ALBERT

Description: αIIbβ3-mediated outside-in signaling following platelet adhesion to fibrinogen affects platelet adhesion, degranulation, and spreading, but the precise mechanisms involved remain incompletely understood. In this study, we demonstrate that the character of αIIbβ3 signaling is dependent on the surface density of the adsorbed ligand. Time-lapse total internal reflection fluorescence microscopy (TIR-FM) allowed us to visualize the interactions between fibrinogen and the αIIbβ3 integrins on the basal membrane of adhering platelets using a fluorescently-labeled antibody specific for β3. We observed significant morphological and biochemical differences in the initial interaction between αIIbβ3 and fibrinogen depending on the fibrinogen surface density. These included differences in the kinetics of filopodia and lamellipodia formation. Although filopodia started to form at the same time after initial platelet contact with both low-density (adsorbed from 3 μg/ml) fibrinogen (~ 40–50 s), new filopodia formed for a significantly shorter period of time on low- than on high-density fibrinogen [120 s (n = 79) vs. 235 s (n = 62); medians; p〈 0.001]. Lamellipodia started to form in platelets adhering to low-density fibrinogen significantly later than in platelets on high-density fibrinogen (140 s vs. 110 s; p = 0.04). Moreover, once lamellipodia started to appear, formation of new filopodia ceased in platelets adhering to low-density fibrinogen. In contrast, in platelets on high-density fibrinogen new filopodia continued to form even in the presence of lamellipodia for another 155 s. We also observed that a higher percentage of platelets adherent to high-density fibrinogen (52 ± 6 %) failed to develop a cytoplasmic Ca2+ signal compared to platelets adherent to low-density fibrinogen (13 ± 11 %; n = 3; p

Description: The ability to generate multiple cell types from human embryonic stem cells (hESC) in culture offers an unprecedented opportunity to produce an unlimited supply of cells for research and clinical purposes. Megakaryocytes comprise only 0.02–0.05% of the nucleated cell population in the bone marrow, thus making them a difficult cell to isolate and study. The ES cell technology provides a resource for generating megakaryocyte progenitors for in vitro and in vivo analyses. For this approach to be successful, reproducible differentiation schemes that generate sufficient numbers of cells are necessary. With an embryoid body (EB)-based protocol, in serum-free media we are able to generate hematopoietic populations with megakaryocyte potential from two different hESC lines; H1 and HES2. Using CD41 as an early marker of definitive hematopoiesis, we found that 4 to 12% of the day 11–12 EB population expressed high levels of the marker (CD41Hi) whereas 10 to 20% expressed low levels of the marker (CD41Lo). Cells from the CD41Hi but not the CD41Lo population expressed the megakaryocyte marker GPIb. To further analyze these populations, both were isolated from day 12 EBs by cell sorting and cultured in media containing TPO and SCF. Following several days in culture, the CD41Hi population expressed increasing levels of GPIb, as determined by flow cytometry and real time PCR. PF4 was detected in the sorted CD41Hi population and levels increased 10-fold following culture. These cells also adhered to fibrinogen and collagen and stained positive for von Willebrand factor (vWF) and P-selectin. Following activation by PAR1, the cells bound soluble fibrinogen and expressed P-selectin on their surface. Few to no progenitors were detected in the CD41Hi fraction by colony assays suggesting that this population contained cells that were already committed to the megakaryocyte lineage. Incubation of the CD41Lo population of cells in the presence of TPO resulted in 70–80% of the cells expressing increased levels of CD41 and GPIb. Immunostaining revealed vWF and P-selectin expression following adhesion of the cells to fibrinogen and collagen. Both fibrinogen binding and P-selectin surface expression were detected following activation with PAR1. Quantitative PCR analysis demonstrated low levels of PF4 message in the sorted population with an increase of 3-fold following incubation in the presence of TPO. Progenitor cells were detected in colony assays suggesting that this population of cells contained megakaryocyte-like progenitors. Taken together, these finding demonstrate the efficient and reproducible generation of megakaryocytes from hESC using a serum-free EB protocol.

Description: The role of the β3 MIDAS in αIIbβ3 ligand binding is well established, but the role of the nearby ADMIDAS is less well defined. Thus, we studied HEK293 cells expressing normal αIIbβ3 (normal cells) or the ADMIDAS mutants β3 D126A and D127A (mutant cells). Both mutant cells adhered as well or better than normal cells to immobilized fibrinogen under static conditions in the presence of either Ca2+/Mg2+ or Mn2+. Under low shear flow conditions (0.15 dyne/cm2), adhesion of normal cells and D126A mutant cells to fibrinogen was similar in the presence of either Ca2+/Mg2+ or Mn2+. Adherent D126A mutant cells, however, demonstrated greater resistance to detachment at increasing shear rates in the presence of Ca2+/Mg2+ (e.g., at 20.4 dynes/cm2, only 40 ± 10% of normal cells remained vs 85 ± 8% of D126A mutant cells; mean ± SD; p

