Journal articles on the topic 'Fibronectin Aggregation in Collagen'
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1
Moon, DG, JE Kaplan, and JE Mazurkewicz. "The inhibitory effect of plasma fibronectin on collagen-induced platelet aggregation." Blood 67, no. 2 (February 1, 1986): 450–57. http://dx.doi.org/10.1182/blood.v67.2.450.450.
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Abstract Plasma fibronectin (Fn) has been proposed to have an antithrombotic effect, protecting against platelet and fibrinogen consumption after injury. The current study was designed to determine the effect of plasma fibronectin on collagen-induced platelet aggregation. In vitro aggregometry using an isolated homologous rat system, demonstrated a significant (P less than .05) inhibitory effect of 120 micrograms/mL Fn on platelet aggregation as induced by 60 micrograms/mL fibrillar collagen (type I). The inhibition was evidenced by a threefold increase in lag time and a significant decrease in the rate and extent of aggregation. The hypothesis was also tested using an in vivo model of collagen-induced platelet aggregation. The model used was intravenous injection of 2 mg/kg of homologous type I collagen into anesthetized Sprague-Dawley rats. Injection of collagen preincubated with 4 mg/kg Fn resulted in significantly less thrombocytopenia and fibrinogen consumption as compared with injection of collagen alone. The results of both the in vitro and in vivo studies are consistent with the proposed antithrombotic effect of plasma fibronectin.
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Moon, DG, JE Kaplan, and JE Mazurkewicz. "The inhibitory effect of plasma fibronectin on collagen-induced platelet aggregation." Blood 67, no. 2 (February 1, 1986): 450–57. http://dx.doi.org/10.1182/blood.v67.2.450.bloodjournal672450.
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Plasma fibronectin (Fn) has been proposed to have an antithrombotic effect, protecting against platelet and fibrinogen consumption after injury. The current study was designed to determine the effect of plasma fibronectin on collagen-induced platelet aggregation. In vitro aggregometry using an isolated homologous rat system, demonstrated a significant (P less than .05) inhibitory effect of 120 micrograms/mL Fn on platelet aggregation as induced by 60 micrograms/mL fibrillar collagen (type I). The inhibition was evidenced by a threefold increase in lag time and a significant decrease in the rate and extent of aggregation. The hypothesis was also tested using an in vivo model of collagen-induced platelet aggregation. The model used was intravenous injection of 2 mg/kg of homologous type I collagen into anesthetized Sprague-Dawley rats. Injection of collagen preincubated with 4 mg/kg Fn resulted in significantly less thrombocytopenia and fibrinogen consumption as compared with injection of collagen alone. The results of both the in vitro and in vivo studies are consistent with the proposed antithrombotic effect of plasma fibronectin.
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Eynard, A. R., M. E. Pasqualini, and R. A. Rovasio. "Exogenous fibronectin modifies the aggregation of collagen-stimulated human platelets." Experientia 46, no. 7 (July 1990): 680–82. http://dx.doi.org/10.1007/bf01939932.
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O'Shea, K. S. "Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo." Development 99, no. 4 (April 1, 1987): 509–19. http://dx.doi.org/10.1242/dev.99.4.509.
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The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.
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Nitkin, R. M., and T. C. Rothschild. "Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR aggregation." Journal of Cell Biology 111, no. 3 (September 1, 1990): 1161–70. http://dx.doi.org/10.1083/jcb.111.3.1161.
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Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrin-induced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches colocalized with AChR patches, raising the possibility of a causal relationship between matrix patching and AChR accumulations. However, patching of AChRs (complete within a few hours) preceded that of laminin or HSPG (not complete until 15-20 h), making it unlikely that matrix accumulations initiate AChR patching at agrin-induced sites. Conversely, when AChR patching was blocked by treatment with anti-AChR antibody mAb 35, agrin was still able to effect patching of laminin and HSPG. Taken together, these findings suggest that agrin-induced accumulations of AChR and laminin/HSPG are not mechanistically linked.
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Huynh, Khon C., Huong T. T. Nguyen, Volker R. Stoldt, Marianna Gyenes, and Rudiger E. Scharf. "Shear-Induced Fibrillar-like Supramolecules of Plasma Fibronectin: A New Form of Fibronectin with Enhanced Activity in Platelet Adhesion and Aggregation." Blood 124, no. 21 (December 6, 2014): 4174. http://dx.doi.org/10.1182/blood.v124.21.4174.4174.
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Abstract Introduction: Plasma fibronectin (FN) is synthesized by hepatocytes and secreted into the circulation in a soluble, compact and non-fibrillar form. Plasma FN is assembled by cells or adherent platelets into functional fibrils. Reports have indicated that the process to incorporate FN into multimer fibrils can also occur in cell-free models in vitro by incubation with denaturants, reducing agents, or anastellin (FN peptidic fragment). Here, we report on (1) the formation of insoluble fibrillar-like supramolecules of plasma FN (FN fibrils) by exposing the molecules to increasing shear rates and (2) the functional characterization in platelet adhesion and aggregation. Methods: To induce the formation of FN fibrils, 1 ml of plasma FN solution (100 μg/ml) was added to plates pre-coated with FN (100 μg/ml). Subsequently, the FN solutions were exposed to shear (50 to 5000 s-1 within 5 min and subsequently 5000 to 50 s-1 within 5 min) generated by a cone-plate rheometer (Haaka Rheostress 1; Thermo Scientific). Viscosities of FN solutions were recorded. To quantify the formation of FN fibrils, FN solutions after exposure to shear were collected and incubated with 2% deoxycholate (DOC). The DOC-insoluble pellets containing FN fibrils were isolated by centrifugation at 19,000 g for 20 min at 4°C and resuspended in 1% SDS buffer for Western blot analyses. For adhesion experiments, washed platelets (107/ml) in HEPES Tyrode’s buffer were labeled with 10 μM 5-chloromethylfluorescein diacetate and placed on 96-well plates pre-coated with FN or FN fibrils (25 µg/ml) for 30 min at 37°C. In parallel experiments, platelets resuspended in FN-depleted plasma (107/ml) were placed onto immobilized collagen, fibrinogen, FN (10 µg/ml) in the presence of FN (300 µg/ml) or FN fibrils (10 µg/ml). For aggregation experiments, FN (5, 10, 300 µg/ml) or FN fibrils (5, 10 µg/ml) was added to platelet-rich plasma (PRP) or platelets resuspended in FN-depleted plasma (2.5 × 108/ml). Aggregation was induced by 400 nM PMA (Phorbol 12-myristate 13-acetate), or 10 µg/ml collagen. Results: The initial viscosities (mPa's) of FN solutions were 7.62 ± 0.98. Upon exposure to dynamic shear for 10 min, viscosities increased to 10.98 ± 1.81 (p = 0.02, n = 4), suggesting conformational changes of FN. Western blot analyses of DOC-insoluble fractions revealed bands of FN in the range of 220 – 250 kDa (reducing condition), indicative of the formation of insoluble fibrils in FN solutions after exposure to shear. Platelet adhesion and aggregation experiments were performed to compare the activity of FN fibrils with normal plasma FN. For adhesion experiments, washed platelets in HEPES Tyrode’s buffer were placed onto immobilized FN or FN fibrils (25 µg/ml). The adhesion rates (mean fluorescence signal ± SD) of washed platelets were higher onto surfaces coated with FN fibrils (0.5 ± 0.06) than onto surfaces coated with plasma FN (0.4 ± 0.01) (p = 0.04, n = 3). In parallel adhesion experiments using platelets resuspended in FN-depleted plasma, addition of plasma FN (300 µg/ml) increased platelet adhesion rates onto immobilized collagen (from 0.14 ± 0.005 to 0.2 ± 0.01, p = 0.0007), fibrinogen (from 0.16 ± 0.03 to 0.22 ± 0.01, p = 0.03), and FN (from 0.14 ± 0.01 to 0.18 ± 0.02, p = 0.04) (n = 3). Addition of FN fibrils at low concentration of 10 µg/ml had a similar supportive effect. FN showed an inhibitory effect in platelet aggregation. Activation by 400 nM PMA induced aggregation of PRP by 81% (amplitude). In the presence of plasma FN at 5, 10, 300 µg/ml, platelet aggregation was reduced to 50 %, 41 %, and 29.5 %, respectively. A stronger inhibition on platelet aggregation was seen when FN fibrils were added. PRP aggregated by 35.4 % and 17 % in the presence of 5 and 10 µg/ml FN fibrils, respectively. The same phenomenon was observed in aggregation assays using platelets resuspended in FN-depleted plasma and collagen (10 µg/ml) as activating agonist. Conclusion: Our study shows that dynamic shear rates induce the formation of insoluble fibrillar-like form of plasma FN in cell-free model in vitro. Fibril formation of FN can be monitored by measuring viscosities of FN solutions during exposure to shear and quantified by Western blot. Shear-induced formed FN fibrils have an explicitly stronger activity in supporting platelet adhesion and inhibiting platelet aggregation than normal plasma FN. This finding emphasizes the importance of FN assembly on its activity in platelet functions. Disclosures No relevant conflicts of interest to declare.
