Synergistic Adhesive Interactions and Signaling Mechanisms Operating between Platelet Glycoprotein Ib/IX and Integrin αIIbβ3
2000; Elsevier BV; Volume: 275; Issue: 52 Linguagem: Inglês
10.1074/jbc.m005590200
ISSN1083-351X
AutoresCindy L. Yap, Sascha C. Hughan, Susan L. Cranmer, Warwick S. Nesbitt, Michael M. Rooney, Simon Giuliano, Suhasini Kulkarni, Sacha M. Dopheide, Yuping Yuan, Hatem H. Salem, Shaun P. Jackson,
Tópico(s)Blood groups and transfusion
ResumoThis study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin αIIbβ3. These include the following: 1) examining the sufficiency of GPIb/IX and integrin αIIbβ3 to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin αIIbβ3 activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin αIIbβ3 activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin αIIbβ3, we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin αIIbβ3mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin αIIbβ3. Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin αIIbβ3 activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150–1800 s−1). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin αIIbβ3 activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin αIIbβ3 activation. This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin αIIbβ3. These include the following: 1) examining the sufficiency of GPIb/IX and integrin αIIbβ3 to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin αIIbβ3 activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin αIIbβ3 activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin αIIbβ3, we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin αIIbβ3mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin αIIbβ3. Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin αIIbβ3 activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150–1800 s−1). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin αIIbβ3 activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin αIIbβ3 activation. glycoprotein von Willebrand factor human von Willebrand factor bovine von Willebrand factor protein kinase C adenosine 5′-O-(1-thiotriphosphate) acetoxymethyl 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester monoclonal antibody antibody fluorescence-activated cell sorter fluorescein isothiocyanate Chinese hamster ovary differential interference contrast adenosine 3′-phosphate 5′-phosphosulfate bisindolylmaleimide I thromboxane A2 The integrin family of cell surface adhesion receptors mediates cell-cell and cell-matrix interactions responsible for mammalian development, inflammation, immunity, and hemostasis (1Hynes R.O. Cell. 1987; 48: 549-554Abstract Full Text PDF PubMed Scopus (3100) Google Scholar, 2Ruoslathi E. Pierschbacher M.D. Science. 1987; 238: 491-497Crossref PubMed Scopus (3860) Google Scholar). For circulating cells such as platelets and leukocytes, integrins play an indispensable role in anchoring these cells to the luminal surface of blood vessels at sites of vascular injury and inflammation. However, a limitation of integrin function is their relative inefficiency at forming adhesion contacts under conditions of blood flow. This has been most clearly demonstrated from studies of leukocyte adhesion to post-capillary venules, in which the formation of integrin adhesion contacts requires an initial cell-tethering step dependent on one or more selectin family members (3Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1876) Google Scholar, 4Ley K. Gaehtgens P. Fennie C. Singer M.S. Lasky L.A. Rosen S.D. Blood. 