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Blood, 15 December 2004, Vol. 104, No. 13, pp. 3979-3985. Prepublished online as a Blood First Edition Paper on August 19, 2004; DOI 10.1182/blood-2004-04-1411. This Article Abstract Full Text (PDF) All Versions of this Article: 2004-04-1411v1 104/13/3979    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Litvinov, R. I. Articles by Weisel, J. W. Search for Related Content PubMed PubMed Citation Articles by Litvinov, R. I. Articles by Weisel, J. W. Related Collections Hemostasis, Thrombosis, and Vascular Biology Cell Adhesion and Motility Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Functional and structural correlations of individual IIb3 molecules Rustem I. Litvinov, Chandrasekaran Nagaswami, Gaston Vilaire, Henry Shuman, Joel S. Bennett, and John W. Weisel From the Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia; the Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia; and the Hematology-Oncology Division of the Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   The divalent cation Mn2+ and the reducing agent dithiothreitol directly shift integrins from their inactive to their active states. We used transmission electron microscopy and laser tweezers-based force spectroscopy to determine whether structural rearrangements induced by these agents in the integrin IIb3 correlate with its ability to bind fibrinogen. Mn2+ increased the probability of specific fibrinogen-IIb3 interactions nearly 20-fold in platelets, and both Mn2+ and dithiothreitol increased the probability more than 2-fold using purified proteins. Of 3 IIb3 conformations, closed with stalks touching, open with stalks separated, and globular without visible stalks, Mn2+ and dithiothreitol induced a significant increase in the proportion of open structures, as well as structural changes in the IIb3 headpiece. Mn2+ also increased the number of complexes between fibrinogen and purified IIb3 molecules, all of which were in the open conformation. Finally, Mn2+ induced the formation of IIb3 clusters that resulted from interactions exclusively involving the distal ends of the stalks. These results indicate that there is a direct correlation between IIb3 activation and the overall conformation of the molecule. Further, they are consistent with the presence of a linked equilibrium between single inactive and single active IIb3 molecules and active IIb3 clusters. (Blood. 2004;104:3979-3985)    Introduction Top Abstract Introduction Materials and methods Results Discussion References   The activity of circulating platelets is tightly regulated to prevent the spontaneous formation of platelet aggregates.1 Thus, circulating platelets are inactive until they adhere to exposed subendothelial matrix or are stimulated by soluble agonists such as adenosine diphosphate (ADP) and thrombin. Each activating event is associated with a change in platelet shape, reorganization of the platelet cytoskeleton, secretion of platelet granules, and an increase in the affinity of the integrin IIb3 for soluble ligands such as fibrinogen and von Willebrand factor. The latter is responsible for platelet aggregation when the macromolecular ligands bind to activated IIb3 and bridge adjacent platelets.2 Although the activation state of IIb3 is normally regulated by agonist-generated "inside-out" signaling,1 IIb3 can also be activated experimentally by perturbing the conformation of its extracellular domain using Mn2+ ions3,4 or the reducing agent dithiothreitol (DTT).5,6 Thus, in platelets, Mn2+ has been reported to activate v3 and IIb3, thereby promoting their interaction with ligands such as fibrinogen, von Willebrand factor, vitronectin, and osteopontin, mimicking the consequences of conventional inside-out signaling.3,4 Mn2+ also stabilizes platelet-fibrinogen interactions.7 Moreover, in experiments using purified integrins, Mn2+ affects the binding kinetics, affinity, and specificity toward synthetic and natural ligands.8-11 Mn2+-induced changes in integrin function have been attributed to specific conformational rearrangements in the integrin ectodomain,12 a suggestion supported by electron microscope studies showing that Mn2+ promotes the opening of integrin molecules into extended structures.13-15 Millimolar concentrations of DTT also induce platelet aggregation by directly stimulating ligand binding to IIb3.5,6 How DTT activates IIb3 is unclear. 