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Prepublished online as a Blood First Edition Paper on June 19, 2003; DOI 10.1182/blood-2003-01-0213.
Blood, 1 October 2003, Vol. 102, No. 7, pp. 2491-2497
Disruption of the
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IIb
3, is a noncovalent heterodimer of glycoproteins IIb and IIIa. This work was aimed at elucidating the role played by the carboxy-terminal extracellular, trans-membrane, and cytoplasmic regions of the glycoprotein 3 in the formation of functional complexes with subunits. Progressive carboxy-terminal deletions of 3 revealed that surface exposure of IIb3 or v3 could not occur in the absence of the transmembrane domain of 3. In contrast, internal deletions 616 to 690 of the carboxy-terminal regions of the 3 ectodomain led to surface exposure of constitu tive active receptors in CHO cells, as indicated by the enhanced rate of cell adhesion to immobilized ligands and spontaneous binding to soluble fibrinogen or activation-dependent antibody PAC-1. The functional analysis of cysteine mutations within the 616 to 690 region of 3 or chimeric 3-7 subunits revealed that disruption of the C663-C687 disulfide bridge endows constitutive activity to the IIb3 receptor. It is concluded that the carboxy-terminal tail of the 3 ectodomain, so-called tail domain (TD), is not essential for cell surface expression of 3 receptors. However, a basal, nonactivated, low ligand-affinity state of the 3 integrins demands a normal conformation of this domain. (Blood. 2003;102:2491-2497) |
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IIb3, is a calcium-dependent, noncovalent heterodimer formed by GPIIb and GPIIIa. This complex is found in the plasma membrane of megakaryocytes, platelets, and some tumor tissues1-3 and functions as a receptor for fibrinogen and other adhesive proteins like the von Willebrand factor, fibronectin, or vitronectin.4 The 3 subunit may also complex the GP v to form the vitronectin receptor (integrin v3) that shares with IIb3 the binding of fibrinogen although with different affinity.5 The platelet IIb3 complex is essential to maintain a normal hemostasis. Unlike other platelet receptors that are constitutively active, the IIb3 is maintained in a low-affinity state for its ligands. Disruption of the vascular endothelium and exposure of platelets to the action of agonists and adhesive proteins from the subendothelial matrix induces a cellular activation. The activated cells interact with adhesive proteins from the extracellular matrix,6,7 and the IIb3 receptors are able to bind fibrinogen with high affinity (inside out signaling), resulting in platelet aggregation.8 Conversely, ligand-bound IIb3 propagates signals to the interior of the cell (outside-in signaling) leading to enhanced interaction with the cytoskeleton, clustering of receptors (increased ligand avidity), and formation of focal contacts rich in signaling complexes.9,10 The agonist-induced increase in ligand affinity of IIb3 is thought to be the result of conformational changes of the heterodimer11-13 initiated by the interaction of the cytoplasmic tails of and 3 subunits with cytosolic proteins. Despite the pathophysiologic importance of the platelet IIb3 receptor, the knowledge of the mechanisms controlling its state of activation is rather limited.
Unlike previous reports,14 recent work from our laboratory15 showed that a truncated form of 3 lacking the transmembrane and cytosolic domains failed to associate with IIb. The present work was aimed at further investigating the role played by the carboxy-terminal domain of 3 in the surface expression and function of 3 heterodimers. The results obtained in this study indicate that surface expression of IIb3 could not occur in the absence of the transmembrane domain of 3. The present study has also revealed that either deletion of the carboxy-terminal region of the 3 ectodomain or disruption of the 663-687 disulfide bridge confers constitutive activity to the 3 integrins, suggesting that this region is involved in maintaining the IIb3 receptor in a resting, nonactivated state.