1991; 77: 2553-2555Crossref PubMed Google Scholar, 5von Andrian U.H. Chambers J.D. McEvoy L.M. Bargatze R.F. Arfors K.E. Butcher E.C. Proc. Natl. Acad. Sci. 1991; 88: 7538-7542Crossref PubMed Scopus (900) Google Scholar). There is now strong evidence that platelets also utilize a multistep adhesion mechanism, involving glycoprotein (GP)1 Ib/IX and one or more surface integrins, to mediate stable adhesion at sites of vascular injury (6Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar, 7Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-686Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar). Platelet tethering involves the binding of the GPIb/IX complex to subendothelial von Willebrand factor (vWf). This multivalent adhesive interaction is unique in that it can tether platelets at high shear stresses (6Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar), a key requirement for the ability of platelets to secure hemostasis throughout the arterial circulation. vWf binding to GPIb/IX also induces platelet activation, converting the major platelet integrin, αIIbβ3, from a low affinity to a high affinity receptor capable of engaging the C1 domain of vWf (8De Marco L. Girolami A. Russell S. Ruggeri Z.M. J. Clin. Invest. 1985; 75: 1198-1203Crossref PubMed Scopus (113) Google Scholar, 9Gralnick H.R. Williams S.B. Collerm B.S. J. Clin. Invest. 1985; 75: 19-25Crossref PubMed Google Scholar, 10Ruggeri Z.M. Curr. Opin. Cell Biol. 1993; 5: 898-906Crossref PubMed Scopus (28) Google Scholar). This latter adhesive interaction is essential for stable platelet adhesion on vWf and also for subsequent cytoskeletal reorganization leading to platelet spreading (11Savage B. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 11227-11233Abstract Full Text PDF PubMed Google Scholar, 12Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar). A major unresolved issue is the mechanism by which the vWf-GPIb interaction induces activation of integrin αIIbβ3 under physiological flow conditions. In particular, it is unclear whether GPIb/IX induces integrin αIIbβ3 activation directly, through the generation of intracellular second messengers, or involves an indirect pathway dependent on the release of ADP and/or the generation of thromboxane A2 (TXA2). Evidence favoring the latter mechanism has been suggested from studies of shear-induced platelet aggregation using a cone-and-plate viscometer (13Moritz M.W. Reimers R.C. Baker R.K. Sutera S.P. Joist J.H. J. Lab. Clin. Med. 1983; 101: 537-544PubMed Google Scholar, 14Moake J.L. Turner N.A. Stathopoulos N.A. Nolasco L. Hellums J.D. Blood. 1988; 71: 1366-1374Crossref PubMed Google Scholar), in which the exposure of platelets in suspension to pathological levels of shear induces platelet activation in an ADP-dependent manner. According to this model (Fig. 1), shear-induced binding of soluble vWf to GPIb induces transmembrane calcium influx through an unidentified surface channel functionally linked to the GPIb/IX complex. The subsequent rise in intracellular calcium promotes secretion of dense granule ADP, which in turn engages one or more purinergic receptors (15MacKenzie A.B. Mahaut-Snith M.P. Sage S.O. J. Biol. Chem. 1996; 271: 2879-2881Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 16Daniel J.L. Dangelmaier C. Jin J. Ashby B. Smith J.B. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2024-2029Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 17Jin J. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2030-2034Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 18Hechler B. Leon C. Vial C. Vigne P. Frelim C. Cazenave J. Gachet C. Blood. 1998; 92: 152-159Crossref PubMed Google Scholar) on the cell surface leading to integrin αIIbβ3 activation (19Gachet C. Hechler B. Leon C. Vial C. Leray C. Ohlmann P. Cazenave J. Thromb. Haemostasis. 1997; 78: 271-275Crossref PubMed Scopus (103) Google Scholar). Thus under pathological levels of shear, the vWf-GPIb interaction has been postulated to induce integrin αIIbβ3activation by an indirect mechanism critically dependent on transmembrane calcium influx and the release of ADP.Figure 1Proposed model for vWf-induced integrin αIIbβ3activation under pathological shear conditions. Exposure of platelets in suspension to pathological levels of shear using a cone-and-plate viscometer induces platelet aggregation via a process dependent on GPIb/IX, integrin αIIbβ3, vWf, extracellular calcium, and ADP (33Ikeda Y. Handa M. Kamata T. Kawano K. Kawai Y. Watanabe K. Kawakami K. Sakai K. Fukuyama M. Itagaki I. Yoshioka A. Ruggeri Z.M. Thromb. Haemostasis. 