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Diehl-Seifert, B., B. Kurelec, R. K. Zahn, A. Dorn, B. Jericevic, G. Uhlenbruck, and W. E. Muller. "Attachment of sponge cells to collagen substrata: effect of a collagen assembly factor." Journal of Cell Science 79, no. 1 (November 1, 1985): 271–85. http://dx.doi.org/10.1242/jcs.79.1.271.
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Collagen, isolated from the sponge Geodia cydonium in the absence of denaturing agents, had the typical amino acid composition and was associated with the carbohydrates galactose and glucose. The resulting individual fibrils with a diameter of 23 nm, displayed a 19.5 nm periodicity with one intraperiod band. A collagen assembly factor (CAF) was identified in and partially purified from the extracellular space. The CAF reacted with antibodies against intact Geodia cells but not with antibodies against Geodia lectin and Geodia aggregation factor. In the presence of the CAF, the collagen fibrils reconstituted collagen bundles in an ordered sequence of events, which were followed by electron-microscopical and biochemical methods. Bundle formation was not dependent on the presence of the homologous lectin, glycoconjugates or aggregation factor. Homologous cells (Geodia archaeocytes) were determined to attach only to those Geodia collagen substrates that contained CAF. The attachment of these cells did not require fibronectin or Geodia lectin. Homologous glycoconjugates or NaOH-treated collagen inhibited cell attachment. Collagen from the sponge Chondrosia reniformis, even in the presence of Geodia CAF, was no appropriate substrate for Geodia cell attachment. Whether collagen is a component of cell-matrix interactions in sponge systems also in vivo is discussed.
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Parmentier, S., B. Catimel, L. McGregor, LL Leung, and JL McGregor. "Role of glycoprotein IIa (beta 1 subunit of very late activation antigens) in platelet functions." Blood 78, no. 8 (October 15, 1991): 2021–26. http://dx.doi.org/10.1182/blood.v78.8.2021.2021.
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Abstract Very late activation antigens (VLAs) are glycoproteins (GPs) that play a major role in platelet adhesion to extracellular matrix. These GPs, members of the integrin family, are heterodimer complexes with different alpha subunits noncovalently associated with a common beta 1 subunit known as GPIIa. GPIa-IIa (also known as VLA2), GPIc-IIa (VLA5), and GPIc*-IIa (VLA6) are involved, respectively, in platelet adhesion to collagen, fibronectin, and laminin. At this stage, very little is known about the role of GPIIa in platelet adhesive functions. In this study, we have generated a monoclonal antibody (MoAb) (LYP22) directed against GPIIa. Immunoaffinity chromatography using LYP22 combined with two-dimensional nonreduced-reduced sodium dodecyl sulfate- polyacrylamide gel electrophoresis shows that the antibody brings down all VLA subunits. Western blots indicate that the binding site of LYP22 on GPIIa is disulfide bridge-dependent. The number of LYP22 binding sites is not increased on stimulation with thrombin and is in the range of what is observed with another anti-GPIIa MoAb (A-1A5). LYP22 is the first anti-GPIIa MoAb to inhibit aggregation and secretion of washed platelets stimulated with collagen, thrombin, or arachidonic acid. Moreover, the lag-phase usually observed on collagen stimulation is significantly prolonged (by 60 seconds) in the presence of LYP22. This lag-phase, mediated by LYP22, is also observed in the presence of plasma proteins and is coupled with a reduced effect on collagen- induced platelet aggregation. In addition, LYP22 affects the adhesion of resting platelets to type III collagen, but not to fibronectin, laminin, or type I collagen. These results strongly indicate that the site on GPIIa, bearing the LYP22 epitope, is an active participant in signal transduction controlling platelet functions.
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Parmentier, S., B. Catimel, L. McGregor, LL Leung, and JL McGregor. "Role of glycoprotein IIa (beta 1 subunit of very late activation antigens) in platelet functions." Blood 78, no. 8 (October 15, 1991): 2021–26. http://dx.doi.org/10.1182/blood.v78.8.2021.bloodjournal7882021.
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Very late activation antigens (VLAs) are glycoproteins (GPs) that play a major role in platelet adhesion to extracellular matrix. These GPs, members of the integrin family, are heterodimer complexes with different alpha subunits noncovalently associated with a common beta 1 subunit known as GPIIa. GPIa-IIa (also known as VLA2), GPIc-IIa (VLA5), and GPIc*-IIa (VLA6) are involved, respectively, in platelet adhesion to collagen, fibronectin, and laminin. At this stage, very little is known about the role of GPIIa in platelet adhesive functions. In this study, we have generated a monoclonal antibody (MoAb) (LYP22) directed against GPIIa. Immunoaffinity chromatography using LYP22 combined with two-dimensional nonreduced-reduced sodium dodecyl sulfate- polyacrylamide gel electrophoresis shows that the antibody brings down all VLA subunits. Western blots indicate that the binding site of LYP22 on GPIIa is disulfide bridge-dependent. The number of LYP22 binding sites is not increased on stimulation with thrombin and is in the range of what is observed with another anti-GPIIa MoAb (A-1A5). LYP22 is the first anti-GPIIa MoAb to inhibit aggregation and secretion of washed platelets stimulated with collagen, thrombin, or arachidonic acid. Moreover, the lag-phase usually observed on collagen stimulation is significantly prolonged (by 60 seconds) in the presence of LYP22. This lag-phase, mediated by LYP22, is also observed in the presence of plasma proteins and is coupled with a reduced effect on collagen- induced platelet aggregation. In addition, LYP22 affects the adhesion of resting platelets to type III collagen, but not to fibronectin, laminin, or type I collagen. These results strongly indicate that the site on GPIIa, bearing the LYP22 epitope, is an active participant in signal transduction controlling platelet functions.