3 has been reported to contain an extracellular redox site that is associated with the presence of 2 unpaired cysteines in inactive IIb3 and 6 unpaired cysteines following exposure of IIb3 to DTT.16 However, the identity of the putatively unpaired cysteines in either inactive or active forms of IIb3 has not been determined, and it has been proposed that IIb3 activation by DTT may involve disulfide bond rearrangement of the originally unpaired cysteines, as well as overall bond reduction.16,17 The ability of Mn2+ and DTT to enhance integrin function, as well as perturb the conformation of integrin ectodomains, provides an opportunity to test the hypothesis that there is an equilibrium between inactive and active integrin activation states that is a consequence of a reversible structural rearrangement of the entire integrin molecule. To address this hypothesis, we used transmission electron microscopy to probe for structural differences between IIb3 in the presence of Ca2+, Mn2+, and DTT and laser tweezers-based force spectroscopy to measure the fibrinogen-binding function of the integrin in the presence of each at the single molecule level. We found that both Mn2+ and DTT increase the probability of specific interactions between IIb3 and fibrinogen, but they do so without changing the average yield strength of fibrinogen binding. Both agents also induce a change in the shape of the IIb3 headpiece, shift IIb3 from a closed conformation with stalks touching to an open conformation with stalks separated, and stimulate the formation of clusters of the open IIb3 conformer. These results indicate that there is a direct correlation between the activation of IIb3 and the overall conformation of the molecule. Further, they are consistent with the presence of a linked equilibrium between inactive and active IIb3 molecules and IIb3 clusters.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Laser tweezers measurements Using laser tweezers to measure integrin function on platelets has been described previously in detail.18 Briefly, we used a custom-built laser tweezers setup assembled from a Nikon Diaphot 300 inverted microscope (Nikon, Mellville, NY), 100 x 1.3NA Fluor lens and a Spectra-Physics (Mountain View, CA) FCBar Nd:YAG laser to measure the strength of fibrinogen binding to human platelets or purified IIb3 in the presence of either Ca2+ or Mn2+. For these measurements, human fibrinogen (American Diagnostica, Stamford, CT) was covalently bound to 0.93 µm carboxylate-modified latex beads using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride as a cross-linking agent in a 2-step procedure described in the TechNote no. 205 issued by Bangs Laboratories (Fishers, IN). Before use, the fibrinogen-coated beads were disaggregated by mild sonication and used at a concentration of approximately 107/mL. For studies using purified human II3, the purified integrin (Enzyme Research Laboratories, South Bend, IN) at a concentration of 1 mg/mL in 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 7.4, containing 60 mM octyl-glucoside was bound covalently to polyacrylamide-coated 1.40 µm silica pedestals using glutaraldehyde as previously described.18 Prior to immobilization, the IIb3 was preincubated with either 1 mM CaCl2, 1 mM MnCl2, or 5 mM DTT/1 mM CaCl2 in the same binding buffer at 37°C for 30 minutes. Interactions between fibrinogen and purified IIb3 were studied in 0.1 M HEPES buffer, pH 7.4, containing 2 mg/mL bovine serum albumin, 0.1% Triton X-100, and either 1 mM CaCl2, 1 mM MnCl2, or 5 mM DTT/1 mM CaCl2. To measure fibrinogen binding to IIb3 on living platelets, an individual platelet was trapped from a suspension of gel-filtered human platelets containing approximately 5 x 106 platelets/mL and approximately 105/mL fibrinogen-coated beads and manually attached to a 5-µm diameter silica pedestal coated with polylysine.18 All experiments with unstimulated platelets were performed in a 4 mM HEPES gel-filtration buffer, pH 7.4, containing 135 mM NaCl, 2.7 mM KCl, 5.6 mM glucose, 1 mM CaCl2, 3.3 mM NaH2PO4, and 0.35 mg/mL bovine serum albumin. To measure the rupture force between fibrinogen and either purified IIb3 or IIb3 on platelets, a fibrinogen-coated bead, trapped by the laser light, was brought to a distance of 2 to 3 µm from the IIb3-coated pedestal or immobilized platelet. After oscillation of the bead was initiated at 5 Hz or 50 Hz with 0.8 µm peak-to-peak amplitude, the bead was brought into contact with the platelet or the pedestal by micromanipulation using a keyboard-controlled piezoelectric stage. Data collection was initiated at the first contact between the bead and the platelet or pedestal. Rupture forces following repeated contacts between the platelet or the pedestal and the bead were collected for periods of several seconds to 1 minute and were displayed as normalized force histograms for each experimental condition. Because only a small percentage of contact/detachment cycles result in effective receptor-ligand binding/unbinding, data from 10 to 22 experiments, representing 3 x 103 to 105 individual measurements, were combined. Individual forces measured during each contact-detachment