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Restriction enzymes were obtained from Boehringer (Mannheim, Germany). The pcDNA3 expression vector was from Invitrogen (San Diego, CA). Oligonucleotides were synthesized by Invitrogen (Paisley, Scotland). Most other reagents were purchased from Sigma Chemical (St Louis, MO), Merck (Darmstadt, Germany), or Calbiochem-Novabiochem (San Diego, CA). Monoclonal antibodies (mAbs) anti-IIb (2bc1) and anti-3 (H1a) were obtained in our laboratory; the anti-v3 MAB1976 was purchased from Chemicon (Temecula, CA), and fluorescein isothiocyanate (FITC)-PAC-1 was obtained from Becton Dickinson (San Jose, CA). WOW-1 F(ab')2 and PAC-1 F(ab')2 were obtained from Dr S. Shattil (The Scripps Research Institute, La Jolla, CA).
Construction of expression vectors with normal or mutant 3 cDNAs
Wild 3-cDNA cloned in EcoRI and HindIII sites of pBluescript II-KS (Stratagene, La Jolla, CA) was digested with XhoI and partially with BamHI, and subcloned in pcDNA3. pcDNA3-3
616 construct was prepared as described previously.15 To generate truncated 3 at 638, 657, 675, and 693 amino acids, stop codons were introduced by polymerase chain reaction (PCR) using pcDNA3-3 as template, and oligonucleotide 3-sense (1670-1689): 5'-ACTGCAACTGTACCACGCGT-3' and the antisense primers 3-AS-638-stop-XhoI: 5'-TATCTCGAGCTAGTCACGGCAG-3', 3-AS-657-stop-XhoI: 5'-TATCTCGAGTTAGGTACAATTCAC-3', 3-AS-675-stop-XhoI: 5'-TAACTCGAGTCAACTAGAATCTTC-3', and 3-AS-693-stop-XhoI: 5'-TATCTCGAGTTAGTCAGGGCCC-3', respectively. Mutations introduced to generate stop codons are underlined. PCR products were digested with NotI and XhoI to replace the normal fragment from pcDNA3-3.
cDNAs encoding [608Ala]3, [614Ala]3, [617Ala]3, [663Ala]3, and [687Ala]3 mutants were prepared by the splicing by overlap extension PCR procedure using the oligonucleotide primers 3-sense (1670-1689) and 3-AS-(2319-2296)-XhoI: 5'-TGATAATGACTCGAGGATGACTGC-3', and the overlapped pairs of the following primers: [608Ala]3-S: 5'-CCAGATGCCGCCACCTTTAAG-3' and [608Ala]3-AS: 5'-CTTAAAGGTGGCGGCATCTGG-3'; [614Ala]3-S: 5'-AAGAAAGAAGCTGTGGAGTGT-3' and [614Ala]3-AS: 5'-ACACTCCACAGCTTCTTTCTT-3'; [617Ala]3-S: 5'-TGTGTGGAGGCTAAGAAGTTTGAC-3' and [617Ala]3-AS: 5'-GTCAAACTTCTTAGCCTCCACACA-3'; [663Ala]3-S: 5'-GAGGATGACGCTGTCGTCAGA-3' and [663Ala]3-AS: 5'-TCTGACGACAGCGTCATCCTC-3'; [687Ala]3-S: 5'-GAGCCAGAGGCTCCCAAGGGC-3' and [687Ala]3-AS: 5'-GCCCTTGGGAGCCTCTGGCTC-3'. The [608Ala]3, [614Ala]3, [617Ala]3, and [663Ala]3 cDNAs were used as template to generate [608Ala/655Ala]3, [614Ala/635Ala]3, [617Ala/631Ala]3, and [663Ala/687Ala]3 mutant cDNAs, respectively, and the overlapped pairs of primers: [655Ala]3-S: 5'-GCAGTGAATGCTACCTATAAG-3' and [655Ala]3-AS: 5'-CTTATAGGTAGCATTCACTGC-3'; [635Ala]3-S: 5'-AACCGTTACGCCCGTGACGAG-3' and [635Ala]3-AS: 5'-CTCGTCACGGGCGTAACGGTT-3'; [631Ala]3-S: 5'-GACGAAAATACCGCCAACCGTTAC-3' and [631Ala]3-AS: 5'-GTAACGGTTGGCGGTATTTTCGTCATG-3'; and [687Ala]3-S and [687Ala]3-AS, respectively. Final PCR products carrying the desired mutations were digested with XhoI and MluI and ligated into pcDNA3 carrying normal 3 cDNA digested with XhoI and partially digested with MluI.