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Baldwin, Samuel P., Christine E. Krewson, and W. Mark Saltzman. "PC12 CELL AGGREGATION AND NEURITE GROWTH IN GELS OF COLLAGEN, LAMININ AND FIBRONECTIN." International Journal of Developmental Neuroscience 14, no. 3 (June 1996): 351–64. http://dx.doi.org/10.1016/0736-5748(96)00018-4.
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Huynh, Khon C., Volker R. Stoldt, Marianna Gyenes, Abdelouahid El-Khattouti, and Rudiger E. Scharf. "Fibronectin Unfolding by Platelets and Its Effect on Platelet Adhesion and Aggregation." Blood 118, no. 21 (November 18, 2011): 2209. http://dx.doi.org/10.1182/blood.v118.21.2209.2209.
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Abstract Abstract 2209 Introduction: Fibronectin (Fn), a dimeric adhesive glycoprotein of 230 to 250 kDa monomers, is present both in plasma and the extracellular matrix. Fn has been suggested to interact with platelets, subsequently being unfolded and forming fibrillar-like networks that contribute to platelet adhesion and aggregation. In our study, we examined the effect of Fn isolated from plasma on platelet adhesion and aggregation in vitro. Specifically, we explored the effect of Fn unfolding while interacting with platelets. Methods: For adhesion experiments, mepacrine-labeled washed platelets in the absence or presence of exogenous Fn (100 μg/ml) were incubated in wells pre-coated with collagen type I, fibrinogen (Fg) or Fn (10 μg/ml each) for 30 min at 37°C. For aggregation experiments, washed platelets were stimulated with 40 nM PMA or 10 μg/ml collagen in the absence or presence of Fn (300 μg/ml). For fluorescence resonance energy transfer (FRET) experiments, Fn isolated from human plasma was doubly conjugated with alexa fluor 488 and 546. Labeled Fn was mixed with 10-fold excess of unlabeled Fn to prevent energy transfer between adjacent protein molecules. Fn mixtures (20 or 100 μg/ml) were incubated for 3 h at 22°C with washed platelets in suspension (108/ml) or with platelets adherent onto immobilized Fn (50 μg/ml). In both settings, platelets were stimulated by 40 nM PMA. In some experiments, platelets were pre-incubated with the monoclonal antibodies LM609 or 10E5 (10 μg/ml) to block αvβ3 or αIIbβ3, respectively, prior to the addition of labeled Fn. For control, FRET signals of Fn mixtures without platelets were recorded. Results: Upon addition of soluble Fn (100 μg/ml) to washed platelets and subsequent co-incubation in wells pre-coated with collagen, Fg, or Fn (10 μg/ml) for 30 min, the percentage (mean % ± SD) of platelets adherent onto one of the immobilized ligands increased significantly by 228±33 (p=0.0112, n=3), 249±42 (p=0.005, n=3), or 198±21 (p=0.0017, n=3), respectively, as compared to adhesion experiments without addition of soluble Fn. By contrast, Fn had an opposing effect on platelet aggregation. Thus, addition of Fn (300 μg/ml) to washed platelets resulted in a reduction of 25 % or 50 % in platelet aggregation induced by PMA (40nM) or collagen (10 μg/ml), respectively. To determine Fn unfolding, the protein was doubly labeled with alexa fluor 488 (donor) randomly at 7–9 amine residues and alexa fluor 546 (acceptor) specifically at 4 free cysteine residues for FRET analyses. To access the sensitivity of FRET for conformational changes in Fn, we exposed labeled Fn to increasing concentrations of GdnHCl (1–4 M) and measured FRET. FRET signals, defined by the ratio of acceptor to donor fluoresecence intensity, varied over the range of GdnHCl concentrations indicating the conformational changes in Fn from its compact to its unfolded state. Fn in its compact conformation (0 M GdnHCl) had a FRET signal of 0.55 (100%) which decreased to 0.34 (63%), as Fn extended in 1 M GdnHCl solution. Further unfolding of Fn in 2 M, 3M and 4 M GdnHCl reduced the FRET signal to 0.27 (50%), 0.23 (44%) and 0.21 (39%), respectively. Addition of labeled Fn to PMA-activated platelets adherent onto immobilized unlabled Fn caused a slow but progressive decrease in FRET signal by 4% at 1 h, 5 % at 2 h and 6% at 3 h incubation. The decrease in FRET signal was reduced to 2% when platelet αvβ3 was blocked by LM609. By contrast, FRET remained unchanged in control experiments without platelets. The same was true when labeled Fn was incubated with PMA-activated platelets in suspension or in the presence of 10E5 (blocking αIIbβ3). Conclusion: Our in vitro studies strongly suggest that fibronectin can play a dual role in hemostasis by promoting platelet adhesion onto immobilized ligands but reducing platelet aggregation. We also demonstrate that activated adherent but not suspended platelets can indeed progressively unfold fibronectin, thereby inducing profound conformational changes that may explain its oppositional effects in platelet adhesion and aggregation. Moreover, our data suggest that unfolding of fibronectin caused by adherent platelets is governed by β3 integrins. Hereby, αIIbβ3 plays a predominant role in comparison to αvβ3. Disclosures: No relevant conflicts of interest to declare.
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Kozhina, K. V., E. N. Volkova, I. N. Saburina, Sergey G. Morozov, I. M. Zurina, N. V. Kosheleva, A. A. Gorkun, and A. A. Grigorieva. "The influence of peptide bioregulators on skin aging in 3D culture model." Russian Journal of Skin and Venereal Diseases 19, no. 1 (February 15, 2016): 58–63. http://dx.doi.org/10.18821/1560-9588-2016-19-1-58-63.
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He effect of mesotherapy injection (Meso-Wharton R199TM) on the dermal fibroblasts culture, simulating condition of (mature) aging skin cells are studied. Material and methods. The culture of 4th passage fibroblasts (P4), that corresponds to young skin fibroblasts (control) and the culture of 18th passage fibroblasts (P18), that has all the signs of aging dermal fibroblasts (predominance of large cells, slow cell division) were used. Bioactivity was assessed by cell morphology, epithelium-mesenchyme plasticity and expression of fibroblasts markers: cytokeratin 19, elastin, a-smooth muscle actin (aSMA), PCNA (proliferation marker), collagen types I, III, IV and fibronectin. The formation of spheroids occur when fibroblasts P18 are cultivating with the injection medication, on terms comparable to the formation of spheroids from P4 young fibroblasts. From culture of fibroblasts P18, that was cultured without medication, does not form the full spheroid, but aggregation of cells and their gradual destruction with necrotic masses within the unit are observed. The presence of the medication stimulates the “rejuvenation” of cells and subsequent recovery of the mesenchyme-epithelial plasticity of cultured fibroblasts due to the reduced ability to synthesize sufficient to establish the amount of intercellular contacts the extracellular matrix components (fibronectin and collagen), which affects the ability to form spheroids. Culturing spheroids formed with the medication stimulates expression of elastin, collagen type IV, fibronectin extracellular matrix protein that supports the skin elasticity and superficial cells actively express cytokeratin 19. The study results clearly demonstrate the effectiveness of mesotherapeutic treatment for skin rejuvenation.
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Cree, R. G. A., P. Aleljung, M. Paulsson, W. Witte, W. C. Noble, A. Ljungh, and T. Wadström. "Cell surface hydrophobicity and adherence to extra–cellular matrix proteins in two collections of methicillin–resistantStaphylococcus aureus." Epidemiology and Infection 112, no. 2 (April 1994): 307–14. http://dx.doi.org/10.1017/s0950268800057721.
