SDS-polyacrylamide gels. Proteins present in the fractions were detected by transferring samples to nitrocellulose and incubating the resulting Western blots with antibodies against proteins of interest. Antigen-antibody complexes were detected by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech). Chinese hamster ovary (CHO) cells were obtained from ATCC (Rockville, MD) and stably transfected with the cDNA encoding GPIbα, GPIbβ, and GPIX along with psv2-neo plasmid containing resistance to neomycin as described previously (30.Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). To obtain CHO cells co-expressing αIIbβ3, the cDNA's encoding the amino acid sequences for αIIb and β3 were subcloned into pDX vector. CHO cells expressing GPIb-IX complex were transfected with 10 μg of each of the plasmids along with 1 μg of psv2-hph vector DNA containing hygromycin B resistance gene (ATCC). For some experiments, cells containing GPIb-IX-V complex were transfected with αIIb and β3 cDNAs. Transfections were performed using LipofectAMINE Plus reagents (Life Technologies, Inc.) according to the manufacturer's protocol. Stable CHO cells were selected in 500 μg/ml G418 (Life Technologies, Inc.) and 250 μg/ml hygromycin B (Roche Molecular Biochemicals) containing growth medium. The expression of αIIbβ3 on the cell surface was analyzed by flow cytometry using αIIbβ3 complex-specific antibody (Immunotech), and the expression of GPIb-IX was analyzed using a monoclonal antibody against the complex GPIb-IX (mAb SZ1, Immunotech). Clones of transfected cells were selected using a FACS Vantage cell sorter (Becton Dickinson, San Jose, CA) after labeling of the cells with antibodies directed against GPIb-IX or αIIbβ3. Platelet suspensions were allowed to settle onto dishes that had been previously coated with 5 μg/ml vWf (American Bioproducts, Parsippany, NJ) in the presence or absence of 1 mg/ml ristocetin (Sigma). In some experiments, platelets were preincubated for 30 min with an antibody against GPIbα (mAb Ib-23) (10 μg/ml) which inhibits the interaction between GPIb-IX and vWf or with an antibody against αIIbβ3 (10 μg/ml). In other experiments, as indicated, platelets were preincubated for 30 min with 4 mm Arg-Gly-Asp-Ser (RGDS) peptide (Sigma) or for 30 min with 2 units/ml of apyrase (Sigma) and 1 mm aspirin (Sigma). After 30 min of spreading, platelets were fixed, permeabilized with 0.5% Triton X-100, and actin filaments stained with 1 μg/ml tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin (Sigma) as described previously (30.Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 37.Meyer S.C. Fox J.E.B. J. Biol. Chem. 1995; 270: 14693-14699Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Transfected cells were incubated with 5 μg/ml botrocetin (Pentapharm, Basel, Switzerland) in serum-free media and plated on Lab-Tek glass chamber slides (Nunc, Inc., Naperville, IL) that had been previously coated with 5 μg/ml vWf and saturated with 3% bovine serum albumin. In some experiments, cells were preincubated for 30 min at 37 °C with 4 mm RGDS, 2 mm EDTA, or 20 μg/ml antibodies against αvβ3 or F(ab′)2 fragment of antibody against αIIbβ3. After 30 min to 2 h at 37 °C, the cells were fixed, permeabilized with 0.5% Triton X-100, and actin filaments stained with TRITC-labeled phalloidin. Fluorescence microscopy was performed using a Leica DMR-HC fluorescence microscope or a Leica TCS-SP Laser Scanning Confocal Microscope (Leica, Heidelberg, Germany) equipped with a Micromax cooled CCD camera (Princeton Instruments, Inc., Trenton, NJ) and Image Pro Plus software (Media Cybernetics, Silver Spring, MD). Platelet suspensions were incubated for 1 min at 37 °C with fluorescently labeled PAC-1 monoclonal antibody in the presence or absence of 1 mg/ml ristocetin and 10 μg/ml vWf. Platelets were diluted in phosphate-buffered saline, and the binding of PAC-1 was quantitated by flow cytometry. Binding of PAC-1 to GPIb-IX/αIIbβ3-expressing CHO cells was assessed in the same way except that incubations were performed for 15 min and the cells washed and fixed in 1% paraformaldehyde prior to analysis by flow cytometry. GPIb-IX can bind to vWf in the blood vessel wall but is not able to bind to vWf immobilized on glass unless a modulator such as ristocetin or botrocetin is present (25.Read M.S. Smith S.V. Lamb M.A. Brinkhous K.M. Blood. 