Thrombospondin-bound Integrin-associated Protein (CD47) Physically and Functionally Modifies Integrin αIIbβ3 by Its Extracellular Domain
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m302194200
ISSN1083-351X
AutoresTetsuro‐Takahiro Fujimoto, Shinya Katsutani, Takeshi Shimomura, Kingo Fujimura,
Tópico(s)Platelet Disorders and Treatments
ResumoIntegrin-associated protein (IAP/CD47) is a receptor for the C-terminal cell binding domain of thrombospondin (TS). A peptide from the C-terminal cell binding domain, KRFYVVMWKK (4N1K) binds to IAP and stimulates the integrin-dependent cell functions, including platelet aggregation. We investigated the mechanism by which TS-bound IAP modulates the affinity of platelet integrin, αIIbβ3. Platelet aggregation induced by 4N1K was not completely inhibited by energy depletion with sodium azide and 2-deoxy-d-glucose, although ADP or collagen-induced platelet response was completely inhibited. The binding of ligand-mimetic antibody PAC1 to αIIbβ3 was also induced in the energy-depleted platelets. In the transfected Namalwa cells, 4N1K induced activation of the αIIbβ3 with mutated β3 (Ser-752 to Pro), which is a non-responsive form to inside-out signaling, as well as wild type αIIbβ3. The truncated form of IAP with only the extracellular immunoglobulin-like (Ig) domain was sufficient for the activation of αIIbβ3 in Chinese hamster ovary cells, although the IAP-mediated intracellular signaling was abolished, which was monitored by the absence of down-regulation of mitogen-activated protein kinase phosphorylation. Furthermore, the soluble recombinant Ig domain of IAP induced PAC1 binding to αIIbβ3 on Chinese hamster ovary cells when added with 4N1K. Physical association between the soluble recombinant Ig domain of IAP and purified αIIbβ3 was detected in the presence of 4N1K. These data indicate that the extracellular Ig domain of IAP, when bound to TS, interacts with αIIbβ3 and can change αIIbβ3 in a high affinity state without the requirement of intracellular signaling. This extracellular event would be a novel mechanism of affinity modulation of integrin. Integrin-associated protein (IAP/CD47) is a receptor for the C-terminal cell binding domain of thrombospondin (TS). A peptide from the C-terminal cell binding domain, KRFYVVMWKK (4N1K) binds to IAP and stimulates the integrin-dependent cell functions, including platelet aggregation. We investigated the mechanism by which TS-bound IAP modulates the affinity of platelet integrin, αIIbβ3. Platelet aggregation induced by 4N1K was not completely inhibited by energy depletion with sodium azide and 2-deoxy-d-glucose, although ADP or collagen-induced platelet response was completely inhibited. The binding of ligand-mimetic antibody PAC1 to αIIbβ3 was also induced in the energy-depleted platelets. In the transfected Namalwa cells, 4N1K induced activation of the αIIbβ3 with mutated β3 (Ser-752 to Pro), which is a non-responsive form to inside-out signaling, as well as wild type αIIbβ3. The truncated form of IAP with only the extracellular immunoglobulin-like (Ig) domain was sufficient for the activation of αIIbβ3 in Chinese hamster ovary cells, although the IAP-mediated intracellular signaling was abolished, which was monitored by the absence of down-regulation of mitogen-activated protein kinase phosphorylation. Furthermore, the soluble recombinant Ig domain of IAP induced PAC1 binding to αIIbβ3 on Chinese hamster ovary cells when added with 4N1K. Physical association between the soluble recombinant Ig domain of IAP and purified αIIbβ3 was detected in the presence of 4N1K. These data indicate that the extracellular Ig domain of IAP, when bound to TS, interacts with αIIbβ3 and can change αIIbβ3 in a high affinity state without the requirement of intracellular signaling. This extracellular event would be a novel mechanism of affinity modulation of integrin. Integrins are heterodimeric transmembrane receptor complexes involved in numerous physiological processes such as angiogenesis, immune response, and hemostasis (1Hynes R.O. Nat. Med. 2002; 8: 918-921Crossref PubMed Scopus (487) Google Scholar, 2Liddington R.C. Ginsberg M.H. J. Cell Biol. 2002; 158: 833-839Crossref PubMed Scopus (254) Google Scholar). They