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SUMMARYNon-specific and specific mechanisms of adherence have been examined in two collections of methicillin–resistantStaphylococcus aureus(MRSA). Determination of hydrophobicity by salt aggregation, hydrophobicity indices and of adherence to the extra–cellular matrix proteins fibronectin, vitronectin, laminin and collagen type 1 have failed to reveal any correlation with phage-type, plasmid profile or antibiogram. Further, the strain collections, made over a period of years in two countries, differ markedly in their adherence characteristics; MRSA are heterogeneous in this respect. Such heterogeneity may explain the polarization of views on the epidemicity or ‘virulence’ of MRSA. With the exception of adherence to collagen a small group of methicillin sensitiveS. aureushad characteristics intermediate between the two groups of MRSA.
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Peerschke, E. I., K. B. Reid, and B. Ghebrehiwet. "Identification of a novel 33-kDa C1q-binding site on human blood platelets." Journal of Immunology 152, no. 12 (June 15, 1994): 5896–901. http://dx.doi.org/10.4049/jimmunol.152.12.5896.
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Abstract The constitutive expression of a 60-kDa platelet membrane protein (cC1qR) recognizing the collagen-like amino terminal of C1q was previously described. Recently, a novel 33-kDa C1q receptor (gC1qR) that interacts with the globular head region of C1q was identified on Raji cells, as well as PBLs, neutrophils, and eosinophils. The present study demonstrates that polyclonal Abs directed against this novel C1q-binding protein also recognize a 33-kDa platelet membrane constituent on Western blots. Interestingly, Ab reactivity with platelets in suspension was minimal, but increased nearly 10-fold after platelet adhesion to collagen, fibrinogen, or fibronectin-coated surfaces. Similar increases in Ab reactivity were not achieved after platelet stimulation in suspension, even with strong agonists such as thrombin or A23187. Platelet function studies, however, demonstrated that both the globular C-terminal domain of C1q and the collagen-like N-terminal region participate in platelet aggregation in response to C1q multimers. Moreover, a synthetic 18 amino acid peptide (X18) corresponding to the amino terminal sequence of the cloned Raji cell gC1qR inhibited both platelet adhesion to immobilized C1q and aggregated C1q-induced platelet aggregation. Aggregated C1q-induced platelet aggregation was also inhibited by a mAb (1B4) directed against the recombinant gC1qR. The data support the involvement of both carboxy- and amino-terminal regions of C1q in platelet-C1q interactions, and suggest a role for the gC1qR in this process.
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Reheman, Adili, Hong Yang, Guangheng Zhu, Wuxun Jin, Feng He, Christopher M. Spring, Xufang Bai, Peter L. Gross, John Freedman, and Heyu Ni. "Plasma fibronectin depletion enhances platelet aggregation and thrombus formation in mice lacking fibrinogen and von Willebrand factor." Blood 113, no. 8 (February 19, 2009): 1809–17. http://dx.doi.org/10.1182/blood-2008-04-148361.
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Abstract We previously showed that platelet aggregation and thrombus formation occurred in mice lacking both fibrinogen (Fg) and von Willebrand factor (VWF) and that plasma fibronectin (pFn) promoted thrombus growth and stability in injured arterioles in wild-type mice. To examine whether pFn is required for Fg/VWF-independent thrombosis, we generated Fg/VWF/conditional pFn triple-deficient (TKO; Cre+, Fnflox/flox, Fg/VWF−/−) mice and littermate control (Cre−, Fnflox/flox, Fg/VWF−/−) mice. Surprisingly, TKO platelet aggregation was not abolished, but instead was enhanced in both heparinized platelet-rich plasma and gel-filtered platelets. This enhancement was diminished when TKO platelets were aggregated in pFn-positive control platelet-poor plasma (PPP), whereas aggregation was enhanced when control platelets were aggregated in pFn-depleted TKO PPP. The TKO platelet aggregation can be completely inhibited by our newly developed mouse anti–mouse β3 integrin antibodies but was not affected by anti–mouse GPIbα antibodies. Enhanced platelet aggregation was also observed when heparinized TKO blood was perfused in collagen-coated perfusion chambers. Using intravital microscopy, we further showed that thrombogenesis in TKO mice was enhanced in both FeCl3-injured mesenteric arterioles and laser-injured cremaster arterioles. Our data indicate that pFn is not essential for Fg/VWF-independent thrombosis and that soluble pFn is probably an important inhibitory factor for platelet aggregation.
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Duband, J. L., and J. P. Thiery. "Distribution of laminin and collagens during avian neural crest development." Development 101, no. 3 (November 1, 1987): 461–78. http://dx.doi.org/10.1242/dev.101.3.461.
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The distribution of type I, III and IV collagens and laminin during neural crest development was studied by immunofluorescence labelling of early avian embryos. These components, except type III collagen, were present prior to both cephalic and trunk neural crest appearance. Type I collagen was widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin shared a common distribution primarily in the basal surfaces of epithelia and in close association with developing nerves and muscle. In striking contrast with the other collagens and laminin, type III collagen appeared secondarily during embryogenesis in a restricted pattern in connective tissues. The distribution and fate of laminin and type I and IV collagens could be correlated spatially and temporally with morphogenetic events during neural crest development. Type IV collagen and lamin disappeared from the basal surface of the neural tube at sites where neural crest cells were emerging. During the course of neural crest cell migration, type I collagen was particularly abundant along migratory pathways whereas type IV collagen and laminin were distributed in the basal surfaces of the epithelia lining these pathways but were rarely seen in large amounts among neural crest cells. In contrast, termination of neural crest cell migration and aggregation into ganglia were correlated in many cases with the loss of type I collagen and with the appearance of type IV collagen and laminin among the neural crest population. Type III collagen was not observed associated with neural crest cells during their development. These observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities during neural crest ontogeny. (i) Type I collagen associated with fibronectins is a major component of the extracellular spaces of the young embryo. Together with other components, it may contribute to the three-dimensional organization and functions of the matrix during neural crest cell migration. (ii) Type III collagen is apparently not required for tissue remodelling and cell migration during early embryogenesis. (iii) Type IV collagen and laminin are important components of the basal surface of epithelia and their distribution is consistent with tissue remodelling that occurs during neural crest cell emigration and aggregation into ganglia.
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Nievelstein, PF, and JJ Sixma. "Glycoprotein IIb-IIIa and RGD(S) are not important for fibronectin- dependent platelet adhesion under flow conditions." Blood 72, no. 1 (July 1, 1988): 82–88. http://dx.doi.org/10.1182/blood.v72.1.82.82.
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Abstract Previous studies have indicated that activated blood platelets interact with fibronectin through binding of fibronectin to the glycoprotein IIb- IIIa complex (GPIIb-IIIa). The cell attachment site of fibronectin with its crucial arg-gly-asp(-ser) [RGD(S)]sequence is involved in these bindings. We studied the importance of these interactions for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a nonreactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin-induced aggregation and adhesion under static conditions at 0.1 mmol/L. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mmol/L had a significant inhibitory effect at 1,500 s-1, but not at the lower shear rates of 800 and 300 s-1 where platelet adhesion is also fibronectin dependent. On the matrix of cultured human umbilical vein endothelial cells, 1 mmol/L GRGDSP had no effect on platelet adhesion. The relation between GPIIb- IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann's thrombasthenia and monoclonal antibodies to GPIIb-IIIa using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann's thrombasthenia showed a similar inhibition of adhesion in the presence of fibronectin-free plasma after the ECMs had been preincubated with antifibronectin F(ab')2 fragments. Incubation of platelets with anti-GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed even though separate experiments had shown that these anti-GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin-activated platelets. These data suggest the existence of another binding system for the interaction of platelets with fibronectin that may only appear when fibronectin is present on a surface.