1993; 69: 496-502Crossref PubMed Scopus (161) Google Scholar, 46Chow T.W. Hellums J.D. Moake J.L. Kroll M.H. Blood. 1992; 80: 113-120Crossref PubMed Google Scholar, 55Oda A. Yokoyama K. Murata M. Tokuhira M. Nakamura K. Handa M. Watanabe K. Ikeda Y. Thromb. Haemostasis. 1995; 74: 736-742Crossref PubMed Scopus (46) Google Scholar). The schematic model demonstrates shear-induced binding of soluble vWf to GPIb leading to transmembrane calcium influx through an unidentified surface channel functionally linked to the GPIb/IX complex (33Ikeda Y. Handa M. Kamata T. Kawano K. Kawai Y. Watanabe K. Kawakami K. Sakai K. Fukuyama M. Itagaki I. Yoshioka A. Ruggeri Z.M. Thromb. Haemostasis. 1993; 69: 496-502Crossref PubMed Scopus (161) Google Scholar). This increase in cytosolic calcium promotes secretion of dense granule ADP (55Oda A. Yokoyama K. Murata M. Tokuhira M. Nakamura K. Handa M. Watanabe K. Ikeda Y. Thromb. Haemostasis. 1995; 74: 736-742Crossref PubMed Scopus (46) Google Scholar), which in turn induces integrin αIIbβ3 activation through engagement of one or more purinergic receptors on the cell surface (15MacKenzie A.B. Mahaut-Snith M.P. Sage S.O. J. Biol. Chem. 1996; 271: 2879-2881Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 16Daniel J.L. Dangelmaier C. Jin J. Ashby B. Smith J.B. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2024-2029Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 17Jin J. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2030-2034Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). This model suggests that under pathological shear conditions, the vWf-GPIb interaction induces integrin αIIbβ3 activation via an indirect signaling mechanism. The bottom left panel schematically depicts platelet aggregates forming in a cone-and-plate viscometer. Thebottom right panel highlights the critical role of vWf multimers in mediating platelet aggregation through interaction with GPIb/V/IX and integrin αIIbβ3. Thetop panel demonstrates the signaling processes linking the vWf-GPIb interaction to integrin αIIbβ3activation.View Large Image Figure ViewerDownload (PPT) The mechanism by which platelets become activated on an immobilized vWf matrix under physiological flow conditions has been less clearly defined. This is a potentially important issue given the critical role played by vWf in promoting platelet-vessel wall and platelet-platelet adhesion contacts under flow. A recent study by Kuwahara et al. (20Kuwahara M. Sugimoto M. Tsuji S. Miyata S. Yoshioka A. Blood. 1999; 94: 1149-1155Crossref PubMed Google Scholar) has raised the interesting possibility that integrin αIIbβ3 activation on the surface of platelets adhering to immobilized vWf occurs in a calcium-independent manner. In their studies, adhesion of platelets to vWf under physiological flow conditions was not associated with detectable changes in the cytosolic concentration of calcium, and furthermore, chelating intracellular calcium did not inhibit integrin αIIbβ3-dependent stationary platelet adhesion. Several other reports have begun to challenge the hypothesis that calcium influx is indispensable for GPIb/IX-dependent signaling. For example, Francesconiet al. (21Francesconi M.A. Deana R. Girolami A. Pontara E. Casonato A. Thromb. Haemostasis. 1993; 70: 697-701Crossref PubMed Scopus (19) Google Scholar, 22Francesconi M. Casonato A. Pontara E. Via L.D. Girolami A. Deana R. Biochem. Biophys. Res. Commun. 