cycle were collected into 10 pN-wide bins. The number of events in each bin was plotted against the average force for that bin after normalizing for the total number of interaction cycles. The percentage of events in a particular force range (bin) represents the probability of rupture events at that tension. Optical artifacts observed with or without trapped latex beads produce signals that appeared as forces below 10 pN.18 Accordingly, rupture forces in this range were not considered when these data were analyzed. Transmission electron microscopy Rotary-shadowed samples were prepared using a modification of standard procedures19,20 by spraying a dilute solution of molecules in a volatile buffer (0.05 M ammonium formate) and glycerol (30%-50%) onto freshly cleaved mica and shadowing with tungsten in a vacuum evaporator (Denton Vacuum, Cherry Hill, NJ).21 All specimens were examined in a FEI/Philips 400 electron microscope (Philips Electronic Instruments, Mahwah, NJ), operating at 80 kV and at a magnification of x 60 000. The molecular dimensions for approximately 100 individual images from the various groups of IIb3 molecules were measured after scanning negative prints using Adobe Photoshop 7.0.1. Measurements were made with a Photoshop tool calibrated to have 1.6 nm resolution. Molecular dimensions of the digitized images were corrected for a shell of tungsten by subtracting 1 nm on each side of a measured molecule.    Results Top Abstract Introduction Materials and methods Results Discussion References   Interaction of Mn2+-treated platelets with fibrinogen When probed by laser tweezers-based force spectroscopy, the vast majority of the interactions between surface-bound fibrinogen and unstimulated platelets in the presence of Ca2+ were characterized by rupture forces that ranged from 10 pN to several tens of pN. Moreover, the probability of detecting these rupture forces decreased exponentially as the rupture force increased (Figure 1A). However, the same platelets incubated with 1 mM MnCl2 for 5 minutes at 25°C were highly reactive with fibrinogen-coated beads, producing a peak in the histogram of rupture forces that ranged from 60 to 110 pN (Figure 1B). Thus, the cumulative probability of detecting rupture forces greater than 60 pN, which we previously found to be specific for fibrinogen binding to IIb3,18 increased nearly 20-fold. To confirm that the Mn2+-induced peak of rupture forces resulted from fibrinogen binding to IIb3, measurements were repeated in the presence of either of the IIb3 antagonists, tirofiban, or abciximab.18 As shown in Figure 1C-D, each antagonist abrogated the effect of Mn2+ and returned the force histograms to that of unstimulated platelets incubated with Ca2+. View larger version (17K): [in this window] [in a new window]   Figure 1.. Force distribution histograms of fibrinogen binding to Mn2+ -activated platelets as measured using laser tweezers. The histograms represent rupture forces greater than 60 pN, previously shown to be specific fibrinogen binding to IIb3.18 The average yield strength in pN and the cumulative probability of specific fibrinogen binding (%) for each histogram are also shown. (A) Unstimulated platelets. (B) Platelets incubated with 1 mM Mn2+ for 5 minutes at 25°C. (C) Platelets incubated with 1 mM Mn2+ in the presence of 20 µM tirofiban. (D) Platelets incubated with 1 mM Mn2+ in the presence of 100 µg/mL abciximab.   Interaction of Mn2+-treated IIb3 preparations with fibrinogen To verify that the increase in rupture force we observed between platelets and fibrinogen-coated beads in the presence of Mn2+ resulted from an increase in the affinity of individual IIb3 molecules and was independent of possible Mn2+-induced changes in the platelet membrane, we measured rupture forces between fibrinogen-coated beads and purified IIb3 that had been preincubated with 1 mM Mn2+ or 1 mM Ca2+. Consistent with our previously reported laser tweezers measurements using purified IIb3,18 we found that the cumulative probability of detecting rupture forces greater than 60 pN was 2.1% in the presence of 1 mM Ca2+, indicating that some of the purified IIb3 was in an active conformation (Figure 2A). Others have observed that approximately 10% of the IIb3 isolated from platelets is in an active conformation.22 Nonetheless, as shown in Figure 2B, the Mn2+-treated preparations were much more reactive with fibrinogen-coated surfaces, such that the cumulative probability of detecting rupture forces greater than 60 pN increased to 4.5%. The ability of the IIb3 antagonists tirofiban and abciximab to decrease this probability to 0.4% confirmed that rupture forces greater than 60 pN resulted from fibrinogen bound to IIb3 (Figure 2C-D). It is noteworthy that, despite the presence of Mn2+, the yield strength