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Nievelstein, PF, and JJ Sixma. "Glycoprotein IIb-IIIa and RGD(S) are not important for fibronectin- dependent platelet adhesion under flow conditions." Blood 72, no. 1 (July 1, 1988): 82–88. http://dx.doi.org/10.1182/blood.v72.1.82.bloodjournal72182.
Full textAbstract:
Previous studies have indicated that activated blood platelets interact with fibronectin through binding of fibronectin to the glycoprotein IIb- IIIa complex (GPIIb-IIIa). The cell attachment site of fibronectin with its crucial arg-gly-asp(-ser) [RGD(S)]sequence is involved in these bindings. We studied the importance of these interactions for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a nonreactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin-induced aggregation and adhesion under static conditions at 0.1 mmol/L. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mmol/L had a significant inhibitory effect at 1,500 s-1, but not at the lower shear rates of 800 and 300 s-1 where platelet adhesion is also fibronectin dependent. On the matrix of cultured human umbilical vein endothelial cells, 1 mmol/L GRGDSP had no effect on platelet adhesion. The relation between GPIIb- IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann's thrombasthenia and monoclonal antibodies to GPIIb-IIIa using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann's thrombasthenia showed a similar inhibition of adhesion in the presence of fibronectin-free plasma after the ECMs had been preincubated with antifibronectin F(ab')2 fragments. Incubation of platelets with anti-GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed even though separate experiments had shown that these anti-GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin-activated platelets. These data suggest the existence of another binding system for the interaction of platelets with fibronectin that may only appear when fibronectin is present on a surface.
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Kunicki, TJ, R. Orchekowski, D. Annis, and Y. Honda. "Variability of integrin alpha 2 beta 1 activity on human platelets." Blood 82, no. 9 (November 1, 1993): 2693–703. http://dx.doi.org/10.1182/blood.v82.9.2693.2693.
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Abstract The activity and surface antigenicity of alpha 2 beta 1 on platelets from 27 normal subjects were found to vary significantly. A fourfold range of surface antigen correlates with a 20-fold variation in the ability of nonactivated, washed platelets to adhere to type I collagen and a fivefold variation in the adhesion of platelets to type III collagen. These differences in surface receptor are reflected in significant variation in the lag time required for type I collagen- induced platelet aggregation in platelet-rich plasma. Among the same individuals, no difference was observed in surface levels or activities of two other platelet integrins, the fibronectin receptor alpha 5 beta 1 and the fibrinogen receptor alpha IIb beta 3. In all cases studied, we observed complimentary differences in the incorporation of 125I into surface alpha 2 beta 1, in quantity of surface alpha 2 beta 1 antigens, and in alpha 2 beta 1 collagen receptor activity. Despite variations in these parameters, there was no difference in the electrophoretic mobility or isoelectric point of either integrin subunit among the individuals studied. The wide range of activity and antigenicity of this platelet collagen receptor may result from polymorphism(s) in the alpha 2 beta 1 genes, or the activity of alpha 2 beta 1 may be variably regulated by another gene product. The heterogeneity of platelet alpha 2 beta 1 that we describe in this report certainly explains previous discrepancies concerning the contributions of this integrin to platelet adhesion to collagens. Most importantly, differences in surface collagen receptor activity may correlate with a long-term risk toward thrombosis, impaired hemostasis, and/or cardiovascular disease.
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Kunicki, TJ, R. Orchekowski, D. Annis, and Y. Honda. "Variability of integrin alpha 2 beta 1 activity on human platelets." Blood 82, no. 9 (November 1, 1993): 2693–703. http://dx.doi.org/10.1182/blood.v82.9.2693.bloodjournal8292693.
Full textAbstract:
The activity and surface antigenicity of alpha 2 beta 1 on platelets from 27 normal subjects were found to vary significantly. A fourfold range of surface antigen correlates with a 20-fold variation in the ability of nonactivated, washed platelets to adhere to type I collagen and a fivefold variation in the adhesion of platelets to type III collagen. These differences in surface receptor are reflected in significant variation in the lag time required for type I collagen- induced platelet aggregation in platelet-rich plasma. Among the same individuals, no difference was observed in surface levels or activities of two other platelet integrins, the fibronectin receptor alpha 5 beta 1 and the fibrinogen receptor alpha IIb beta 3. In all cases studied, we observed complimentary differences in the incorporation of 125I into surface alpha 2 beta 1, in quantity of surface alpha 2 beta 1 antigens, and in alpha 2 beta 1 collagen receptor activity. Despite variations in these parameters, there was no difference in the electrophoretic mobility or isoelectric point of either integrin subunit among the individuals studied. The wide range of activity and antigenicity of this platelet collagen receptor may result from polymorphism(s) in the alpha 2 beta 1 genes, or the activity of alpha 2 beta 1 may be variably regulated by another gene product. The heterogeneity of platelet alpha 2 beta 1 that we describe in this report certainly explains previous discrepancies concerning the contributions of this integrin to platelet adhesion to collagens. Most importantly, differences in surface collagen receptor activity may correlate with a long-term risk toward thrombosis, impaired hemostasis, and/or cardiovascular disease.
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Scudder, Lesley, Susan Smyth, Dimitrios Tsakiris, and Barry Coller. "Structure and Function of Murine αIIbβ3 (GPIIb/IIIa): Studies Using Monoclonal Antibodies and β3-null Mice." Thrombosis and Haemostasis 84, no. 12 (2000): 1103–8. http://dx.doi.org/10.1055/s-0037-1614177.
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SummaryThe αIIbβ3 receptor (GPIIb/IIIa) is the only platelet-specific integrin receptor and the most abundant adhesion/aggregation receptor on the surface of human platelets. Since mice are increasingly being used as models of human disease, we analyzed the structure and function of murine platelet αIIbβ3, utilizing both β3 integrin-deficient mice, who have a phenotype that resembles Glanzmann thrombasthenia, and our hamster monoclonal antibody (mAb) 1B5 to murine αIIbβ3. By immunoblot analysis, flow cytometry, and mAb binding studies, mouse platelets express abundant amounts of αIIbβ3 (60-80,000 copies/platelet). Like their human counterparts, murine αIIb and β3 exhibit different electrophoretic motilities under nonreducing (αIIb 135k Da; β3 92k Da) and reducing (αIIb 120k Da; β3 108k Da) conditions, and the αIIbβ3 complex is dissociated by EDTA at pH 8 and 37 ºC. Murine β3 is less susceptible to proteolysis by plasmin than is human β3. In addition to defective platelet aggregation, mouse platelets lacking αIIbβ3 and αVβ3 are unable to adhere to fibrinogen and prothrombin, but retain the ability to adhere to fibronectin and collagen. Following platelet activation, β3-null platelets express slightly less P-selectin than do wild-type mouse platelets. Moreover, β3-null platelets have altered tyrosine phosphorylation patterns following thrombinand collagen-induced aggregation. These results suggest fundamental similarities between human and mouse platelet activation and aggregation, but delineate subtle differences that need to be considered when comparing studies from mice and humans.