1995; 214: 102-109Crossref PubMed Scopus (27) Google Scholar) failed to detect transmembrane calcium influx following vWf binding to GPIb/IX. Moreover, Kermode et al. (23Kermode J.C. Zheng Q. Cook E.P. Blood Cells Mol. Dis. 1996; 22: 238-253Crossref PubMed Scopus (11) Google Scholar) have suggested that previously observed changes in cytosolic calcium initiated by vWf binding to GPIb/IX may be artifactual, resulting from extrusion of the indicator dyes from loaded platelets. Our recent studies also do not support an indispensable role for calcium influx in GPIb/IX-dependent signaling, as pretreating platelets or GPIb/IX-transfected CHO cells with extracellular calcium chelators did not prevent GPIb/IX-induced cytoskeletal remodeling (24Yuan Y. Kulkarni S. Ulsemer P. Cranmer S.L. Yap C.L. Nesbitt W.S. Harper I. Mistry N. Dopheide S.M. Hughan S.C. Williamson D. de la Salle C. Salem H.H. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 36241-36251Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The reason for the apparent discrepancies between different studies in terms of GPIb/IX signaling is not immediately evident but may reflect methodological differences that influence the platelet activation process. For example, studies in a cone-and-plate viscometer primarily examine the effects of pathological levels of shear on the formation of platelet-platelet adhesion contacts (aggregation) in suspension, whereas studies in flow chambers principally examine platelet adhesion onto reactive protein surfaces. In this study we have examined several aspects of the adhesive and signaling relationship operating between GPIb/IX and integrin αIIbβ3 using an in vitroflow-based adhesion assay. First, we have examined whether vWf engagement of GPIb/IX and integrin αIIbβ3is sufficient to mediate irreversible cell adhesion under flow. Second, we have examined whether the vWf-GPIb interaction can transduce signals directly to regulate the ligand binding status of integrin αIIbβ3. Third, we have examined whether there is a second messenger role for calcium in linking the vWf-GPIb interaction to integrin αIIbβ3 activation. Our studies indicate that the sequential binding of vWf to GPIb/IX and integrin αIIbβ3 is sufficient to mediate irreversible cell adhesion under flow and that GPIb/IX can transduce signals directly to regulate the ligand binding function of integrin αIIbβ3. In addition, we have demonstrated that intracellular calcium mobilization and activation of protein kinase C (PKC) are two key signaling events linking the vWf-GPIb interaction to integrin αIIbβ3 activation over the full range of shear forces experienced by platelets in vivo. FITC-conjugated phalloidin, adenosine 3′-phosphate 5′-phosphosulfate (A3P5PS), and acetylsalicylic acid were purchased from Sigma. ATPαS, calphostin C, bisindolylmaleimide I (BIM), and the calcium chelators, EGTA-AM and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), were from Calbiochem. 5,5′-Dimethyl-BAPTA, AM, Oregon Green 488 BAPTA-1, AM, and Fura Red, AM, were from Molecular Probes Inc. (Eugene, OR). Aggrastat was from Merck & Co. Inc. (Whitehouse Station, NJ). Apyrase was purified from potatoes according to the method of Molnar and Lorand (25Molnar J. Lorand L. Arch. Biochem. Biophys. 1961; 93: 353-363Crossref PubMed Scopus (164) Google Scholar). Human von Willebrand factor (HvWf) and bovine von Willebrand factor (BvWf) were purified to homogeneity from plasma cryoprecipitate according to the method of Montgomery and Zimmerman (26Montgomery R.R. Zimmerman T.S. J. Clin. Invest. 1978; 61: 1498-1507Crossref PubMed Scopus (76) Google Scholar). Botrocetin was a generous gift from Prof. Michael Berndt (Baker Medical Research Institute, Melbourne, Australia). AR-C69931MX was generously supplied by AstraZeneca R & D Charnwood (Leicestershire, UK) (27Ingall A.H. Dixon J. Bailey A. Coombs M.E. Cox D. McInally J.I. Hunt S.F. Kindon N.D. Teobald B.J. Willis P.A. Humphries R.G. Leff P. Clegg J.A. Smith J.A. Tomlinson W. J. Med. Chem. 1999; 42: 213-220Crossref PubMed Scopus (268) Google Scholar). All other reagents were obtained from sources described previously (28Jackson S.P. Schoenwaelder S.M. Yuan Y. Rabinowitz I. Salem