Description: Although the role of the β3 MIDAS metal ion in ligand binding to αIIbβ3 is well established, serving as the site of interaction of the ligand Asp residue, the role of the nearby LIMBS metal ion is less well defined. Previous studies suggested a role for the LIMBS in ligand binding. We confirmed this by showing that HEK293 cells expressing normal αIIbβ3 adhered to both immobilized fibrinogen and the RGD-containing venom echistatin in the presence of either Mg++/Ca++ or Mn++, whereas two different αIIbβ3 LIMBS mutants (β3 N215A and D217A) failed to adhere to either protein. In addition, we found that both mutations also increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the β3 PSI domain (normal 7±4% vs N215A 46±12% and D217A 41±20% of mAb anti-αIIb (HIP8) binding; mean±SD, n=6, p

Description: Most studies of αIIbβ3 biogenesis have been conducted in transfected HEK293 and CHO cells or megakaryocyte-like cell lines, but the protein processing mechanisms in native megakaryocytes may differ. To address this issue we have studied αIIbβ3 biogenesis in human megakaryocyte-like cells derived from umbilical cord blood (UCB) and in 293 cells stably expressing αIIbβ3, and developed a mathematical model to express the kinetics. The leukocyte pool of whole UCB units was separated by Dextran and Ficoll sedimentation and enriched for CD34+ progenitor cells by negative selection using a commercial antibody panel. These cells were then cultured in the presence of 20 ng/ml thrombopoietin. By day 10 of culture approximately 90% of cells expressed αIIbβ3, 80% expressed GPIb, 45% expressed α2β1 and 55% expressed P-selectin. The cells adhered to fibrinogen and collagen, spread in a manner similar to platelets, and formed focal adhesions as judged by vinculin and phalloidin (F-actin) staining. While αIIbβ3 on the surface of 293 cells cannot undergo inside-out activation, 18% of the UCB-derived cells exhibited increased binding of PAC-1, an activation-specific monoclonal antibody, in response to TRAP, and the majority bound PAC-1 after adhesion to collagen. The ploidy of the UCB-derived cells ranged from 2 to 128. The dynamics of αIIbβ3 biosynthesis was analyzed by pulse-chase analysis with 35S-Cys/Met followed by immunoprecipitation, SDS-PAGE, and densitometry of exposed x-ray film. Using non-linear regression, curves were fitted to the observed data and a 3-compartment mathematical model (pro-αIIb, mature αIIb in complex with β3, and degraded αIIb) was used to describe the αIIb dynamics. There were important similarities and differences between the two types of cells. In both cell types, pro-αIIb decreased over time in a pattern best described by a simple exponential function, P = (a)exp(-bt), where P is the amount of pro-αIIb, a is the initial amount of pro-αIIb, and b is a rate constant. The half-lives (ln2/b) of pro-αIIb in the UCB-derived cells and the 293 cells were similar (120 and 140 min, respectively). In both cell types, mature αIIb increased over time with a pattern best described by a sigmoidal function, M = c/(1+exp(−(x−x0)/d), where M is the measured amount of mature αIIb, c is the asymptotic value of αIIb, d is the rate constant, and x0 is the time to half maximum mature αIIb. In the megakaryocyte-like cells, the time to half maximum (x0) was 120 min, while in the 293 cells it was only 55 min. The amount of pro-αIIb that has been degraded at any time point (D) can be calculated by subtracting the amounts of pro-αIIb and mature αIIb at that time point from the initial amount of pro-αIIb (D = a-P-M). In the UCB-derived cells ~ 25% of pro-αIIb is degraded without being processed to mature αIIb, while in 293 cells ~ 40% is degraded. In both cell types there is a ~90 minute lag time before the onset of net αIIb degradation. These findings suggest that while the ability to degrade αIIb may be similar between megakaryocytes and 293 cells, the folding, complex formation, and quality control mechanisms are quite different. These observations have implications for the study of integrin dynamics and may be useful in analysis of the molecular mechanisms of integrin biogenesis.