3(685-690del), 3(654-690del), and 3(616-690del) were obtained by PCR amplification from the plasmid pBJ1-3, with antisense primers located just before the region to delete: 3-AS-(2055-2030): 5'-TGGCTCTTCTACCACATACGGATGG-3', 3-AS-(1959-1938): 5'-CACTGCATCCTTGCCAGTGTCC-3' and 3-AS-(1845-1819): 5'-CACACATTCTTTCTTAAAGGTGCAGGC-3', respectively, and the sense primer 3-S-(2071-2092): 5'CCTGACATCCTGGTGGTCCTGC-3', located after the fragment to be removed. The PCR products were phosphorylated and ligated to obtain pBJ1-3(685-690del), pBJ1-3(654-690del), and pBJ1-3(616-690del) that were used as template for another PCR performed with oligonucleotides 3-S-(1670-1689) and 3-AS-(2319-2296)-XhoI. The resulting PCR products were digested with NotI and XhoI to replace the normal fragment from pcDNA3-3.
A 3-7 chimera was obtained as follows. The 2078-2323 fragment of 7 was amplified by PCR with the oligonucleotides 7-S-(2078-2095): 5'-ACACCATGGCTAGCACACCGGGAC-3' and 7-AS-(2323-2298): 5'-CGTGTGGTATCGATCCTTTTCTTGG-3', containing a NheI and a ClaI site, respectively. The PCR product was digested with NheI and ClaI and ligated to pcDNA3-3 where the homologous fragment had been deleted. For this, NheI and ClaI were previously introduced in the 3 sequence using the overlapped oligonucleotide primers 3-S (2137-2160)-ClaI: 5'-TGTCCCAAGGATCGATACATCCTG-3' and 3-AS-(2160-2137)-ClaI: 5'-CAGGATGTATCGATCCTTGGGACA-3'. The cDNA so obtained was used as a template to introduce the NheI site with the following overlapped oligonucleotides: 3-S (1918-1939)-NheI: 5'-TGTGTGGAGCTAGCGAAGTTTGAC-3' and 3-AS (1939-1918)-NheI: 5'-GTCAAACTTCGCCTAGCTCCACACA-3'. The final PCR product carrying the NheI and ClaI sites was digested with XhoI and MluI and ligated into pCDNA3-3 cDNA digested with XhoI and partially digested with MluI. The DNA sequence of each construct was verified. The IIb cDNA was cloned into the HindIII site of pcDNA3, as previously described.16
Cell culture and transfection
CHO cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells were transiently cotransfected by the diethylaminoethyl (DEAE)-dextran method17 with normal or mutated pcDNA3-3 constructs and, when indicated, with pcDNA3-IIb. Forty-eight hours after transfection the cells were harvested and the surface expression of IIb3 complexes was assessed by flow cytometry analysis or immunoprecipitation.
CHO cell lines stably expressing 3 or 3(616-690del) were established by transfection with 15 µg pcDNA3-3 or pcDNA3-3(616-690del), using the calcium phosphate precipitation procedure. The transfected cells were fed with medium containing 800 µg/mL G-418 every 3 to 4 days, and clones of cells stably expressing 3 were selected by flow cytometry analysis. Cells stably expressing 3 or 3(616-690del) were transfected with pcDNA3-IIb, and cells expressing IIb were cloned by cell sorting with fluorescence-activated cells sorting (FACS Vantage; Becton Dickinson) with argon laser tuned to 488 nm of excitation and specific fluorescence signals collected by a 530-bp filter.