function in cell adhesion and signaling by interacting with an extracellular matrix or cellular counter receptors. The adhesive function is subject to rapid regulation referred to as inside-out signaling. A prototypical example of integrin modulation is the transition of platelet αIIbβ3 (GPIIb-IIIa complex) from a low affinity/avidity state to a state in which it can effectively bind soluble ligands such as fibrinogen (3Phillips D.R. Prasad K.S. Manganello J. Bao M. Nannizzi Alaimo L. Curr. Opin. Cell Biol. 2001; 13: 546-554Crossref PubMed Scopus (92) Google Scholar, 4Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar, 5Woodside D.G. Liu S. Ginsberg M.H. Thromb. Haemostasis. 2001; 86: 316-323Crossref PubMed Scopus (65) Google Scholar). Recent evidence suggests that physiologically relevant signals are transduced to the integrin through its cytoplasmic domains by intracellular associated factors, such as β3-endonexin, calcium integrin-binding protein, or cytoskeletal protein, talin (6Kashiwagi H. Schwartz M.A. Eigenthaler M. Davis K.A. Ginsberg M.H. Shattil S.J. J. Cell Biol. 1997; 137: 1433-1443Crossref PubMed Scopus (115) Google Scholar, 7Naik U.P. Patel P.M. Parise L.V. J. Biol. Chem. 1997; 272: 4651-4654Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 8Vinogradova O. Velyvis A. Velyviene A. Hu B. Haas T. Plow E. Qin J. Cell. 2002; 110: 587-597Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Studies of gene-targeted murine platelets or cultured megakaryocytes have defined the requirements for intracellular signaling molecules including Syk, phosphatidylinositol 3-kinase, vasodilator-stimulated phosphoprotein, calpain, and Rap1b (9Hauser W. Knobeloch K.P. Eigenthaler M. Gambaryan S. Krenn V. Geiger J. Glazova M. Rohde E. Horak I. Walter U. Zimmer M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8120-8125Crossref PubMed Scopus (193) Google Scholar, 10Azam M. Andrabi S.S. Sahr K.E. Kamath L. Kuliopulos A. Chishti A.H. Mol. Cell. Biol. 2001; 21: 2213-2220Crossref PubMed Scopus (215) Google Scholar, 11Bertoni A. Tadokoro S. Eto K. Pampori N. Parise L.V. White G.C. Shattil S.J. J. Biol. Chem. 2002; 277: 25715-25721Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In addition, cell surface membrane proteins including urokinase plasminogen activator receptor, CD98, platelet endothelial cell adhesion molecule-1, and integrin-associated protein (IAP/CD47) 1The abbreviations used are: IAP, integrin-associated protein; srIAP, soluble recombinant form of IAP; TS, thrombospondin; FITC, fluorescein isothiocyanate; MAP, mitogen-activated protein; CHO, Chinese hamster ovary; IL-2, interleukin 2; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. are also likely involved in the affinity modulation (12Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (260) Google Scholar). Thus, the control of platelet αIIbβ3 seems to be complex. Recent crystallographic determination of the structure of integrin has shown that a bent integrin conformation has low affinity, and an extended structure is linked to high affinity access of macromolecular ligands to its contact site in the integrin head (13Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar, 14Arnaout M.A. Goodman S.L. Xiong J.P. Curr. Opin. Cell Biol. 2002; 14: 641-651Crossref PubMed Scopus (170) Google Scholar). Such a dynamic conformational rearrangement in the integrin extracellular domains can be induced by ligand mimetic peptides, Mn2+, and also by inside-out signaling. However, the molecular basis for regulation of its extracellular conformation is not entirely clear. IAP is a 50-kDa membrane glycoprotein that has 5 transmembrane-spanning regions and 1 immunoglobulin (Ig)-like extracellular domain (15Brown E.J. Frazier W.A. Trends Cell Biol. 2001; 11: 130-135Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 16Brown E.J. Curr. Opin. Cell Biol. 2002; 14: 603-607Crossref PubMed Scopus (51) Google Scholar). It was originally reported to be physically associated with certain integrins including α2β1, αVβ3, and αIIbβ3 (17Chung J. Wang X.Q. Lindberg F.P. Frazier W.A. Blood. 