Description: The conventional description of platelet interactions with collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets. To obtain dynamic information, we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22°C. With untreated human and wild-type mouse platelets, soon after the initial adhesion of a small number of “vanguard” platelets, “follower” platelets attached to the spread-out vanguard platelets. Follower platelets then adhered to and spread onto nearby collagen or over the vanguard platelets. Thus, thrombi formed as a concerted process rather than as sequential processes. Treatment of human platelets with monoclonal antibody (mAb) 7E3 (anti–GPIIb/IIIa (αIIbβ3) + αVβ3) or tirofiban (anti–GPIIb/IIIa) did not prevent platelet adhesion but nearly eliminated the deposition of follower platelets onto vanguard platelets and platelet thrombi. Similar results were obtained with Glanzmann thrombasthenia platelets. Wild-type mouse platelets in the presence of mAb 1B5 (anti–GPIIb/IIIa) and platelets from β3-null mice behaved like human platelets in the presence of 7E3 or tirofiban. Deposition patterns of untreated human and wild-type mouse platelets were consistent with random distributions under a Poisson model, but those obtained with 7E3- and tirofiban-treated human platelets, 1B5-treated mouse platelets, or β3-null platelets demonstrated a more uniform deposition than predicted. Thus, in this model system, absence or blockade of GPIIb/IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition.

Description: To test the hypothesis that platelet activation contributes to tumor dissemination, we studied metastasis in mice lacking Gαq, a G protein critical for platelet activation. Loss of platelet activation resulted in a profound diminution in both experimental and spontaneous metastases. Analyses of the distribution of radiolabeled tumor cells demonstrated that platelet function, like fibrinogen, significantly improved the survival of circulating tumor cells in the pulmonary vasculature. More detailed studies showed that the increase in metastatic success conferred by either platelets or fibrinogen was linked to natural killer cell function. Specifically, the pronounced reduction in tumor cell survival observed in fibrinogen- and Gαq-deficient mice relative to control animals was eliminated by the immunologic or genetic depletion of natural killer cells. These studies establish an important link between hemostatic factors and innate immunity and indicate that one mechanism by which the platelet-fibrin(ogen) axis contributes to metastatic potential is by impeding natural killer cell elimination of tumor cells.

Description: The search for novel inhibitors of the platelet αIIbβ3 receptor continues with the dual goals of better defining structure-function relationships and developing second generation oral agents. We previously reported on a novel small compound (Compound 1; RUC-1) identified by high throughput screening that inhibits human αIIbβ3. RUC-1 did not inhibit αVβ3, suggesting that it interacts with αIIb, and molecular docking studies supported this speculation. RUC-1 also induced less extensive changes in αIIbβ3 conformation than existing small molecule inhibitors, which may be therapeutically desirable. We have now studied RUC-1’s effects on murine and rat platelets, which are less sensitive than human to inhibition by RGD peptides due to differences in the αIIb sequences contributing to the binding pocket. We found that RUC-1 (100 μM) was much less potent in inhibiting platelet aggregation of murine and rat platelets than human platelets or the platelets of a mouse expressing a hybrid receptor composed of human αIIb and murine β3 (hαIIb/mβ3) (mouse, 6±6%, n=4; rat, 0±15%, n=3; human, 97±2% n=3; hαIIb/mβ3 99±1%, n=4). RUC-1 also inhibited fibrinogen binding to murine platelets expressing the hybrid hαIIb/mβ3 receptor (94± 2%, n=4), but not a hybrid receptor composed of murine αIIb and human β3 (mαIIb/hβ3; 0%, n=4). Molecular docking studies of RUC-1 were consistent with the functional data with RUC-1 binding entirely within the β-propeller. αIIb In vivo studies of RUC-1 administered intraperitoneally (IP) at a dose of 26.5 mg/kg demonstrated antithrombotic effects in the FeCl3 carotid artery model in mice expressing hαIIb/mβ3 (Figure 1A), but did not protect WT mice from thrombotic occlusion at the same dose (Figure 1B). Collectively, these data support RUC-1’s specificity for αIIb, provide new insights into the αIIb ligand-binding pocket, and establish RUC-1’s anti-thrombotic effects in vivo. In addition, the hαIIb/mβ3 mice provide a convenient model for testing low molecular weight αIIbβ3 antagonist drugs such as RUC-1 for toxicity and therapeutic potential. Figure 1. RUC-1 protects hαIIb/mβ3 mice, but not WT mice from FeCl3-induced carotid artery thrombotic occlusion. A. Mice expressing hαIIb/mβ3 receptor were injected IP with vehicle control (n=4), an inactive congener of RUC-1 (RUC-1-piperidine; n=4; 26.5 mg/kg), or RUC-1 (n=7; 26.5 mg/kg) 25 min before carotid artery injury with 20% FeCl3 for 3 min. Blood flow through the carotid artery was monitored for 30 min with a Doppler flow probe. Kaplan-Meier analysis was calculated from the time the FeCl3 was applied to the artery until complete occlusion. The control curve contains the combined data from the mice treated with vehicle and RUC-1-piperidine. B. WT mice (n=4) were given RUC-1 (26.5 mg/kg) and their data are compared to those reported in panel A for mice expressing hαIIb/mβ3. Figure 1. RUC-1 protects hαIIb/mβ3 mice, but not WT mice from FeCl3-induced carotid artery thrombotic occlusion. A. Mice expressing hαIIb/mβ3 receptor were injected IP with vehicle control (n=4), an inactive congener of RUC-1 (RUC-1-piperidine; n=4; 26.5 mg/kg), or RUC-1 (n=7; 26.5 mg/kg) 25 min before carotid artery injury with 20% FeCl3 for 3 min. Blood flow through the carotid artery was monitored for 30 min with a Doppler flow probe. Kaplan-Meier analysis was calculated from the time the FeCl3 was applied to the artery until complete occlusion. The control curve contains the combined data from the mice treated with vehicle and RUC-1-piperidine. B. WT mice (n=4) were given RUC-1 (26.5 mg/kg) and their data are compared to those reported in panel A for mice expressing hαIIb/mβ3.