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Skuja, Sandra, Nityanand Jain, Marks Smirnovs, and Modra Murovska. "Alcohol-Induced Alterations in the Vascular Basement Membrane in the Substantia Nigra of the Adult Human Brain." Biomedicines 10, no. 4 (April 1, 2022): 830. http://dx.doi.org/10.3390/biomedicines10040830.
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The blood–brain barrier (BBB) represents a highly specialized interface that acts as the first line of defense against toxins. Herein, we investigated the structural and ultrastructural changes in the basement membrane (BM), which is responsible for maintaining the integrity of the BBB, in the context of chronic alcoholism. Human post-mortem tissues from the Substantia Nigra (SN) region were obtained from 44 individuals, then grouped into controls, age-matched alcoholics, and non-age-matched alcoholics and assessed using light and electron microscopy. We found significantly less CD31+ vessels in alcoholic groups compared to controls in both gray and white matter samples. Alcoholics showed increased expression levels of collagen-IV, laminin-111, and fibronectin, which were coupled with a loss of BM integrity in comparison with controls. The BM of the gray matter was found to be more disintegrated than the white matter in alcoholics, as demonstrated by the expression of both collagen-IV and laminin-111, thereby indicating a breakdown in the BM’s structural composition. Furthermore, we observed that the expression of fibronectin was upregulated in the BM of the white matter vasculature in both alcoholic groups compared to controls. Taken together, our findings highlight some sort of aggregation or clumping of BM proteins that occurs in response to chronic alcohol consumption.
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Kinlough-Rathbone, Raelene L., Marian A. Packham, Maria A. Guccione, Mary Richardson, Elizabeth J. Harfenist, and J. Fraser Mustard. "Characteristics of Thrombin-Degranulated Human Platelets: Development of a Method that Does not Use Proteolytic Enzymes for Deaggregation." Thrombosis and Haemostasis 65, no. 04 (1991): 403–10. http://dx.doi.org/10.1055/s-0038-1648161.
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SummaryA method has been developed for preparing suspensions of washed human platelets that have lost as much as 90% of their dense granule and alpha granule contents as a result of stimulation by thrombin (0.9 U/ml for 3 min at 37° C), and recovering the platelets without using a proteolytic erwyme. Glycyl-Lprolyl-Larginyl-L-proline (GPRP) was used to prevent polymerization of released fibrinogen and arginyl-glycyl-aspartyl-serine (RGDS) to block the interaction of released fibrinogen, vWf or fibronectin with the glycoprotein IIb/IIIa complex. The thrombin used to degranulate the platelets was neutralized with D-phenylalanyl-Lprolyl-L-arginine chlororhethyl ketone (FPRCH2CI) and prostaglandin E1 was added to return the platelets towards a disc shape. The degranulated platelets aggregated in response to ADR platelet activating factor, arachidonate and the thromboxane 42 mimetic, U46619 in the presence of added fibrinogen; the platelets changed shape but did not aggregate in response to collagen. Thrombin and the calcium ionophore, A23L87, caused aggregation without added fibrinogen. Synergism between pairs of aggregating agents at low concentrations was observed. Little TXB2 was formed when the platelets were reaggregated by thrombin. RGDS and F(ab’)2 fragments of an antibody to fibrinogen inhibited reaggregation induced by thrombin and A23187 indicating that small amounts of fibrinogen at the platelet surface may support aggregation by strong agonists. Adherence of thrombin-degranulated platelets to a collagen-coated surface was less than for controls, but spreading was more extensive. Electron-microscopic immunogold cytochemistry with anti-human fibrinogen IgG showed numerous gold particles in platelet vacuoles. Thrombin-degranulated platelets can be used to study pathways involved in platelet aggregation without the complicating effects of released granule contents including ADR and to study indirectly the factors released from platelets that contribute to the stability of platelet aggregates.
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Aoshiba, K., S. I. Rennard, and J. R. Spurzem. "Cell-matrix and cell-cell interactions modulate apoptosis of bronchial epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 272, no. 1 (January 1, 1997): L28—L37. http://dx.doi.org/10.1152/ajplung.1997.272.1.l28.
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Apoptosis is an important process maintaining cell number and tissue structure. To determine whether cell-extracellular matrix (ECM) and cell-cell interactions modulate apoptosis in bronchial epithelium, we cultured human bronchial epithelial cells in different conditions and evaluated the cells for apoptosis. We found that plating cells in conditions that prevent cell-ECM adhesion induced apoptosis. Plating cells on type I collagen, fibronectin, and biosynthesized matrix prevented apoptosis, due at least in part to integrin-mediated adhesion. When cells were cultured at high density but under conditions preventing cell-substratum adhesion, aggregation occurred. Apoptosis was inversely correlated with aggregation. Cell-cell adhesion in these conditions was mediated at least partly by integrins containing alpha v. Cell aggregation was not associated with activation of a signaling pathway that is usually activated by cell-ECM adhesion, phosphorylation of focal adhesion kinase, but was associated with Bcl-2 protein expression, consistent with the concept that Bcl-2 protects against apoptosis. We conclude that both cell-ECM and cell-cell interactions, likely mediated in part by integrins, modulate apoptosis in bronchial epithelium.
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Siddiqua, Ashia, Michael Wilkinson, Vijay Kakkar, Yatin Patel, Salman Rahman, and Kalwant Authi. "Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets." Thrombosis and Haemostasis 79, no. 01 (1998): 177–85. http://dx.doi.org/10.1055/s-0037-1614247.
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SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.
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Stoldt, Volker R., Matthias Abtmeier, Khon C. Huynh, and Rudiger E. Scharf. "Platelets Can Attack Candida Albicans Hypha Opsonized by Fibronectin." Blood 118, no. 21 (November 18, 2011): 2210. http://dx.doi.org/10.1182/blood.v118.21.2210.2210.