H.H. Mitchell C.A. J. Biol. Chem. 1994; 269: 27093-27099Abstract Full Text PDF PubMed Google Scholar, 29Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 30Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The anti-β3chimeric Fab fragment of the monoclonal antibody (mAb) 7E3 (c7E3 Fab abciximab) was from Eli-Lilly (Centocor, Leiden, Netherlands). The complex-specific anti-αIIbβ3 antibody, P2, was from Coulter/Immunotech (Marseille, France). The anti-human integrin αvβ3 antibody, LM609, was from Chemicon International, Inc. (Temecula, CA). Anti-GPIbα mAb, AK2, was generously donated by Prof. Michael Berndt (Baker Medical Research Institute, Melbourne, Australia). PAC-1 mAb was from Becton Dickinson (Victoria, Australia), and FITC-conjugated anti-mouse IgM (α-IgM) antibody was from Southern Biotechnology Associates, Inc. (Birmingham, AL). The eukaryotic expression vector pcDNA3 containing the cDNAs encoding for αIIb or β3 was generously provided by Dr. Peter Newman (Blood Research Institute, Milwaulkee, WI). The pZeoSV vector and ZeocinTM were from Invitrogen (San Diego, CA). Whole blood (anticoagulated with 15 mm trisodium citrate, pH 7.4) was collected from healthy volunteers who had not received any anti-platelet medication in the preceding 2 weeks. Washed platelets were prepared as described previously (29Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and resuspended in modified Tyrode's buffer (10 mm Hepes, 12 mm NaHCO3, pH 7.4, 137 mm NaCl, 2.7 mm KCl, 5 mm glucose) containing 1 mm CaCl2 and/or 1 mmMgCl2 where indicated. Autologous red blood cells were obtained by an initial centrifugation of anticoagulated whole blood at 200 × g for 30 min. The platelet-rich plasma was removed, and the red blood cells were pelleted by further centrifugation at 2000 × g for 10 min. The red blood cells were washed three times with washing buffer (10 mmHepes, pH 7.4, 140 mm NaCl, 5 mm glucose) prior to reconstitution with washed platelets (50% (v/v) autologous packed red blood cells), in the presence of 0.4 units/ml apyrase (ADPase activity). The concentrations of apyrase, ATPαS, AR-C69931MX, and A3P5PS required to inhibit completely the platelet-activating effects of exogenous ADP (10 μm) were determined by monitoring the presence of platelet aggregates and/or platelet shape change in washed platelets. Platelet aggregation in whole blood, or washed platelets reconstituted with red blood cells, was determined by reduction in single platelet count (Cell-Dyn, Abbott). Similarly, the concentration of aspirin required to inhibit the effects of arachidonic acid (1.5 mm) was determined by monitoring platelet aggregation in platelet-rich plasma or by the reduction in single platelet count in whole blood. Transfection of CHO.K1 cells with GPIb/IX (CHO-Ib/IX) and/or integrin αIIbβ3was performed as described previously using calcium phosphate precipitation (30Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Cells were placed under selection pressure after 48 h using 500 μg/ml Zeocin, and clones were screened for GPIb/IX and integrin αIIbβ3 expression by flow cytometry (FACSCalibur, Becton Dickinson) using the the anti-GPIbα mAb, AK2, or the complex-specific anti-αIIbβ3 antibody, P2. Cells expressing both GPIb/IX and integrin αIIbβ3 were designated CHO-Ib/IX-αIIbβ3. Control cells transfected with pDX alone were designated CHO-pDX. Static adhesion assays were performed using a modified method of Yuan et al. (29Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Briefly, glass coverslips were coated with HvWf (10 μg/ml) for 2 h at room temperature or overnight at 4 °C. Platelets were allowed to adhere and spread on the matrix for 30–60 min unless otherwise indicated and visualized using phase contrast microscopy or differential interference contrast (DIC) microscopy. Images were captured, and the surface area of adherent platelets was determined using MCIDTM software (Imaging Research Inc., Canada). The