Description: An elevated plasma fibrinogen level is a risk factor for thrombotic cardiovascular disease, but which of fibrinogen's functions is responsible for the increased risk is unknown. To define better the contribution of fibrinogen to large vessel thrombus formation, we studied carotid artery thrombosis in wild-type mice, mice lacking fibrinogen (fbg–/–), mice treated with 7E9 (a blocking antibody to the fibrinogen γ-chain C-terminus), and mice expressing a mutant fibrinogen (γΔ5) that lacks the γ-chain platelet-binding motif QADGV. In control mice, thrombus formation resulted in occlusion in 8 ± 2 minutes (mean ± SD). In fbg–/– mice, thrombi grew to large sizes, but then they abruptly embolized, confirming previous observations by others in an arteriolar thrombus model. In contrast, mice treated with 7E9 and γΔ5 mice developed only small, nonoclusive mural thrombi and embolization was limited. These findings reveal that a fibrinogen antibody, 7E9, or a fibrinogen mutant retaining clotting function, can limit thrombus formation more effectively than the complete absence of fibrinogen. We hypothesize that the smaller thrombi in these animals result from the ability of fibrin to bind and sequester thrombin and/or the ability of the altered fibrinogen molecules, which cannot recruit platelets, to bind to and passivate the surface.

Description: Two separate conformational changes have been proposed to accompany activation of platelet αIIbβ3: 1) leg separation leading to extension of the head region composed of the αIIb propeller and β3 βA (I-like) domains, and 2) a swing-out motion at the junction of the β3 βA (I-like) and hybrid domains. Small molecule inhibitors of αIIbβ3 competitively block the RGD ligand binding site and variably induce conformational changes in αIIbβ3 as judged by the binding of ligand-induced binding site (LIBS)-specific monoclonal antibodies. In an attempt to identify molecules that may inhibit αIIbβ3 activation without initiating the conformational changes associated with ligand binding, we screened 33,264 compounds from four different chemical libraries (Prestwick, Chembridge, Cerep and ChemDiv) for their ability to inhibit the adhesion of washed platelets in HEPES-modified Tyrode’s buffer with 1 mM Ca2+/0.5 mM Mg2+ to immobilized fibrinogen adsorbed from a 50 μg/ml solution. When tested at a final concentration of 16 μM, a total of 102 compounds (0.31%) demonstrated greater than 50% inhibition of platelet adhesion, and two of these (Figure 1) demonstrated 〉30% inhibition of the initial wave of ADP-induced aggregation of platelets in citrated platelet-rich plasma. IC50s for inhibition of ADP (5 μM)-induced platelet aggregation for compounds 1 and 2 were 13 ± 4.5 and 17 ± 5 μM (n=3), respectively. Compounds 1 and 2 also inhibited fibrinogen binding to platelets induced by the activating LIBS antibody AP5 with IC50s of 27 and 30 μM, and 20 and 27 μM, respectively, in two experiments. Since AP5 binds to and directly activates αIIbβ3, it is likely that the compounds’ inhibitory effects are due to direct binding to αIIbβ3 rather than inhibition of signal transduction. In two separate experiments, compound 1 at 15 - 20 μM produced variable increases in the binding of LIBS mAbs AP5, PMI-1 or LIBS1 to unactivated and ADP-activated platelets, whereas tirofiban (20 μM) consistently increased the binding of each mAb. Compound 2 did not increase the binding of any of the mAbs. Neither compound contains a negatively charged carboxyl group, which mediates the interaction of the Asp group in RGD ligands with the β3 MIDAS metal ion, but compound 1 has a carbonyl group that may potentially interact with the MIDAS metal ion. Compound 1 resembles 1,2-fused pyrimidine derivatives that have previously been demonstrated to inhibit platelet aggregation (Roma et al., Bioorg. Med. Chem. 2003, 11, 123). We conclude that high throughput screening of molecular libraries can identify novel compounds that inhibit αIIbβ3 and that one of them appears to inhibit αIIbβ3 without inducing conformational changes in the receptor. Figure Figure