1999; 94: 642-648Crossref PubMed Google Scholar, 18Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (186) Google Scholar, 19Chung J. Gao A.G. Frazier W.A. J. Biol. Chem. 1997; 272: 14740-14746Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Blockade of IAP with monoclonal antibody inhibits some aspects of integrin function, and ligation of IAP with activating antibodies induces the modulation of integrin-dependent cell adhesion. A gene-targeting study showed that IAP plays a key role in host defense by participation in migration and activation of leukocytes in response to bacterial infection (20Lindberg F.P. Bullard D.C. Caver T.E. Gresham H.D. Beaudet A.L. Brown E.J. Science. 1996; 274: 795-798Crossref PubMed Scopus (300) Google Scholar). Subsequently, it was reported that IAP is a receptor for the C-terminal cell binding domain of thrombospondin (TS) (21Gao A.G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). TS, its C-terminal cell binding domain, and a peptide from the C-terminal cell binding domain, KRFYVVMWKK (4N1K), all stimulate the integrin-dependent adhesion, spreading, and motility of the cells including endothelial cells, leukocytes, and smooth muscle cells (18Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (186) Google Scholar, 22Wang X.Q. Frazier W.A. Mol. Biol. Cell. 1998; 9: 865-874Crossref PubMed Scopus (139) Google Scholar). On the other hand, IAP is a ligand for the transmembrane signal-regulatory protein (23Seiffert M. Brossart P. Cant C. Cella M. Colonna M. Brugger W. Kanz L. Ullrich A. Buhring H.J. Blood. 2001; 97: 2741-2749Crossref PubMed Scopus (153) Google Scholar). In this case, IAP likely plays a role in macrophage function. The role on hematopoiesis (24Furusawa T. Yanai N. Hara T. Miyajima A. Obinata M. J. Biochem. 1998; 123: 101-106Crossref PubMed Scopus (23) Google Scholar) or adhesion of sickle red blood cell (25Brittain J.E. Mlinar K.J. Anderson C.S. Orringer E.P. Parise L.V. Blood. 2001; 97: 2159-2164Crossref PubMed Scopus (51) Google Scholar) was also reported. In platelets, the functional role of IAP was hardly detected using only a monoclonal antibody against IAP (26Fujimoto T. Fujimura K. Noda M. Takafuta T. Shimomura T. Kuramoto A. Blood. 1995; 86: 2174-2182Crossref PubMed Google Scholar). However, the peptide 4N1K induces platelet aggregation (27Dorahy D.J. Thorne R.F. Fecondo J.V. Burns G.F. J. Biol. Chem. 1997; 272: 1323-1330Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and spreading on immobilized fibrinogen or collagen (19Chung J. Gao A.G. Frazier W.A. J. Biol. Chem. 1997; 272: 14740-14746Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The ability of the peptide depends on its interaction with IAP because platelet response was decreased by treatment with an antifunctional IAP antibody or in platelets from IAP-deficient mice (17Chung J. Wang X.Q. Lindberg F.P. Frazier W.A. Blood. 1999; 94: 642-648Crossref PubMed Google Scholar). There are reports showing that the binding of 4N1K to IAP initiates intracellular signaling that would result in affinity modulation of αIIbβ3 (19Chung J. Gao A.G. Frazier W.A. J. Biol. Chem. 1997; 272: 14740-14746Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). 4N1K induces tyrosine phosphorylation of several proteins such as Syk and focal adhesion kinase. The effect of 4N1K is inhibited by pertussis toxin, indicating the participation of a heterotrimeric Gi protein (19Chung J. Gao A.G. Frazier W.A. J. Biol. Chem. 1997; 272: 14740-14746Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). This sort of stimulation of integrin function via G proteins is similar to that caused by agonists through seven transmembrane spanning receptors, such as ADP, epinephrine, and thrombin. Recently cloned ADP receptor (P2Y12) (28Hollopeter G. Jantzen H.M. Vincent D. Li G. England L. Ramakrishnan V. Yang R.B. Nurden P. Nurden A. Julius D. Conley P.B. Nature. 