1989; 74: 1031-1035Crossref PubMed Google Scholar, 26.Howard M.A. Firkin B.G. Thromb. Diath. Haemorrh. 1971; 26: 362-369PubMed Google Scholar). Thus, if platelets are allowed to settle onto vWf-coated glass they do not adhere unless ristocetin is present (Fig. 1, comparepanels A and B). αIIbβ3 is not able to bind to vWf unless it has become activated (10.Kieffer N. Fitzgerald L. Wolf D. Cheresh D. Phillips D. J. Cell Biol. 1991; 113: 451-461Crossref PubMed Scopus (102) Google Scholar, 41.Ruggeri Z.M. Bader R. De Marco L. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6038-6041Crossref PubMed Scopus (135) Google Scholar); thus all of the binding induced in the presence of ristocetin results from interaction of GPIb-IX with vWf. This can be demonstrated by using antibodies against GPIb-IX (Fig. 1,panel C), which totally inhibit platelet binding. Although αIIbβ3 is not able to bind to vWf when GPIb-IX-vWf interactions are prevented, αIIbβ3 is converted to a form that can bind vWf once GPIb-IX-vWf interactions have taken place. This is not seen as an increased number of adherent platelets, because activation of αIIbβ3 only occurs in platelets that are already adherent through GPIb-IX. However, it is detected by the spreading that occurs once αIIbβ3 has bound ligand and transmits signals leading to cytoskeletal reorganizations (Fig. 1, panel A). The fact that spreading results from αIIbβ3-induced signaling is demonstrated inpanel D in which antibodies against αIIbβ3 prevented spreading or inpanel E in which RGDS (which inhibits the interaction of αIIbβ3 with ligand but does not affect that of GPIb-IX with vWf) markedly reduced spreading. Platelets that lacked αIIbβ3 (from patients with Glanzmann's thrombasthenia) adhered but showed little spreading (data not shown). Both adhesion and spreading occurred even in the presence of aspirin and apyrase (panel F) supporting the idea that integrin activation occurred as a direct consequence of GPIb-IX-induced signaling and was not due to ADP or thromboxane secreted from the platelet. To determine whether the GPIb-IX-induced conversion of αIIbβ3 to a form that can bind vWf results from an altered functional state of the integrin (rather than from a GPIb-IX-induced change in the conformation of vWf) that allows αIIbβ3 to bind to vWf, we determined whether GPIb-IX-induced signaling also altered the ability of αIIbβ3 to bind the fibrinogen mimetic monoclonal antibody PAC-1 (8.Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar) and the soluble ligand fibrinogen. The binding of PAC-1 to platelets in suspension was quantitated by flow cytometry. Because of problems associated with measuring binding of ligand to aggregated cells, the experiments were performed in the absence of stirring. As shown in Fig. 2, we were able to detect an increase in the binding of PAC-1 following incubation with vWf (p = 0.001, unpaired ttest). Moreover, this increase was diminished by the presence of a F(ab′)2 fragment of an αIIbβ3antibody (p = 0.01). A consequence of ligand binding to αIIbβ3in suspensions of aggregating platelets is the incorporation of αIIbβ3 into the insoluble fraction that can be sedimented by centrifugation of Triton X-100 lysates at lowg forces (typically 15,600 × g) (40.Fox J.E.B. Lipfert L. Clark E.A. Reynolds C.C. Austin C.D. Brugge J.S. J. Biol. Chem. 1993; 268: 25973-25984Abstract Full Text PDF PubMed Google Scholar, 42.Kouns W.C. Fox C.F. Lamoreaux W.J. Coons L.B. Jennings L.K. J. Biol. Chem. 1991; 266: 13891-13900Abstract Full Text PDF PubMed Google Scholar,43.Phillips D.R. Jennings L.K. Edwards H.H. J. Cell Biol. 1980; 86: 77-86Crossref PubMed Scopus (248) Google Scholar). Therefore, as another method of determining whether GPIb-IX-induced signaling induced fibrinogen binding in platelet suspensions, we stirred platelets with ristocetin and vWf in the presence and absence of fibrinogen, isolated the detergent-insoluble cytoskeletons, and assayed for the presence of αIIbβ3 in the detergent-insoluble fractions. Because the presence of ristocetin in detergent-lysed platelets is known to affect the sedimentation properties of the platelet