of IIb3-fibrinogen binding was not changed substantially, whereas the cumulative probability of specific rupture forces greater than 60 pN increased more than 2-fold. Thus, these results are consistent with the hypothesis that Mn2+ binding directly induces an increase in the affinity of individual IIb3 molecules for fibrinogen. View larger version (17K): [in this window] [in a new window]   Figure 2.. Force distribution histograms of fibrinogen binding to purified surface-bound IIb3 preincubated with Ca2+ or Mn2+. In the experiments shown, fibrinogen-coated beads were oscillated at 5 Hz touching the IIb3-coated pedestals repeatedly. These data represent rupture forces greater than 60 pN, indicative of specific fibrinogen binding to IIb3.18 The average yield strength in pN and the cumulative probability of specific fibrinogen binding (%) for each histogram are also shown. (A) Fibrinogen binding to IIb3 immobilized in the presence of 1 mM Ca2+. (B) Fibrinogen binding to IIb3 immobilized in the presence of 1 mM Mn2+. (C) Fibrinogen binding to IIb3 treated with 1 mM Mn2+ measured in the presence of 50 µM tirofiban. (D) Fibrinogen binding to IIb3 treated with 1 mM Mn2+ measured in the presence of 100 µg/mL abciximab.   Mn2+-induced changes in the conformation of IIb3 Purified IIb3 molecules from the same preparation used for the laser tweezers experiments were visualized by transmission electron microscopy after rotary shadowing with tungsten. Previous electron microscopy studies indicated that IIb3 molecules are composed of a dense "headpiece" and 2 "stalks," one from each subunit of the heterodimer.23 The headpiece, composed of the amino-terminal portions of the extracellular domains of IIb and 3, contains the ligand-binding site of the integrin, whereas the stalks contain the transmembrane and cytoplasmic domains of each subunit. From the observations of more than 2000 individual images, we were able to distribute IIb3 molecules into 3 groups. Group 1 consists of relatively compact molecules in which the stalks appear to touch ("closed" images) (Figure 3A). Group 2 consists of extended molecules in which the stalks are separated ("open" images) (Figure 3B). Group 3 consists of globular heads in which the stalks are not visualized. View larger version (97K): [in this window] [in a new window]   Figure 3.. Transmission electron microscopy of purified IIb3 in the presence of 1 mM Ca2+ or 1 mM Mn2+. Individual IIb3 molecules were visualized using transmission electron microscopy after rotary shadowing with tungsten. The images could by classified into 3 groups, of which 2 are shown. (A) Closed structures with the tips of the IIb and 3 stalks touching that were obtained in the presence of 1 mM Ca2+. (B) Open structures with the IIb and 3 stalks separated that were obtained in the presence of 1 mM Mn2+. A minority of structures consisting of globular headpieces with no visible stalks were also observed in the presences of both Ca2+ and Mn2+. Magnification, x 170 000.   Although each group was present regardless whether IIb3 had been incubated with Ca2+ or Mn2+, the distribution was significantly different. As shown in Table 1, there was a large increase in the proportion of open (group 2) structures in the presence of Mn2+ (60% ± 4% in the presence of 1 mM Mn2+ versus 11% ± 4% in presence of Ca2+, P < .001), with corresponding decreases in the proportion of group 1 (24% ± 3% in the presence of 1 mM Mn2+ versus 51% ± 12% in the presence of Ca2+, P < .001) and group 3 structures (17% ± 3% in the presence of 1 mM Mn2+ versus 38% ± 10% in presence of Ca2+, P < .001). View this table: [in this window] [in a new window]   Table 1.. Analysis of transmission electron microscope images of purified IIb3 molecules in the absence and presence of Mn2+   We also compared the molecular dimensions of individual IIb3 molecules in the presence of Ca2+ or Mn2+ using digitized images of the electron micrographs. In the presence of Ca2+, there were no significant differences in the dimensions of the IIb3 headpiece when the molecules were in either the open or closed conformation (Table 2). Moreover, the distance separating the IIb and 3 stalks of open molecules did not change when IIb3 was bound to fibrinogen (13.4 ± 4.1 nm versus 12.6 ± 2.7 nm, respectively). The latter measurements suggest that stalk separation of approximately 13 nm is sufficient to identify active IIb3 molecules. By contrast, in the presence of Mn2+, there was an increase in both the length (10.9 ± 1.5 nm versus 12.6 ± 1.5 nm, P < 10-8) and width (7.3 ± 1.5 nm versus 8.4 ± 1.1 nm, P < 10-4) of the IIb3 headpiece when IIb3 shifted from a closed to an open conformation. In addition, the distance separating the stalks of open IIb3 was significantly greater in the presence of Mn2+ than in the