2001; 409: 202-207Crossref PubMed Scopus (1300) Google Scholar) also involves activation of Gi-containing heterotrimeric GTPases. However, these receptors are not necessarily present close to αIIbβ3. Therefore, the functional significance of the association of IAP with the integrin is not clear. In this report we investigate the mechanism by which IAP modulates the affinity of αIIbβ3. We provide evidence that only the extracellular Ig domain of IAP interacts with αIIbβ3 and can change αIIbβ3 to a high affinity state without requirement of intracellular signaling when it binds to TS. These phenomena might correspond with the dynamic structural change of integrin. The extracellular event reported here would be a novel mechanism of affinity modulation of integrin. Materials—Hybridoma cells producing anti-IAP monoclonal antibody B6H121 were purchased from American Type Culture Collection (ATCC; HB-9771, Manassas, VA). The antibody was purified from mouse ascites using HiTrap protein G affinity column (Amersham Biosciences) according to the manufacturer's instructions. T10, a monoclonal antibody that recognizes αIIbβ3 and inhibits fibrinogen binding, and Tab, an antibody that recognizes αIIb, were kindly provided by Dr. R. P. McEver (Oklahoma Medical Research Foundation, Oklahoma City, OK) (29McEver R.P. Bennett E.M. Martin M.N. J. Biol. Chem. 1983; 258: 5269-5275Abstract Full Text PDF PubMed Google Scholar). Anti-LIBS6, an activating anti-β3 antibody, was kindly provided by Dr. M. H. Ginsberg (Scripps Clinic, La Jolla, CA) (30Huang M.M. Lipfert L. Cunningham M. Brugge J.S. Ginsberg M.H. Shattil S.J. J. Cell Biol. 1993; 122: 473-483Crossref PubMed Scopus (159) Google Scholar). FITC-conjugated PAC1, a ligand-mimetic monoclonal antibody (31Shattil S.J. Cunningham M. Hoxie J.A. Blood. 1987; 70: 307-315Crossref PubMed Google Scholar), was purchased from BD Biosciences). M2 anti-FLAG antibody was from Eastman Kodak Co.. Anti-phospho-p44/p42 MAP kinase (Thr-202/Tyr-204) antibody and anti-p44/p42 MAP kinase antibody were from New England Biolabs (Beverly, MA). Control antibodies were from Organon Technica (West Chester, PA). FITC-conjugated goat F(ab′)2 anti-mouse immunoglobulins and horseradish peroxidase-conjugated anti-mouse IgG were from TAGO (Burlingame, CA). Human αIIbβ3 was purified from platelet concentrates as previously described (32Fujimoto T. Fujimura K. Kuramoto A. Thromb. Haemostasis. 1991; 66: 598-603Crossref PubMed Scopus (15) Google Scholar). Anti-αIIbβ3 antibody was raised by immunizing rabbits with purified human αIIbβ3 according to a standard protocol. IgG was purified by protein A CL-4B-Sepharose (Amersham Biosciences) according to the manufacturer's instructions. Specificity of the antibody was confirmed by Western blot using human platelets. ADP was purchased from Biopool (Ventura, CA). Human fibrinogen was purified as previously described (26Fujimoto T. Fujimura K. Noda M. Takafuta T. Shimomura T. Kuramoto A. Blood. 1995; 86: 2174-2182Crossref PubMed Google Scholar). A peptide from the C-terminal cell binding domain of TS, 4N1K (KRFYVVMWKK), and a control scrambled peptide (KVFRWKYVMK) were synthesized by BioSynthesis (Lewisville, TX) and purified by high performance liquid chromatography. CHO-K1 cells and Namalwa cells were grown in Dulbecco's modified Eagle's medium/F-12 and RPMI-1620 (Invitrogen), respectively, and supplemented with 10% fetal bovine serum under 5% CO2 at 37 °C. Expression vectors for αIIb and β3 in pBK-EF and CHO cells stably expressing αIIbβ3 were described previously (26Fujimoto T. Fujimura K. Noda M. Takafuta T. Shimomura T. Kuramoto A. Blood. 1995; 86: 2174-2182Crossref PubMed Google Scholar). Platelet Aggregation—Blood samples were collected from consenting healthy volunteer donors in a 1:10 volume of 3.8% trisodium citrate (w/v). Platelets were isolated from platelet-rich plasma by centrifugation at 800 × g for 10 min in the presence of 0.1 μg/ml prostaglandin E1 and 1 unit/ml apyrase. The pellet was resuspended and washed twice in 85 mm sodium citrate, 111 mm dextrose, 71 mm citric acid, pH 7.0, containing prostaglandin E1 and apyrase and then resuspended at a concentration of 3 × 108 platelets/ml in a modified Tyrode-HEPES buffer (138 mm NaCl, 0.36 mm NaH2PO4, 2.9 mm KCl, 12 mm NaHCO3, 10 mm HEPES, 5 mm glucose, 1 mm MgCl2, and 1 mm CaCl2, pH 7.4). Platelet aggregation was measured by the addition of peptides (4N1K or control peptide) at 37 °C in an aggregometer (Chrono-log, Havertown, PA), with continuous stirring at 1200 rpm. To inhibit the fibrinogen binding or 4N1K binding, 100 μg/ml T10 or B6H12 was added 10 min before aggregation measurements. B6H12 induced direct aggregation of the platelets from some