increase in surface area of spreading platelets was expressed in pixels following subtraction of the surface area of resting platelets. Flow assays were performed using glass microcapillary tubes (Microslides, Vitro Dynamics Inc., NJ) coated with BvWF (10 μg/ml) or HvWf (100 μg/ml), according to a modified method of Cooke et al. (31Cooke B.M. Usami S. Perry I. Nash G.B. Microvasc. Res. 1993; 45: 33-55Crossref PubMed Scopus (174) Google Scholar). For studies using platelets reconstituted with red blood cells, microcapillary tubes were blocked with 10–25% heat-inactivated human serum. Citrate or heparin-anticoagulated whole blood or washed platelets reconstituted with red blood cells were perfused through HvWf-coated microcapillary tubes at shear rates of 150, 600, or 1800 s−1 for up to 5 min. Platelet adhesion was visualized in real time using DIC microscopy (DMIRB Leica microscope), and the first 2 min of flow was video-recorded for off-line analysis. In the indicated experiments, citrated whole blood was incubated with apyrase (8.25 units/ml) alone or in combination with aspirin (1 mm) for 30 min at 37 °C. In other studies, platelets were pretreated with ATPαS (100 μm) or AR-C69931MX with A3P5PS (100 nm and 200 μm, respectively) for 10 min. Alternatively, washed platelets were incubated with EGTA/Mg2+ (1 mm each), EGTA-AM (50 μm), BIM (0.2–0.5 μm), or with calphostin C (1 μm) for 30 min at 22 °C, prior to reconstitution with red blood cells. For CHO cell flow adhesion studies, CHO-Ib/IX and CHO-Ib/IX-αIIbβ3 cells (resuspended in modified Tyrode's buffer (1 × 106/ml)) were perfused through BvWf-coated (10 μg/ml) microcapillary tubes at a shear rate of 150 s−1 for 5 min. Tethered cells were subsequently exposed to incremental increases in shear (every 60 s) from 150 to 750, 3000, and 6000 s−1. Adherent cells were visualized using phase-contrast microscopy (IX70, Olympus, Japan), and images were video-recorded at each shear rate for off-line analysis. In the indicated experiments, CHO-Ib/IX and/or CHO-Ib/IX-αIIbβ3 cells were pretreated with either the anti-GPIbα mAb, AK2 (5 μg/ml), the anti-β3mAb, c7E3 Fab (20 μg/ml), the anti-αvβ3mAb, LM609 (2 μg/ml), or the non-peptide antagonist of integrin αIIbβ3, Aggrastat (200 nm), for 15 min prior to performing flow assays. In all studies, any cell forming an adhesion contact with immobilized vWf for greater than 40 ms was scored as a tethered cell. Stationary adhesion was defined as cells not moving more than a single cell diameter over a 10-s period for platelets and within a 30-s period for transfected CHO cells. In static adhesion assays, washed platelets were allowed to adhere to HvWf-coated coverslips in the presence of PAC-1 (1 μg/ml). In flow adhesion assays, washed platelets reconstituted with red blood cells were perfused through microcapillary tubes for 5 min prior to the perfusion of PAC-1 (1 μg/ml) over adherent platelets for 15 min. Adherent platelets were fixed, incubated with a FITC-conjugated α-IgM antibody, and visualized using confocal microscopy (Leica TCS NT, Netherlands). Fluorescence was quantified by integrated pixel intensity determination (Leica TCS NT software). Washed platelets (1.5 × 109/ml) resuspended in platelet wash buffer (PWB) were incubated with the calcium indicator dyes, Oregon Green 488 BAPTA-1, AM (1 μm), and Fura Red, AM (1.25 μm), for 30 min at 37 °C. The platelets were washed twice with platelet wash buffer, resuspended in Tyrode's buffer containing either extracellular calcium (1 mm) or EGTA/Mg2+ (1 mmeach), and if indicated, incubated with DM-BAPTA, AM (70 μm). Changes in cytosolic calcium concentration were associated with increased or decreased fluorescence emission of Oregon Green and Fura Red, respectively (emission wavelengths of 500–570 nm for Oregon Green and 600–710 nm for Fura Red). These changes were analyzed by confocal microscopy and based on a ratio signal intensity in the Oregon Green