presence of Ca2+ (17.8 ± 3.6 nm versus 13.7 ± 4.1 nm, P < 10-8). These measurements indicate that not only does Mn2+ shift IIb3 from a closed to an open conformation by inducing the separation of IIb and 3 stalks, but it also alters the overall size and shape of the headpiece. View this table: [in this window] [in a new window]   Table 2.. Molecular dimensions of individual IIb3 molecules in the presence of Ca2+ or Mn2+   Besides altering the size and configuration of individual IIb3 molecules, Mn2+ increased their tendency to oligomerize. Whereas oligomers were uncommon in the presence of Ca2+, 38% ± 10% of the open forms of IIb3 in the presence of Mn2+ consisted of dimers, trimers, and higher-order oligomers (Figure 4A-C). Moreover, as shown in Figure 4D, the distribution of monomers, dimers, trimers, and higher-order oligomers could be fit to an exponential function (Figure 4D), as previously described for equilibria involving molecules that self-assemble into oligomers such as actin,24,25 hemoglobin S,26 and prions.27 It is also noteworthy that the oligomers resulted from interactions that exclusively involved the distal ends of the stalks and that oligomers composed of 3 or 5 molecules did not form closed rosettes as one would expect if the stalks underwent homomeric interactions. View larger version (64K): [in this window] [in a new window]   Figure 4.. Transmission electron microscopy of IIb3 oligomers observed in the presence of Mn2+. (A) Most frequently, dimers were observed with 1-tail or 2-tail touching. (B) There were fewer integrin trimers. (C) Tetramers and larger oligomers were rare. (D) Size distribution of IIb3 oligomers. These data were fit to an exponential function using Microsoft Excel. (A-C) Magnification, x 170 000.   Mn2+-induced complex formation between IIb3 and fibrinogen Previously, we observed that IIb3 bound to fibrinogen tended to have an open conformation with separated stalks.23 To quantify these observations and to measure the distance separating the stalks when IIb3 was bound to fibrinogen, we incubated fibrinogen with IIb3 in the presence of 1 mM Mn2+ at 37°C for 30 minutes before the mixture was sprayed onto mica and rotary-shadowed with tungsten. As illustrated by Figure 5A, fibrinogen displayed its typical trinodular structure with 2 lateral D nodules and a central E nodule, and IIb3 complexes were present in the 3 different conformations described in the previous section. Although the majority of individual fibrinogen and IIb3 molecules were separated from each other, a minor fraction formed bimolecular and trimolecular complexes of 2 possible stoichiometric ratios, either 1:1 or 1:2 of IIb3 to fibrinogen (Figure 5B-D). As we reported previously,23 IIb3 was always spatially oriented so that its headpiece was attached to the end of a fibrinogen molecule, and the stalks of 2 integrins attached to 1 fibrinogen were oriented in opposite directions. The orientation was such that the entire complex had a 2-fold axis of symmetry through the center of the fibrinogen, ie, a rotation of 180° about this axis brings one IIb3 into the other one. All of the IIb3 molecules interacting with fibrinogen were in the open conformation, implying that this conformation represents its activated state. View larger version (91K): [in this window] [in a new window]   Figure 5.. Transmission electron microscopy of IIb3-fibrinogen complexes formed in the presence of Mn2+. (A) Separate IIb3 and fibrinogen molecules. (B-C) Bimolecular and trimolecular complexes of IIb3 and fibrinogen. It is noteworthy that the IIb3 molecules involved in these complexes all were in the open conformation. (D) Fibrinogen bound to an IIb3 oligomer. Magnification, x 170 000.   Because IIb3 was in excess, quantitative analysis of complex formation was based on the relative proportion of fibrinogen molecules participating in the complexes versus those remaining free. Comparison of the fractions of free fibrinogen molecules in the presence and in the absence of Mn2+ clearly showed the promoting effect of manganese ions on fibrinogen binding to IIb3. Thus, in the absence of Mn2+, 23% ± 8% of the fibrinogen molecules were bound to IIb3. In the presence of Mn2+, the percentage increased significantly to 66% ± 6%. Further, we found that the distance separating the stalks of open IIb3 increased from 17.8 ± 3.6 nm in the presence of Mn2+ alone to 19.9 ± 4.7 nm (P = 10-3) when Mn2+-treated IIb3 was bound to fibrinogen (Table 2). DTT-induced changes in the activity and conformation of IIb3 IIb3 on platelets and transfected tissue culture cells is activated by incubating the cells with DTT.5,6,28 Therefore, to determine whether the changes in the conformation of IIb3 induced by Mn2+ are unique to this cation, we repeated the measurements described in the