Adhesion of CHO cell lines to immobilized fibrinogen and vitronectin
The 96-well flat-bottomed plates were coated with 10 µg/mL fibrinogen in phosphate-buffered saline (PBS) pH 7.2 (100 µL/well) for 2 hours at 37°C and then washed with PBS, blocked with 200 µL 1% bovine serum albumin (BSA) for 1 hour at 37°C, and washed with PBS. CHO cell lines were labeled with 10 µM calcein-AM (Molecular Probes, Leiden, The Netherlands) for 10 minutes, washed by centrifugation, and resuspended in serum-free DMEM, plated onto dishes in duplicate at a density of 4 x 104 cells/well and incubated for different periods of time at 37°C. Nonadherent cells were removed by carefully washing twice with 200 µL PBS. Cell adhesion was quantitated by cytofluorometry. Basal adherence to BSA (cell binding to BSA-coated wells was always < 1%) was subtracted from attachment values. To study adhesion of cell lines to vitronectin, cytomatrix cell adhesion strips with human vitronectin (Chemicon) were used following the manufacturer's recommendations. At the end of the assays, adherent cells were examined with a phase-contrast Nikon TMS microscope (Barcelona, Spain) and micrographs were taken with a digital camera.
Soluble fibrinogen-dependent aggregation of CHO cell lines
CHO cells were incubated with 1 mg/mL fibrinogen for 15 minutes at room temperature at a density of 2.5 x 106 cells/mL. Then, 250 µL cell suspension was plated onto wells of a 24-well culture dish precoated with 1 mg/mL BSA. Ten to 15 minutes after plating, cell aggregates were examined by phase-contrast microscopy as described. In some cases, cells were preincubated with 10 µg anti-3 antibody H1a or 10 mM EDTA (ethylenediaminetetraacetic acid).
Flow cytometric analysis of IIb and 3
Transfected cells were harvested using 0.5 mM EDTA in PBS, washed with PBS, resuspended at a density of 106 cells/100 µL, and incubated for 20 minutes at 4°C with mAbs specific to IIb (2bc1), 3 (H1a), or v3 (MAB1976). Next, cells were washed and exposed to FITC-F(ab')2 fragment of rabbit antimouse Ig (Dako, Glostrup, Denmark) at 4°C for 20 minutes, and the surface fluorescence was analyzed in a Coulter flow cytometer, model EPICS XL (Miami, FL).
Binding of fibrinogen and PAC-1 to transfected CHO cells
Human fibrinogen was labeled with FITC as described previously.16 CHO cells (5 x 105) coexpressing normal or mutated 3, and either human IIb or endogenous v were resuspended in Tyrode buffer (5 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 2 mM MgCl2, 0.3 mM NaH2PO4, 3 mM KCl, 134 mM NaCl, 12 mM NaHCO3, 0.1% glucose, 0.1% BSA, and 1 mM CaCl2, pH 7.0) with 10 µg FITC-fibrinogen and incubated for 25 minutes at room temperature. After washing, cells were resuspended and analyzed by flow cytometry. When indicated, cells were preincubated for 30 minutes at 4°C with mAbs directed against activated v3 (WOW-1) or activated IIb3 (PAC-1), 2 mM MnCl2, or 10 mM EDTA.
To assess the binding of PAC-1, cells were resuspended in Tyrode buffer, incubated at room temperature for 15 minutes with FITC-PAC-1, and then cell fluorescence was determined by flow cytometry.
Metabolic labeling and immunoprecipitation analysis of IIb3 complexes
Metabolic labeling of transfected CHO cells was performed 48 hours after transfection. First, cells were incubated in medium without methionine and cysteine for 30 minutes and, then, with [35S]-methionine/cysteine (400 µCi/mL; 14.8 MBq) for 3 hours. Cells were washed 3 times with PBS and extracted in 0.5 mL lysis buffer (50 mM Tris [tris(hydroxymethyl)aminomethane], pH 7.4, 150 mM NaCl, 2 mM phenylmethylsulfonyl fluoride [PMSF], 1% Triton X-100, 0.05% Tween 20, and 0.03% sodium azide). Precleared lysates were immunoprecipitated with either anti-3 or anti-IIb. The immunoprecipitates were then bound to protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech, Uppsala, Sweden), washed, and eluted by incubating 10 minutes at 100°C in 50 µL reducing loading buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 5% -mercaptoethanol, 4% sodium dodecyl sulfate [SDS], and 0.002% bromophenol blue) and, finally, resolved by electrophoresis. The gels were vacuum dried and exposed to hypersensitive x-ray film for 48 hours.