subjects depending on the polymorphism of Fc receptor, FcγRII. Therefore, the effect of B6H12 was tested using the platelets from "non-responders." To block the intracellular metabolism, platelets were incubated with 0.4% sodium azide and 4 mg/ml 2-deoxy-d-glucose at 37 °C for 1 h. Human fibrinogen was added at a final concentration of 200 μg/ml before the addition of several agonists (100 μm peptides, 5 μg/ml collagen, or 5 μm ADP). Flow Cytometric Analysis—The affinity state of αIIbβ3 was determined by flow cytometric analysis using ligand-mimetic monoclonal antibody PAC1. PAC1 is an IgM monoclonal antibody that binds only to the activated form of αIIbβ3 in the same manner as the physiological ligand (31Shattil S.J. Cunningham M. Hoxie J.A. Blood. 1987; 70: 307-315Crossref PubMed Google Scholar). Platelets were suspended at a concentration of 2 × 107/ml in a modified Tyrode-HEPES buffer and stimulated with peptides or agonists in the presence of 20 μg/ml FITC-conjugated PAC1 for 20 min at room temperature without stirring. Fifty μl of each aliquot was then diluted with 500 μl of the buffer, and the mixture was directly analyzed by a flow cytometer, Epics XL (Coulter, Fullerton, CA). Single platelets were gated and analyzed. In the case of cultured Namalwa and CHO cells, cells were harvested and resuspended at 1 × 106 cells/ml in a modified Tyrode-HEPES buffer. Cells were incubated with 100 μm peptides and/or antibodies (50 μg/ml LIBS6 for stimulation and 100 μg/ml T10 or B6H12 for inhibition) in the presence of 20 μg/ml FITC-PAC1 for 30 min at room temperature. After washing, cells were stained with 1 μg/ml propidium iodide, and propidium iodide-negative cells were gated to exclude permeabilized dead cells. To estimate the expression of αIIbβ3 or IAP, cells were incubated with the first antibody, T10 or B6H12, (10 μg/ml) in Hanks' balanced salt solution with Ca2+ containing 1% fetal bovine serum and 0.1% NaN3. After washing, they were incubated again with FITC-conjugated goat F(ab′)2 anti-mouse immunoglobulins. Each incubation was performed on ice for 1 h. Cells were finally washed and analyzed in the buffer containing propidium iodide. Because the expression levels of wild type and mutant forms of IAP in CHO cells were different, PAC1 binding was standardized by the mean fluorescence intensity of B6H12 binding to each cell type. Mutational Analysis—Mutant cDNAs of αIIbβ3 and IAP were constructed by PCR according to the strategy previously described (33Fujimoto T. Stroud E. Whatley R.E. Prescott S.M. Muszbek L. Laposata M. McEver R.P. J. Biol. Chem. 1993; 268: 11394-11400Abstract Full Text PDF PubMed Google Scholar). To mutate Ser-752 of β3, PCR was performed using cDNA of wild type β3 as a template. The sense primer contained the BamHI site at base 1500 within the β3 cDNA, and the antisense primer contained the Ser-752 to Pro mutation. After gel purification, the PCR product was used for the second PCR. The second PCR was performed with the same sense primer and another antisense primer that overlapped with the first antisense primer and contained the stop codon of β3 cDNA and the HindIII site for cloning. The PCR fragment was digested by BamHI and HindIII, isolated by gel electrophoresis, and then used to replace the fragment of the wild type β3 cDNA extending from the BamHI site in the cDNA to the HindIII site in the multicloning site of the expression vector. IAP cDNA in the expression vector was previously described (26Fujimoto T. Fujimura K. Noda M. Takafuta T. Shimomura T. Kuramoto A. Blood. 1995; 86: 2174-2182Crossref PubMed Google Scholar). Wild type IAP in this study was identical to the so-called form 2, which is the most abundant isoform (15Brown E.J. Frazier W.A. Trends Cell Biol. 2001; 11: 130-135Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar). Two truncated mutants were constructed. In the first construct (IAP Δ291) a stop codon was introduced just after the 5th transmembrane domain, resulting in deletion of the C-terminal cytoplasmic tail after Lys-291. In the second (IAP Δ163) a stop codon was created after the first transmembrane domain, which resulted in deletion after Lys-163. In both cases PCR was performed with the sense primer containing the