previous section using DTT as the stimulus for IIb3 activation. First, we used laser tweezers to measure rupture forces between fibrinogen-coated beads and purified IIb3 that had been preincubated with 5 mM DTT for 30 minutes in the presence of 1 mM Ca2+. Similar to Mn2+, DTT increased the cumulative probability of detecting rupture forces greater than 60 pN in the presence of Ca2+ from 1.9% ± 0.5% to 3.2% ± 0.6% (Table 3). Moreover, there was no difference between the average yield strength of fibrinogen binding to IIb3 in the presence of Mn2+ or DTT, suggesting that the IIb3 activation state was similar under both sets of conditions. View this table: [in this window] [in a new window]   Table 3.. Comparison of Mn2+-and DTT-induced fibrinogen binding to IIb3 measured using laser tweezers   DTT-treated IIb3 molecules were then visualized by transmission electron microscopy after rotary shadowing with tungsten. Like Mn2+, DTT treatment resulted in a nearly 3-fold increase in the number of IIb3 molecules in the open, rather than closed, conformation and in the formation of IIb3 clusters. Further, DTT induced changes in the molecular dimensions of individual IIb3 molecules that were similar to the changes induced by Mn2+. Thus, the distance separating the IIb and 3 stalks of open molecules significantly increased from 13.1 ± 2.8 nm in the presence of Ca2+ to 18.1 ± 3.9 nm in the presence of DTT and Ca2+ (P = 4.8 x 10-23) (Table 4). Moreover, there were significant increases in both the head length (11.9 ± 1.9 nm versus 13.4 ± 2.0 nm, P < 10-6) and width (8.7 ± 1.4 nm versus 9.2 ± 1.1 nm, P < .034) when IIb3 was treated with DTT. Thus, these measurements confirm that agents that activate IIb3 by perturbing its extracellular domain induce separation of the IIb and 3 stalks. They also indicate that these agents can alter the overall size and shape of the IIb3 headpiece as well. View this table: [in this window] [in a new window]   Table 4.. Molecular dimensions of individual IIb3 molecules in the presence of Ca2+ or DTT      Discussion Top Abstract Introduction Materials and methods Results Discussion References   Integrins can be activated in vitro by cleavage with specific proteases,29 stabilization of their activated states using monoclonal antibodies,30 exposure to reducing agents such as DTT,28 and incubation with the divalent cation Mn2+.31 How each of these treatments alters integrin activation states is not entirely clear. For example, although Ca2+ or Mg2+ are required for ligand binding to integrins, neither by themselves activate integrins, whereas Mn2+, at millimolar concentrations, induces integrin-mediated cell adhesion,4,9,31,32 binding of isolated integrins to immobilized ligands,12,33,34 and binding of soluble ligands to integrins on cell surfaces.35-37 The crystal structure of the extracellular portion of the integrin v3 revealed that it contains 8 divalent cation-binding sites.14 Four sites were located in the -propeller domain of v, 1 at the subunit genu (knee), and 3 in the 3 A domain.38 The number of divalent cations bound to the A domain appears to be directly related to the presence or absence of ligand. Thus, in the absence of ligand, only the cation binding site in the A ADMIDAS (adjacent to the metal ion-dependent adhesion site) motif is occupied,14 whereas in the presence of Mn2+ and a cyclic Arg-Gly-Asp (RGD) ligand, each of the remaining 2 A domain binding sites contain a cation. One of these sites is located in the A MIDAS (metal ion-dependent adhesion site), and Mn2+ at this site contacts one of the ligand Asp carboxylate oxygens. A second Mn2+ is located 0.6 nm away from the MIDAS at a site designated as the ligand-induced metal binding site (LIMBS), but does not interact with ligand. Although it has been postulated that Mn2+ affects integrin activation states by antagonizing inhibitory effects of Ca2+,9 analysis of the crystal structure of the v3 extracellular domain suggests that by occupying sites in the MIDAS and LIMBS motifs of the A domain, Mn2+ stabilizes its ligand-occupied conformation.14 It is currently thought that integrins such as IIb3 reside on cell surfaces in a thermodynamic equilibrium between inactive and active conformations that can be perturbed by altering the relative position of an integrin and stalks.13,34 An essential element of this hypothesis is that the equilibrium can also be perturbed by altering the conformation of the integrin extracellular domain. To test this premise, we used laser tweezers-based force spectroscopy and electron microscopy to correlate the functional and ultrastructural consequences of exposing the platelet integrin IIb3 to either Mn2+ or DTT. Laser tweezers are an optical system in which external forces applied to a spherical particle trapped by a laser