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3 on the surface exposure of heterodimers
We have recently reported a nonsense mutation producing a truncated form of 3 (3616) associated with thrombasthenic phenotype (type I Glanzmann thrombasthenia).15 The underlying molecular mechanism for the thrombasthenic phenotype was the failure of 3616 to complex IIb. This observation appeared to be in conflict with previous reports in which 3-truncated proteins complex subunits14,18,19 and the mutated complexes reached the cell surface.14 Because the apparent discrepancy could be due to differences in the lengths of the deleted fragments, we performed a transient transfection analysis of progressive carboxy-terminal or internally deleted forms of 3. Figure 1 depicts the deletion mutants used for transfection analysis (Figure 1A) as well as some structural features of the carboxy-terminal ectodomain of 3 (Figure 1B) based on recent studies on the crystal structure of heterodimeric v and 3 ectodomains.20
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Whether the carboxy-terminal-deleted constructs were transfected alone or with IIb, no detectable surface expression of 3 or 3 heterodimers was appreciated by flow cytometry (Figure 2). In contrast, deletion of the 616-690 carboxy-terminal region of the 3 ectodomain (616-690del) did not prevent the surface expression of v3 or IIb3 heterodimers. According to these observations, a normal level of surface exposure of these receptors demands the presence of the transmembrane and cytosolic regions of 3.
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Immunoprecipitation analysis of metabolically labeled cells cotransfected with IIb3 were performed (Figure 3). When anti-3 was used, immunoprecipitates of the expected size for the normal and mutated forms of 3 were obtained. All the anti-3 immunoprecipitates contain products with apparent molecular size of pro-IIb, whereas mature heavy chains of IIb were detected only in cells transfected with either normal 3 or 3(616-690del). When anti-IIb was used, pro-IIb was precipitated in all cases, and IIb and 3 were only coprecipitated in cells transfected with either normal 3 or 3(616-690del). Because the proteolytic cleavage of pro-IIb occurs only when the subunit is complexed to 3, the absence of the mature heavy chain of GPIIb suggests absent or very low rate of pro-IIb-3 complex formation. Immunoprecipitation of concentrated 48-hour-conditioned medium with either anti-3 or anti-IIb did not yield detectable amounts of truncated 3. Thus, 3 forms are not secreted to the extracellular medium, either alone or associated with heavy chain, at a significant rate; however, it cannot be excluded that the 3 or the IIb3 complexes could have been rapidly degraded.
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Functional features of v3(616-690del) andIIb3(616-690del) complexes
CHO cells expressing recombinant IIb3 receptors adhere spontaneously to solid-phase fibrinogen. Thus, we found it of interest to examine whether the mutant IIb3(616-690del) complexes exhibited a distinct capacity of adhesion onto ligand-coated plates. The CHO-IIb3(616-690del) cells were larger than cells expressing normal complexes and showed enhanced fibrinogen concentration-dependent adherence onto ligand-coated plates (Figure 4A). Because CHO cells express endogenous v, we stably transfected normal 3 or 3(616-690del) and studied the adherence of normal CHO-v3 and CHO-v3(616-690del) cells to plates coated with either vitronectin (Figure 4B) or fibrinogen (Figure 4C). In both conditions the cells expressing the mutant complexes showed enhanced adherence to coated plates.