translation initiation codon and the antisense primer containing the indicated stop codon. In another mutant form (IAP-Tac) the extracellular domain of IAP was fused with the transmembrane and cytoplasmic domains of the IL-2 receptor α chain (Tac antigen/CD25). Primer sets were prepared in which half of the sequence was identical to the end of the extracellular domain of IAP, before Asn-142, and the other half was identical to the beginning of the transmembrane domain of the IL-2 receptor. Two PCR were performed separately to amplify the extracellular domain of IAP and the transmembrane and cytoplasmic domains of the IL-2 receptor. The PCR products were mixed, and the final PCR was performed using the outside primers. All constructs were then cloned into the expression vector, pBK-EF, and verified by nucleotide sequencing of the region encoding the PCR products using an automated DNA sequencer (ABI 310, Applied Biosystems, Foster City, CA). Cells Expressing Recombinant αIIbβ3 and IAP—Namalwa and CHO cells were transfected using DMRIE-C and LipofectAMINE reagents (Invitrogen), respectively, according to the manufacturer's instructions. Permanent transfectants were selected with G418 as described elsewhere. To establish the cells that stably express αIIbβ3 and/or IAP, stable cells were sorted by reactivity to the antibody T10 and/or B6H12 using a cell sorter (Epics Elite, Coulter, Fullerton, CA). Cells were cultured again for 5–7, days and the positive clones were obtained. Immunoprecipitation and Immunoblotting Analysis—To determine the association of αIIbβ3 and IAP, platelets or transfected cells were lysed in an ice-cold lysis buffer (10 mm Tris, pH 7.4, 100 mm NaCl, 1 mm CaCl2, 0.1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 100 μg/ml leupeptin). The lysates were chilled on ice for 1 h followed by centrifugation at 15,000 × g for 10 min. The supernatants were precleared with protein G-agarose (Immunopure Immobilized Protein G, Pierce), and the resultant supernatants were incubated for 1 h with protein G-agarose that was preincubated with antibodies T10, B6H12, or control antibody. Protein G-agarose was washed with the lysis buffer three times. The samples were then separated into two aliquots. One was solubilized with SDS sample buffer under reduced conditions and applied to 7% polyacrylamide electrophoresis gel (PAGE) for the detection of αIIbβ3. The other was incubated with SDS sample buffer under non-reduced conditions at 60 °C for 30 min and applied to 10% PAGE for IAP. The resolved proteins were then electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 10% skim milk in TBS buffer (10 mm Tris, 150 mm NaCl, pH 7.4) and then incubated with anti-αIIbβ3 rabbit polyclonal serum or 1 μg/ml B6H12 for 2 h. After extensive washing with TBS containing 0.1% Tween 20, antibody binding was detected using peroxidase-conjugated anti-rabbit or anti-mouse IgG and visualized with ECL chemiluminescence reaction reagents (Amersham Biosciences). MAP Kinase Phosphorylation—To determine the intracellular signaling events, MAP kinase activation was analyzed in the CHO cells expressing IAPs (34Wang X.Q. Lindberg F.P. Frazier W.A. J. Cell Biol. 1999; 147: 389-400Crossref PubMed Scopus (95) Google Scholar). Cells were incubated with 4N1K or control peptide for 10 min and lysed by the addition of an equal volume of 2× lysis buffer (35Fujimoto T. McEver R.P. Blood. 1993; 82: 1758-1766Crossref PubMed Google Scholar) (20 mm Tris, pH 7.4, 40 mm KH2PO4, 10 mm sodium orthovanadate, 40 mm molybdic acid, 80 mm sodium pyrophosphate, 0.2 mm trifluoroperazine, 2 mm EGTA, 20 mm benzamidine, 2 mm phenylmethylsulfonyl fluoride, 200 μg/ml of leupeptin, and 2% Triton X-100). After removal of the insoluble fraction by centrifugation, cell lysates were subjected to immunoblotting with anti-phospho-MAPK antibody. Immunoblotting was performed as described above, except that blocking was with 2% bovine serum albumin. To reprobe with anti-MAPK antibody to detect the total MAPK levels, membranes were incubated in stripping buffer (62.5 mm Tris, pH 6.7, 100 mm 2-mercaptoethanol, 2% SDS) at 70 °C for 1 h, washed, and then used for the second immunoblotting. Recombinant Soluble Form of IAP and Pull-down Assay—An