can be accurately measured because the angular deflection of the laser beam is directly proportional to the lateral force applied to the particle and are sensitive and accurate at the lower end of the force spectrum (0-150 pN).39,40 Previously, we found that specific binding of fibrinogen to IIb3 resulted in rupture forces ranging from 60 to 150 pN and an average yield strength of 80 to 100 pN.18 Because the specific rupture forces occurred as a single well-defined peak, they likely represent the interaction of individual IIb3 and fibrinogen molecules. Using living platelets and isolated IIb3 molecules, we found that Mn2+ and DTT increased the affinity of IIb3 for fibrinogen and that the rupture forces between fibrinogen and Mn2+- or DTT-stimulated IIb3 were essentially the same as those we measured previously using ADP- and thrombin-related activation peptide (TRAP)-stimulated platelets.18 Thus, like physiologic platelet agonists, Mn2+ and DTT shift IIb3 from an inactive to an active conformation and do so in the absence of IIb3 clustering. However, unlike Mn2+- and DTT-activated 41 that was found to have an affinity for vascular cell adhesion molecule-1 (VCAM-1) or a ligand peptide intermediate between its inactive and fully active state,41-43 we detected only 2 IIb3 activation states. Thus, we found essentially no difference in the spectrum of rupture forces between fibrinogen and IIb3 regardless whether we measured it in the presence of Ca2+, Mn2+, or DTT or whether platelets were stimulated with ADP or TRAP. One might conclude erroneously that there are intermediate activation states when large ensembles of IIb3 molecules are studied because time-averaged mixtures of low- and high-affinity IIb3 molecules are being measured. Moreover, because the activation of integrin by Mn2+ is presumably reversible,12 it is reasonable to assume that there is an equilibrium between the closed and open forms that is shifted toward the open form by Mn2+. Thus, it is not necessary to hypothesize an intermediate conformer since such a conformer can be attributed to the mixture of 2 forms of the integrin. The possibility that there are only 2 IIb3 activation states is supported by the electron microscope images of single IIb3 molecules. Although we detected 3 basic IIb3 structures: open, closed, and globular, it is likely that the latter 2 are related because a comparable fraction of each was converted to the open form by Mn2+ or DTT. Similar images were obtained in earlier electron microscope studies of IIb323 and 51,13,36 regardless of the molecular staining technique. These structures were present in the absence of Mn2+ or DTT, but each agent converted most of the IIb3 molecules to the open form. In electron microscope images of negatively stained v3, Takagi et al13 observed inactive molecules that had a bent conformation, similar to the bent conformation of v3 in crystals,38 and that v3 was both extended and active in the presence of Mn2+. We did not observe bent forms of IIb3, even though our IIb3 preparations clearly contained inactive and active molecules. However, Takagi et al13 studied recombinant v3 molecules containing a carboxyl-terminal clasp, whereas we studied IIb3 molecules isolated from platelets. Accordingly, our studies and those of Takagi et al13 may not be comparable. Takagi et al13 also observed that the Stokes (hydrodynamic) radius of Mn2+-treated v3 in the absence of ligand was intermediate between that of v3 in the presence of Ca2+ and that of v3 complexed with an RGD-containing ligand. However, Mould et al44 observed no gross differences in the conformation of the 51 headpiece in the presence of Ca2+ and Mn2+ by solution x-ray scattering, but their data were also consistent with an opening of the headpiece that involved an outward movement of the 1 hybrid domain and downward swing of the 7 helix in the presence of Mn2+. We did not detect a difference in the dimensions of the closed and open conformations of IIb3 or in the distance separating the IIb and 3 stalks of open and fibrinogen-bound IIb3 in the presence of Ca2+. Thus, these dimensions are at least sufficient to identify an active conformation of IIb3. Moreover, like Takagi et al,13 we found an increase in the size of the IIb3 headpiece and a further increase in the distance separating the IIb and 3 stalks in the presence of Mn2+ and DTT. Nonetheless, x-ray crystallography revealed no changes in the structure of the extracellular portion of v3 when the crystals were soaked with buffer containing MnCl2.14 There are at least 2 possibilities to reconcile the difference between these results. First, contact forces in preexisting crystals may prevent the structural change normally induced by Mn2+. Second, transmembrane and cytoplasmic segments absent in the crystal of