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Normal resting CHO-IIb3 cells in suspension do not bind to soluble fibrinogen at significant rates and, therefore, as expected, no spontaneous aggregation was observed (Figure 5). In contrast, CHO-IIb3(616-690del) cells in suspension underwent receptor and soluble fibrinogen-dependent spontaneous aggregation and this effect was prevented by pretreatment with either EDTA or an anti-3 function blocking mAb (Figure 5). The fibrinogen-dependent aggregation of CHO-IIb3(616-690del) cells suggested that the mutant receptor was constitutively active. To further investigate this point, we studied the spontaneous binding to soluble FITC-labeled fibrinogen of cells expressing either normal or mutated complexes. As shown in Figure 6, CHO-IIb3(616-690del) but not CHO-IIb3 cells bound to soluble fibrinogen. Fibrinogen (Fg) is a ligand for both IIb3 and v3. Thus, to assess the binding of FITC-Fg to each integrin, the cells were incubated with function-blocking, activation-dependent mAbs directed against either IIb3 (PAC-1) or v3 (WOW-1). As observed in Figure 6, the binding of FITC-Fg was partially prevented by both WOW-1 and PAC-1. In agreement with their different sites of action, the combined administration of WOW-1 and PAC-1 showed additive effects, so that the binding of cells to FITC-Fg was virtually abolished (data not shown). The presence of Mn2+ enabled CHO-v3 or CHO-IIb3 cells to bind to soluble fibrinogen. Cells expressing the 3(616-690del) subunit showed spontaneous binding to FITC-Fg that was further stimulated by Mn2+ (Figure 6 lower panels). In all conditions, the binding of cells to FITC-Fg was completely abolished by preincubation with EDTA. We next tried to determine whether 3 subunits containing deletions shorter than 616-690del, such as 654-690del or 685-690del, would confer receptor activity. All the constructs yielded normal rates of cell surface expression of IIb3 complexes (Figure 7A). The cells carrying IIb3 receptors with internally deleted 3 subunit, even one as small as 5 residues, 3(685-690del), exhibited enhanced binding to PAC-1 and FITC-Fg (Figure 7B-C). EDTA prevented the binding of cells to FITC-Fg (Figure 7C).
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A recent crystallographic study on the v3 integrin described a new fold in the carboxy-terminal end 606-690 of the 3 ectodomain, named tail domain (TD), that shows a weak homology to the cytostatin fold and contains 4 disulfide bridges (608Cys-655Cys, 614Cys-635Cys, 617Cys-631Cys, and 663Cys-687Cys).20 Since the smaller deletion conferring receptor activity, 3(685-690del), disrupts the 663-687 disulfide bridge, we next investigated the functional effect of disrupting each of the disulfide bridges of the TD, by replacing the cysteine residues by alanine. None of these mutations altered the normal levels of surface expression of IIb3 complexes (Figure 8A); however, disruption of the 663-687 disulfide bridge enabled the cells to bind spontaneously to soluble fibrinogen and PAC-1 (Figure 8B-C). To further verify the role of this disulfide bridge, we mutated only the 687C>A. The 3(685-690del) construct as well as [663Ala-687Ala]3 and [687Ala]3 mutants were less efficient than 3(616-690del) in enhancing the receptor activity (approximately 50%). The latter observation suggested that both shortening of the carboxy-terminal ectodomain of 3 and disulfide bridge disruption could have contributed to confer constitutive activation to 3(616-690del). To further investigate this point, we prepared a chimeric 3 subunit in which the 616-690 fragment was replaced with the homologous domain of 7 that lacks the cysteines found in 3 at positions 663 and 687. Figure 8B-C shows that heterodimers of the 3-7 chimera bound spontaneously to PAC-1 and fibrinogen to a similar extent as the 663Ala-687Ala and 687Ala 3 mutants, that is, less than 3(616-690del). This observation supports the idea that both disruption of the 663Cys-687Cys disulfide bridge and shortening of the carboxy-terminal ectodomain enhance the activity of 3 heterodimers by different yet additive mechanisms. Although the importance of the TD in maintaining the basal, nonactive state of the 3 receptors is evident under our experimental conditions, the physiologic significance of these findings is difficult to ascertain. The possibility exists that a normal mechanism of receptor activation could involve the sliding of the subunits with the result of a relative shortening of the 3. On the other hand, molecular genetic defects in the TD of 3 might be associated with constitutive active 3 receptors and subsequent pathologic disorders.