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Abstract Abstract 2210 Background and objectives: Candida albicans is a dimorphic fungus that can change between its yeast and hypha form. The ability to germinate and grow in hypha is a relevant factor of virulence. Platelets can bridge innate and adaptive immunity. To attack microorganisms, platelets store microbicidal and immune stimulatory proteins in their α-granules. Platelet integrins and their plasma ligands can govern both binding to the pathogen and activation of platelets with subsequent secretion of their granular constituents. Fibronectin (Fn), a large dimeric glycoprotein, with specific binding sites for integrins and collagens, circulates in plasma and is part of extracellular matrix of the host. Both platelets and C. albicans are known to interact with Fn. Platelet can bind Fn specifically by their integrins α5β1, αIIbβ3, and αvβ3. Several protein species have been identified on the surface of C. albicans also interacting with Fn. Here, we examined whether or not Fn can support the platelet-mediated host defense against C. albicans. Material and methods: Platelet-rich plasma from citrated anticoagulated human blood was washed with phosphate-buffered saline (PBS), pH 6.5, containing 2 U/ml apyrase. The platelet pellet was carefully resuspended in Tyrode buffer, pH 7.3, containing 2 mM MgCl2, 2 mM CaCl2, and 10 μM of the cell tracker dye CMFDA (5-chloromethylfluorescein diacetate, Invitrogen). CMFDA did not affect platelet function, as documented by intact activation and normal adhesion, aggregation, secretion, and thromboxyne A2 formation. Washed and stained platelets were adjusted at a concentration of 2.5 × 108 plt/ml. Platelet aggregation was induced by 40 nM phorbol 12-myristate 13-acetate (PMA) or 10 μg/ml collagen and recorded by changes in light transmission. Fn was purified from human fresh-frozen plasma by gelatine sepharose affinity chromatography and subsequent elution with 3 M urea. The isolated adhesive protein was conjugated with alexa fluor 488 according the manufacturer's instruction (Invitrogen). C. albicans, cultured over night in yeast extract pepton glucose medium at 30°C, was washed twice in PBS, pH 7.3, and starved for 1 h at room temperature in PBS. C. albicans was diluted in PBS supplemented with fetal calf serum (10 %) to adjust a final optical density of 0.4 as a measure of hypha formation and to induce germination at 37°C. Samples were taken at various times of germination. For flow cytometric binding studies, serum was washed away and C. albicans yeast and hypha forms were incubated for 1h with 40 μg/ml Fn-alexa fluor 488 or 1×107 fluorescently CMFDA-stained platelets were added under static conditions. Results: C. albicans germination (mean + SD) at 30 min, 60 min, and 120 min of incubation revealed 5+4 %, 40+9 %, and 90+12 % hypha, as determined by light microscopy and confirmed by flow cytometry, as quantified by their characteristic forward and side scatter profiles. C. albicans hypha induction increased binding of Fn-alexa fluor 488 (40 μg/ml) from 1.2+0.3 % positive yeast cells (baseline), to 9.4+2.9 % at 30 min, 23.0+3.4 % at 60 min, or 45.0+5.1 % at 120 min, respectively (p<0.05, each). As observed by microscopy, C. albicans germination tubes promoted the binding Fn. Upon incubation, germation of C. albicans, obtained after 120 min, increased its binding to washed platelets 5-fold, as compared to the yeast form (p<0.05). Moreover, pretreatment of C. albicans hypha with Fn (40 μg/ml) enhanced their specific binding to platelet integrin αIIbβ3, as abciximab (2 μg/ml) blocked Fn binding to C. albicans completely. Conclusion: Hypha of C. albicans, a relevant factor of virulence, can be attacked by platelets. Fibronectin, a ligand of the β1 and β3 platelet integrins, opsonizes the hypha forms of C. albicans and supports platelet binding. The multiple binding sites in this adhesive protein for collagen and integrins can support the platelet-mediated host defense and may modify adaptive immune responses against C. albicans. Disclosures: No relevant conflicts of interest to declare.
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Oliveira, Luciana S., Maria Inácia Estevão-Costa, Valéria G. Alvarenga, Dan E. Vivas-Ruiz, Armando Yarleque, Augusto Martins Lima, Ana Cavaco, Johannes A. Eble, and Eladio F. Sanchez. "Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergón) Induces Glycoprotein VI Shedding and Impairs Platelet Function." Molecules 24, no. 19 (September 26, 2019): 3489. http://dx.doi.org/10.3390/molecules24193489.
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Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2β1 integrin. Neither the α2β1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.
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Farfan, Mauricio J., Keith G. Inman, and James P. Nataro. "The Major Pilin Subunit of the AAF/II Fimbriae from Enteroaggregative Escherichia coli Mediates Binding to Extracellular Matrix Proteins." Infection and Immunity 76, no. 10 (June 30, 2008): 4378–84. http://dx.doi.org/10.1128/iai.00439-08.
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ABSTRACT Enteroaggregative Escherichia coli (EAEC) adherence to human intestinal tissue is mediated by aggregative adherence fimbriae (AAF); however, the receptors involved in EAEC adherence remain uncharacterized. Adhesion to extracellular matrix proteins is commonly observed among enteric pathogens, so we addressed the hypothesis that EAEC may bind to extracellular matrix proteins commonly found in the intestine. We found that EAEC prototype strain 042 adhered more abundantly to surfaces that were precoated with the extracellular matrix proteins fibronectin, laminin, and type IV collagen. Differences in fibronectin binding of almost 2 orders of magnitude were observed between EAEC 042 and a mutant in the AAF/II major pilin gene, aafA. Purified AafA, refolded as a donor strand complementation construct, bound fibronectin in a dose-dependent manner. Addition of fibronectin to the apical surfaces of polarized T84 cell monolayers augmented EAEC 042 adherence, and this effect required expression of aafA. Finally, increased bacterial adherence was observed when apical secretion of fibronectin was induced by adenosine in polarized T84 cells. Binding to fibronectin may contribute to colonization of the gastrointestinal tract by EAEC.
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Wang, Yiming, Adili Reheman, Jalil Kalantari, Wuxun Jin, Peter L. Gross, Margaret L. Rand, John J. Freedman, and Heyu Ni. "Plasma Fibronectin In Thrombosis and Hemostasis: Exploring the Fibrin Dependent and Independent Mechanisms." Blood 116, no. 21 (November 19, 2010): 484. http://dx.doi.org/10.1182/blood.v116.21.484.484.
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Abstract Abstract 484 Background: Plasma fibronectin (pFn) is an abundant protein in the blood. It has long been suspected that pFn plays a role in thrombosis/hemostasis, but this has remained controversial. Our previous study using pFn deficient mice demonstrated that pFn supports thrombosis (PNAS. 2003; 100: 2415-9). Unexpectedly, depletion of pFn in fibrinogen (Fg) and von Willebrand factor (VWF) double deficient (Fg/VWF−/−) mice enhanced, rather than abolished, platelet aggregation and thrombus formation, revealing a functional switch of pFn in the presence and absence of Fg and VWF (Blood. 2009;113:1809-17). However, the mechanism that controls this switch is not known. Furthermore, the hemostatic function of pFn in VW disease (VWD) or afibrinogenemia is unclear. Methods: To address these questions, we bred pFn conditional knockout mice with VWF−/− or Fg−/− mice, establishing 2 new strains of mice: Fg/pFn−/− and VWF/pFn−/−. We also extended our studies of pFn in the triple knockout (TKO, Fg/VWF/pFn−/−) mice. PolyI-polyC was injected into Cre+ and Cre- mice, which resulted in the depletion of plasma pFn (>98%) and platelet pFn (>80%) in Cre+ mice but not in Cre- littermate controls. Aggregometry, a perfusion chamber system, thromboelastography (TEG), tail vein bleeding assay and intravital microscopy were used to study these mice. Results: We first observed a significantly higher mortality in TKO (25%, P<0.05) mice than their Cre- Fg/VWF−/− littermates within 1–2 weeks following the depletion of pFn. Autopsy of these mice revealed severe subcutaneous or abdominal bleeding at the sites of injection. The tail vein bleeding time in TKO mice was also prolonged (P<0.05). Using a laser injury model of intravital microscopy, we observed rapid deposition of fluorescently-labeled pFn at sites of vessel injury in Fg/VWF−/− mice prior to significant platelet deposition. This suggested pFn is a quick/efficient factor contributing to hemostasis in the absence of Fg/VWF. We further found that the mortality rate in Fg/pFn−/− mice was also higher than their Cre- Fg−/− littermates (29%, P<0.05), demonstrating that pFn is a critical hemostatic factor that prevents fatal hemorrhage in afibrinogenemic mice via a fibrin-independent mechanism. We also found that pFn supports hemostasis in VWF−/− mice, although no significant mortality difference was observed (P>0.05). The tail vein bleeding time was longer in VWF/pFn−/− mice than in Cre- VWF−/− littermates (P<0.05), and significantly smaller thrombi were observed when VWF/pFn−/− whole blood was perfused over a collagen surface under shear rate of 1800s-1 (P<0.05). This suggests that pFn may play a role in VWF deficiency (i.e. in type 3 VWD). pFn was also found to support hemostasis in a fibrin-dependent manner. We first demonstrated with TEG that fibrin clot strength was significantly stronger in Cre- littermates than in pFn−/− mice (P<0.05). Platelet aggregation in gel-filtered platelets induced by thrombin, which converts Fg to fibrin on the platelet surface, was greater in Cre- VWF−/− than VWF/pFn−/− platelets (P<0.05). Very interestingly, in keeping with our earlier observation in TKO mice, pFn also inhibited platelet aggregation when fibrin was absent. In Fg−/− mice, we found that pFn depletion enhanced gel-filtered platelet aggregation induced by both thrombin and thrombin receptor activating peptide (TRAP, AYPGKF; P<0.05). In Cre- VWF−/− mice where Fg is present, pFn depletion also enhanced gel-filtered platelet aggregation induced by TRAP (which cannot convert Fg to fibrin) (P<0.05). pFn therefore plays a dual role in platelet aggregation based on the presence of fibrin (i.e. covalently linked fibrin-pFn supports platelet aggregation, while pFn alone inhibits aggregation). Conclusion: Our data demonstrated that pFn is a critical factor for the survival of Fg−/− mice and supports hemostasis in VWF−/− mice via both fibrin-independent and dependent pathways. We clearly showed that fibrin, likely in the form of covalently-linked fibrin-pFn complexes, is required for pFn to support platelet aggregation. Through inhibition of platelet aggregation, non-fibrin-linked soluble pFn may play an important role in the prevention of excessive thrombus formation at the site of vessel injury and thus maintains blood circulation. pFn is therefore likely a crucial supportive factor in hemostasis (for afibrinogenemic and VWD patients), and an important regulator in thrombosis. Disclosures: No relevant conflicts of interest to declare.