membrane skeleton, we included a control that contained ristocetin alone. Fig. 3 shows that, as expected, the presence of ristocetin caused most of the GPIb-IX, which is associated with the membrane skeleton, to be recovered in the low speed detergent-insoluble pellet (Fig. 3, compare lanes 1 and 2). However, in contrast to GPIb-IX, most of the αIIbβ3 remained in the detergent-soluble fraction unless vWf was present in the incubation (Fig. 3, lanes 3, 4, and 6). The vWf-induced redistribution of αIIbβ3 to the low speed pellet was initiated by interaction between GPIb-IX and vWf as it was prevented by inclusion of antibodies that inhibited this interaction (data not shown). The redistribution was not initiated by thromboxane A2 or by secreted ADP, as similar redistributions were detected in samples in which platelets were preincubated with aspirin and apyrase (lanes 6 of Fig. 3). The finding that αIIbβ3 was incorporated into the cytoskeleton even in the absence of fibrinogen is consistent with the observation in spreading platelets (Fig. 1) that signaling induced by GP-Ib-IX-vWf interactions leads to association of vWf with αIIbβ3. However, inclusion of fibrinogen, which is the preferred ligand for αIIbβ3 in plasma, in the incubations, led to an even greater incorporation of αIIbβ3into the cytoskeleton supporting the idea that GPIb-IX-induced signaling converts αIIbβ3 to a form that can bind soluble ligand (compare lanes 3 and4 of Fig. 3). Quantitation of Western blots by densitometry revealed that the amount of αIIbβ3 present in the low speed cytoskeletal fraction when fibrinogen was present was 1.3-fold that of the amount when only vWf was present (p = 0.01, paired t test, three different experiments). Fibrinogen alone was not sufficient to induce a significant redistribution of αIIbβ3 into the low speed pellet (Fig. 3, lane 5) showing the importance of GPIb-IX-vWf-induced signaling in initiating an event that allows fibrinogen to induce αIIbβ3activation. With the exception of B-lymphocytes, previous attempts to induce integrin activation in transfected cell systems by activation of signaling pathways involved in thrombin-induced activation in platelets have been unsuccessful. To determine whether GPIb-IX-induced signaling pathways could lead to activation of αIIbβ3 in a nucleated cell model, we transfected CHO cells with the cDNA for αIIbβ3 in the presence or absence of the three subunits of the GPIb-IX complex (GPIbα, GPIbβ, and GPIX). Clones expressing similar amounts of αIIbβ3(as assessed by FACS analysis) were selected in order to compare spreading (Fig. 4, panel A). To determine whether GPIb-IX transmitted signals in these cells, cells were allowed to settle onto vWf-coated dishes in the presence of the modulator botrocetin. As shown in Fig. 4(panel B), cells transfected with αIIbβ3 alone showed little adhesion to the vWf-coated dishes; those that settled showed little spreading. In contrast, cells that also contained GPIb-IX attached to vWf and spread (Fig. 4, panel C). Quantitation of 10 random fields taken from two representative experiments showed that 17 ± 4.0 (S.D.) cells adhered/field when GPIb-IX was absent and 54 ± 9.4 (S.D.) cells/field adhered when GPIb-IX was present. The importance of GPIb-IX-vWf interactions in mediating adhesion was also shown by decreased attachment if botrocetin was omitted or if an antibody against GPIbα was included (data not shown). In contrast, the spreading of the adherent cells was induced by signaling across integrins, as shown by in" @default.
• W2001165687 created "2016-06-24" @default.
• W2001165687 creator A5007765285 @default.
• W2001165687 creator A5020003804 @default.
• W2001165687 creator A5031643201 @default.
• W2001165687 creator A5068531626 @default.
• W2001165687 creator A5085917063 @default.
• W2001165687 date "2000-06-01" @default.
• W2001165687 modified "2023-09-30" @default.
• W2001165687 title "Signaling across the Platelet Adhesion Receptor Glycoprotein Ib-IX Induces αIIbβ3 Activation Both in Platelets and a Transfected Chinese Hamster Ovary Cell System" @default.
• W2001165687 cites W1484853482 @default.
• W2001165687 cites W1492746912 @default.
• W2001165687 cites W1531681292 @default.
• W2001165687 cites W1532268531 @default.