expression vector that sequentially introduces a FLAG epitope, His tag, and a stop codon at 3′-end of cDNA in pBK-EF was kindly provided by Dr. K. Fukudome (Saga Medical College, Saga City, Japan) (36Fukudome K. Kurosawa S. Stearns Kurosawa D.J. He X. Rezaie A.R. Esmon C.T. J. Biol. Chem. 1996; 271: 17491-17498Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). To make recombinant soluble IAP (rsIAP), the extracellular domain of IAP until Asn-142 was PCR-amplified, digested, and ligated to this vector. Human 293 cells were transfected and selected with G418. High producing clones were screened in culture dishes by agarose diffusion immunoassay for secreted protein as previously described (37Ushiyama S. Laue T.M. Moore K.L. Erickson H.P. McEver R.P. J. Biol. Chem. 1993; 268: 15229-15237Abstract Full Text PDF PubMed Google Scholar). Immunoassay was performed with B6H12 and the anti-FLAG antibody, M2. The cells producing the highest amount of rsIAP were grown until semi-confluent, and the culture medium was changed to serum-free medium for 293 cells, SFMII (Invitrogen). After 5 days of culture, the medium was collected, and the rsIAP was purified by a His-Trap chelating column (Amersham Biosciences) according to the manufacturer's instructions. The purification procedure was repeated twice, and the protein concentration was determined by BCA protein assay (Pierce). The effects of rsIAP on the affinity state of αIIbβ3 in CHO cells were analyzed by flow cytometer. To analyze the association of αIIbβ3 and rsIAP in vitro, pull down assay was performed. Purified human αIIbβ3 (32Fujimoto T. Fujimura K. Kuramoto A. Thromb. Haemostasis. 1991; 66: 598-603Crossref PubMed Scopus (15) Google Scholar) and rsIAP (10 μg each) were mixed in a buffer containing 50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm CaCl2, 0.5 mm MgCl2, 1% CHAPS, and then 100 μg peptide was added in a total volume of 100 μl for 3 h. Protein G-agarose, which was preincubated with 5 μg of antibodies Tab or M2, was added and further incubated for 1 h. The protein G-agarose was subsequently washed three times with the above buffer. Bound proteins were released in the SDS sample buffer and then determined by immunoblotting. 4N1K Induces the Activation of αIIbβ3 on Human Platelets— 4N1K peptide, which is from the C-terminal cell binding domain of TS, induced aggregation of washed human platelets dose-dependently, with a maximal response at 100 μm. The mutated control peptide did not. Pretreatment with an inhibitory anti-αIIbβ3 antibody, T10, partially blocked the aggregation by ∼50%. A functional antibody for IAP, B6H12, also partially inhibited 4N1K-induced aggregation (Fig. 1A). We next tested platelets in which intracellular response was prevented (energy-depleted). The mechanism of inside-out signaling is not entirely clear, but it is known that the affinity modulation of platelet αIIbβ3 requires metabolic energy and can be abolished by pretreatment with sodium azide and 2-deoxy-d-glucose (6Kashiwagi H. Schwartz M.A. Eigenthaler M. Davis K.A. Ginsberg M.H. Shattil S.J. J. Cell Biol. 1997; 137: 1433-1443Crossref PubMed Scopus (115) Google Scholar). In the treated platelets, aggregation induced by collagen or ADP was completely abolished. However, 4N1K induced aggregation in the presence of fibrinogen, although the aggregation was decreased compared with non-treated platelets. This aggregation was also partially inhibited by antibody T10 or B6H12 (Fig. 1B). This result suggested that 4N1K induced platelet aggregation in part through agglutination, which was not dependent on αIIbβ3 and IAP, as reported recently (38Tulasne D. Judd B.A. Johansen M. Asazuma N. Best D. Brown E.J. Kahn M. Koretzky G.A. Watson S.P. Blood. 2001; 98: 3346-3352Crossref PubMed Scopus (41) Google Scholar), but also indicated the presence of aggregation mediated by αIIbβ3 and IAP. The latter part of the response partially depended on intracellular metabolism. The activation state of αIIbβ3 was estimated by the binding of ligand mimetic antibody, PAC1 (Fig. 2). 4N1K induced PAC1 binding to platelets, whereas control peptide did not. The binding was completely inhibited by T10 and also partia
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