the ectodomain may be critical to integrin activation because they were shown to have a major impact on the ligand-binding activity and the shape of the integrin.45 We found a clear correlation between the ability of IIb3 to bind fibrinogen and the presence of open IIb3 molecules. Thus, all IIb3 molecules bound to fibrinogen, whether in the presence of Ca2+ or Mn2+, had separated stalks. Moreover, we found that fibrinogen binding to IIb3 was enhanced by Mn2+ and DTT in parallel with the increased fraction of open IIb3 conformers. Because there is an equilibrium involving the inactive and active conformations of IIb3, these observations suggest that the differential effects of Ca2+ and Mn2+ on the IIb3 activation state are a function of the ability of each cation to stabilize one conformational state or the other. In the presence of Ca2+, the probability of encountering an active open lIb3 conformation was approximately 10%, but, in the presence of Mn2+, the probability increased to approximately 60%. It is also noteworthy that, as would be expected in a chemical equilibrium, active and inactive molecules coexisted in the presence of either cation. Thus, Mn2+ appears to induce IIb3 activity by stabilizing the active conformation of the IIb3 headpiece, thereby shifting the chemical equilibrium in the active direction and transmitting the conformational change to the stalks. The changes in the dimensions of the IIb3 headpiece are likely a consequence of the differences in size and electronegativity of Ca2+ and Mn2+ ions and of the additional Mn2+ bound to the headpiece in the presence of ligand. Although IIb3 activation by DTT likely involves overall disulfide bond reduction, as well disulfide bond rearrangement,16 the identity of the cysteines involved is not clear. Because IIb3 activation appears to involve changes in the conformation of A and hybrid domains,44,46-47 it would be logical to assume that the relevant cysteines are located in these domains, an assumption consistent with the changes in the dimensions of the IIb3 headpiece that we detect in the presence of DTT. Nonetheless, the free cysteines identified when IIb3 is exposed to mild reducing conditions are located in the epidermal growth factor-like repeats that constitute the 3 stalk,16,38 as is an activating Cys583Tyr mutation.17 Thus, it is likely that the perturbed disulfide bonds that are responsible for IIb3 activation by DTT remain to be identified. Clusters of IIb3 molecules have been observed on the surface of thrombin-stimulated platelets,48 and IIb3 clustering has been induced in vitro as well.49-51 Using Mn2+-activated IIb3 and electron microscopy, we observed that in the absence of membrane or cytoskeletal constraints, isolated IIb3 formed dimers, trimers, and higher-order oligomers that involved the distal ends of IIb and 3 stalks. Thus, these images indicate that directly perturbing the activation state of the IIb3 extracellular domain also results in the formation of IIb3 oligomers in the absence of ligand, as would be predicted from the equilibrium model of integrin regulation. It is also noteworthy that the trimers and pentamers were always open, as would be predicted if the ends of the stalks only undergo homo-oligomerization, ie, the formation of - or - subunit oligomers. Although it is possible that this observation could be the result of steric interference, it is consistent with the hypothesis that homomeric associations involving transmembrane domains are associated with integrin activation and clustering.45 It also suggests that a component of Mn2+-induced modulation of integrin function in cell membranes may result in increased integrin avidity arising from Mn2+-induced aggregation or clustering of integrin molecules. In conclusion, we used laser tweezers and electron microscopy to demonstrate a direct correlation between conformational changes in individual IIb3 molecules and their ligand-binding activity. Using Mn2+ and DTT as activating tools, we found that separation of the IIb and 3 stalks is an integral part of the mechanism leading to the exposure of the fibrinogen binding site in the IIb3 ectodomain. Moreover, because the open conformation induced by Mn2+ and DTT occurred in the absence of ligand binding, it likely represents a primary activating event, perhaps mimicking the consequences of agonist-induced stimulation of IIb3 in platelet membranes.    Footnotes   Submitted April 16, 2004; accepted August 4, 2004. Prepublished online as Blood First Edition Paper, August 19, 2004; DOI 10.1182/blood-2004-04-1411. Supported by the National Institutes of Health (grants HL57407, HL30954, HL40387, and HL62250). The publication costs of this article were defrayed in part by page charge payment. 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