and Fura Red channels. Fluorescence ratios were then converted to calcium concentration units according to Equation1, [Ca2+]=170×(R−Rmin)/(Rmax−R)×(Fmax/Fmin)Equation 1 where 170 = Kd of Oregon Green Ca2+ binding; R = fluorescence ratio;Rmax and Rmin represent the fluorescence ratio of platelets that have been incubated with 50 μmA23187 + 10 mm Ca2+ or 70 μm DM-BAPTA, AM, + 2 mm EGTA, respectively;Fmin and Fmax represent the Oregon Green fluorescence of Rmax andRmin. The calcium concentrations for minimal and maximal calcium fluorescence ratios are calculated based on established methods, and the values obtained are in close agreement with that previously published (20Kuwahara M. Sugimoto M. Tsuji S. Miyata S. Yoshioka A. Blood. 1999; 94: 1149-1155Crossref PubMed Google Scholar, 32Sage S.O. Rink T.J. J. Biol. Chem. 1987; 34: 16364-16369Google Scholar, 33Ikeda Y. Handa M. Kamata T. Kawano K. Kawai Y. Watanabe K. Kawakami K. Sakai K. Fukuyama M. Itagaki I. Yoshioka A. Ruggeri Z.M. Thromb. Haemostasis. 1993; 69: 496-502Crossref PubMed Scopus (161) Google Scholar). Basal cytosolic calcium concentrations were determined in platelets in suspension and compared with calcium levels obtained in platelets adherent to HvWf under static or flow conditions. For population analysis of the calcium response in platelets adherent to a vWf matrix under static conditions, the cytosolic calcium concentration was determined over a 37.5-s time interval, 10 min after adhesion. The calcium concentration in pulsing platelets was determined using MCIDTM software (Imaging Research Inc., Canada) at 7-s intervals over the 37.5-s period. For single cell analysis, the calcium dynamics in individual platelets was monitored every 0.586 s over a 37.5-s time interval (Leica TCS NT software). Under flow conditions, the frequency of a calcium "event" (where event is defined as the recording of a particular calcium concentration) was determined at 7-s intervals over a 37.5-s period. Significant differences were detected using Student's t test and one-way analysis of variance, using the Prism software package (GraphPAD Software for Science, San Diego, CA). The necessity of GPIb/IX and integrin αIIbβ3 for irreversible platelet adhesion on vWf has been clearly established through the use of specific inhibitors against either receptor (6Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar, 12Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar, 34Handa 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, 35Niiya 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) and from the study of platelets with qualitative or quantitative deficiency of GPIb/IX or integrin αIIbβ3 (37Weiss H.J. Rogers J. N. Engl. J. Med. 1971; 285: 369-374Crossref PubMed Scopus (73) Google Scholar, 38Ruggeri Z.M. Zimmerman T.S Semin. Hematol. 1985; 22: 203-218PubMed Google Scholar, 39Weiss H.J. Turitto V.T. Baumgartner H.R. Blood. 1986; 67: 322-330Crossref PubMed Google Scholar, 40Sakariassen K.S. Nievelstein P.F.E.M. Coller B.S. Sixma J.J. Br. J. Haematol. 1986; 63: 681-691Crossref PubMed Scopus (177) Google Scholar, 41Weiss H.J. Ann. N. Y. Acad. Sci. 1991; 614: 125-137Crossref PubMed Scopus (39) Google Scholar). However, it has yet to be established definitively that these two receptors are sufficient to mediate irreversible cell adhesion under flow, independent of other platelet adhesion receptors or endogenous stimuli. To investigate this possibility, we performed studies on CHO cells co-transfected with GPIb/IX and integrin αIIbβ3. We chose this cell line as it does not express GPIb/IX or integrin αIIbβ3, does not contain storage granules for ADP or other activating stimuli, or generate TXA2 following cell stimulation. We have recently demonstrated that CHO cells transfected with the GPIb/IX complex are able to tether and translocate on a vWf matrix but are unable to form irreversible adhesio
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