• W2001165687 cites W1539185722 @default.
• W2001165687 cites W1549296148 @default.
• W2001165687 cites W1549544590 @default.
• W2001165687 cites W1561189761 @default.
• W2001165687 cites W1598021300 @default.
• W2001165687 cites W1601529928 @default.
• W2001165687 cites W1604015258 @default.
• W2001165687 cites W1615731206 @default.
• W2001165687 cites W1629000899 @default.
• W2001165687 cites W1665591202 @default.
• W2001165687 cites W1924175076 @default.
• W2001165687 cites W1983555410 @default.
• W2001165687 cites W1985189094 @default.
• W2001165687 cites W1989887433 @default.
• W2001165687 cites W1993486440 @default.
• W2001165687 cites W2007073199 @default.
• W2001165687 cites W2009967151 @default.
• W2001165687 cites W2011165507 @default.
• W2001165687 cites W2014970594 @default.
• W2001165687 cites W2016566683 @default.
• W2001165687 cites W2026624464 @default.
• W2001165687 cites W2029231027 @default.
• W2001165687 cites W2030665169 @default.
• W2001165687 cites W2031550633 @default.
• W2001165687 cites W2033337088 @default.
• W2001165687 cites W2038366724 @default.
• W2001165687 cites W2043083034 @default.
• W2001165687 cites W2052026152 @default.
• W2001165687 cites W2054584946 @default.
• W2001165687 cites W2062581256 @default.
• W2001165687 cites W2065155390 @default.
• W2001165687 cites W2066395516 @default.
• W2001165687 cites W2075013504 @default.
• W2001165687 cites W2081401313 @default.
• W2001165687 cites W2089270691 @default.
• W2001165687 cites W2090451941 @default.
• W2001165687 cites W2102324547 @default.
• W2001165687 cites W2118277673 @default.
• W2001165687 cites W2168918180 @default.
• W2001165687 cites W2171721261 @default.
• W2001165687 cites W2227357774 @default.
• W2001165687 cites W2291211051 @default.
• W2001165687 cites W2400712319 @default.
• W2001165687 cites W2407760336 @default.
• W2001165687 cites W4254009866 @default.
• W2001165687 cites W4254742516 @default.
• W2001165687 doi "https://doi.org/10.1074/jbc.275.22.16779" @default.
• W2001165687 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10828063" @default.
• W2001165687 hasPublicationYear "2000" @default.
• W2001165687 type Work @default.
• W2001165687 sameAs 2001165687 @default.
• W2001165687 citedByCount "83" @default.
• W2001165687 countsByYear W20011656872012 @default.
• W2001165687 countsByYear W20011656872013 @default.
• W2001165687 countsByYear W20011656872014 @default.
• W2001165687 countsByYear W20011656872016 @default.
• W2001165687 countsByYear W20011656872017 @default.
• W2001165687 countsByYear W20011656872019 @default.
• W2001165687 countsByYear W20011656872021 @default.
• W2001165687 countsByYear W20011656872023 @default.
• W2001165687 crossrefType "journal-article" @default.
• W2001165687 hasAuthorship W2001165687A5007765285 @default.
• W2001165687 hasAuthorship W2001165687A5020003804 @default.
• W2001165687 hasAuthorship W2001165687A5031643201 @default.
• W2001165687 hasAuthorship W2001165687A5068531626 @default.
• W2001165687 hasAuthorship W2001165687A5085917063 @default.
• W2001165687 hasBestOaLocation W20011656871 @default.
• W2001165687 hasConcept C104317684 @default.
• W2001165687 hasConcept C108625454 @default.
• W2001165687 hasConcept C153911025 @default.
• W2001165687 hasConcept C170493617 @default.
• W2001165687 hasConcept C175656101 @default.
• W2001165687 hasConcept C178790620 @default.
• W2001165687 hasConcept C185592680 @default.
• W2001165687 hasConcept C203014093 @default.
• W2001165687 hasConcept C26828662 @default.
• W2001165687 hasConcept C2780009322 @default.
• W2001165687 hasConcept C2993802102 @default.
• W2001165687 hasConcept C3018697912 @default.
• W2001165687 hasConcept C54009773 @default.
• W2001165687 hasConcept C55493867 @default.
• W2001165687 hasConcept C84416704 @default.
• W2001165687 hasConcept C86803240 @default.
• W2001165687 hasConcept C89560881 @default.

• next