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3 on the assembly and surface exposure of IIb3 complexes
We have recently reported a case of Glanzmann thrombasthenia associated with a point mutation that changed Glu616 to a termination codon resulting in a truncated 3 protein that lacks the transmembrane and cytoplasmic regions.15 The lack of surface exposure of IIb3616 was caused by a failure of the truncated protein to complex IIb. This observation appeared to be in conflict with a previous report indicating that a truncated 3 lacking the transmembrane and cytoplasmic domains (3693) allowed assembly and surface exposure of IIb3 complexes.14 Moreover, truncated forms of recombinant IIb and 3 seem to form soluble IIb-3 heterodimers capable of binding to ligands.21 To determine whether the apparent discrepancy could be due to the extension of the 3 truncation in each experimental condition, we analyzed the ability of 3-truncated (3) forms of different lengths to complex IIb. We failed to demonstrate association of any of the 3 forms tested with IIb. The proteolytic cleavage of pro-IIb to yield IIb heavy (IIbH) and light (IIbL) chains requires a previous association of and subunits; therefore, the lack of coprecipitation of IIbH with 3 indicates that pro-IIb failed to form stable complexes with any of the 3 forms analyzed. This assertion agrees with the observation that the Iraqi-Jewish thrombasthenic phenotype associated with the 3650 mutation shows detectable platelet pro-IIb22 and absence of platelet IIb3 and v3 receptors23,24; moreover, the recombinant 3657 does not complex IIb.25 However, truncated 3 preserving the transmembrane domain and 1 or 8 of the 47 cytoplasmic amino acids have been reported to reach the cell surface.26,27 On the other hand, the IIb and 3 ectodomains can form heterodimers that bind ligands and exhibit immunochemical properties similar to the full-length receptor inserted in the plasma membrane.21,28 On these grounds, it seems plausible to conclude that the cell surface exposure of detectable levels of IIb3 demands the presence of the transmembrane region of 3.
State of activation of recombinant v3(616-690del) and IIb3(616-690del) receptors
Platelet IIb3 receptors, about 80 000/cell,8 as well as their main ligands, fibrinogen and von Willebrand factor, are present in blood at high concentrations. Thus, to prevent intravascular aggregation, the receptors must be maintained in a low-affinity state for their ligands. The activation of platelets by physiologic agonists leads to increased fibrinogen binding to IIb3 and the ligand-bound receptors propagate signals into the cell resulting in cytoskeletal organization and receptor clustering.8-10 Domains within the cytoplasmic tails of and subunits seem to be involved in the bidirectional signaling through IIb3.29,30 The precise molecular mechanisms of receptor activation have not been yet elucidated, although early studies on this matter postulated that ligand affinity changes could be the result of conformational changes.11 In agreement with this postulate, recent crystallographic studies of the ectodomains of 3 revealed that the binding of the ligand mimetic RGD peptide is associated with precise conformational changes31-34 and that clasping of the and tails prevents the integrin activation.35 Moreover, analysis of peptide fragments from resting and activated IIb312 and 3-dimensional modeling of IIb3 based on electron microscopy and x-ray crystallographic studies13 reported the existence of domain-specific conformational changes in receptors from activated platelets. Studies of binding of platelets to monoclonal antibodies also suggest specific agonist-induced conformational changes in IIb3.36 Furthermore, point mutations in 3 leading to conformational changes are associated with changes in the state of activation of IIb3.37-42 Thus, a variety of experimental approaches support the contention that integrin activation is accompanied by conformational changes; however, the mechanisms by which the putative conformational changes increase the ligand binding affinity remain unclear.35 The same comment applies to the mechanisms involved in locking the fibrinogen receptor in a low-affinity state. Our observation that deletion of residues 616 to 690 in 3 endows constitutive activity to the IIb
3 663-687 disulfide bridge confers constitutive activity to 
) or 
). (B) Cells were transfected with either normal or mutant forms of 