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Carter, W. G., E. A. Wayner, T. S. Bouchard, and P. Kaur. "The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells." Journal of Cell Biology 110, no. 4 (April 1, 1990): 1387–404. http://dx.doi.org/10.1083/jcb.110.4.1387.
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We have examined cultures of neonatal human foreskin keratinocytes (HFKs) to determine the ligands and functions of integrins alpha 2 beta 1, and alpha 3 beta 1 in normal epidermal stratification and adhesion to the basement membrane zone (BMZ) in skin. We used three assay systems, HFK adhesion to purified extracellular matrix (ECM) ligands and endogenous secreted ECM, localization of integrins in focal adhesions (FAs), and inhibition of HFK adhesion with mAbs to conclude: (a) A new anti-alpha 3 beta 1 mAb, P1F2, localized alpha 3 beta 1 in FAs on purified laminin greater than fibronectin/collagen, indicating that laminin was the best exogeneous ligand for alpha 3 beta 1. However, in long term culture, alpha 3 beta 1 preferentially codistributed in and around FAs with secreted laminin-containing ECM, in preference to exogenous laminin. Anti-alpha 3 beta 1, mAb P1B5, detached prolonged cultures of HFKs from culture plates or from partially purified HFK ECM indicating that interaction of alpha 3 beta 1 with the secreted laminin-containing ECM was primarily responsible for HFK adhesion in long term culture. (b) In FA assays, alpha 2 beta 1 localized in FAs conincident with initial HFK adhesion to exogenous collagen, but not laminin or fibronectin. However, in inhibition assays, anti-alpha 2 beta 1 inhibited initial HFK adhesion to both laminin and collagen. Thus, alpha 2 beta 1 contributes to initial HFK adhesion to laminin but alpha 3 beta 1 is primarily responsible for long-term HFK adhesion to secreted laminin-containing ECM. (c) Serum or Ca2(+)-induced aggregation of HFKs resulted in relocation of alpha 2 beta 1 and alpha 3 beta 1 from FAs to cell-cell contacts. Further, cell-cell adhesion was inhibited by anti-alpha 3 beta 1 (P1B5) and a new anti-beta 1 mAb (P4C10). Thus, interaction of alpha 3 beta 1 with either ECM or membrane coreceptors at cell-cell contacts may facilitate Ca2(+)-induced HFK aggregation. (d) It is suggested that interaction of alpha 3 beta 1 with a secreted, laminin-containing ECM in cultured HFKs, duplicates the role of alpha 3 beta 1 in basal cell adhesion to the BMZ in skin. Further, relocation of alpha 2 beta 1 and alpha 3 beta 1 to cell-cell contacts may result in detachment of cells from the BMZ and increased cell-cell adhesion in the suprabasal cells contributing to stratification of the skin.
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Bai, Xia, Jianxin Fu, Jian Su, Lan Dai, Wei Zhang, Kaiyang Ding, Zhaoyue Wang, and Changgeng Ruan. "Expression and Functional Analysis of Disintegrin from Agkistrodon Actus Venom." Blood 106, no. 11 (November 16, 2005): 3963. http://dx.doi.org/10.1182/blood.v106.11.3963.3963.
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Abstract Disintegrin is a kind of small native peptides derived from the snake venom, containing RGD sequences. Its interaction with integrins, such as aIIbb3, anb3 and a5b1, can inhibit platelet aggregation. hinder the process of angiogenesis and tumor metastasis. In order to further investigate the function of disintegrin in platelet aggregation, cell adhesion and angiogenesis, the sequence encoding the disintegrin domain of agacutin was fused with enhanced green fluorencent protein(eGFP) and inserted in plasmid pQE-30. The recombinant protein was expressed in E.Coli M15 after induction by IPTG, amounting to 38% of the total bacterial protein. The cells expressing integrin such as breast cancer cell line MDA-MB-231 and endothelial cell line EA.hy926 can specifically bind to the recombinant GFP-disintegrin. Flow cytometry showed that the recombinant protein could bind to platelet specfically and could compete with anti-b3 integrin monoclonal antibody CD61. Moreover, the recombinant protein could inhibit platelet aggregation induced by collagen and ADP in a dose-dependent manner, but had no ability to impede the platelet aggregation induced by Ristocetin. Meanwhile, it could inhibit cell (MDA-MB-231 and EA.hy926) adhesion to fibronectin with inhibition rate of 69% and 48%, respectively. Finally, it could induce apoptosis of EA.hy926 endothelial cells and inhibit the angiogenesis on chicken chorioallantoic membrane mode. As a result, it is demonstrated that GFP-disintegrin has the combined quality of its disparate components and can serve as a novel tool for study of tumor and angiogenesis. In addition, the recombinant protein can efficiently inhibit platelet aggregation and angiogenesis, which will be useful for designing the potential drug for anti-thrombosis and anti-tumor metastasis.
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Bee, J. A., and K. von der Mark. "An analysis of chick limb bud intercellular adhesion underlying the establishment of cartilage aggregates in suspension culture." Journal of Cell Science 96, no. 3 (July 1, 1990): 527–36. http://dx.doi.org/10.1242/jcs.96.3.527.
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To examine the mechanism of intercellular adhesion in the establishment of limb skeletal elements we have investigated the process of limb bud cell aggregation in vitro. Limb bud cells are aggregation-competent immediately after their trypsin:collagenase dissociation in the absence of calcium. This aggregation is largely Ca2(+)-independent (CI) and is completely and reversibly inhibited by cycloheximide. In contrast, when limb bud cells are first allowed to recover from Ca2(+)-free trypsin:collagenase dissociation, aggregation of the surviving population is exclusively Ca2(+)-dependent (CD) and completely and reversibly inhibited by cycloheximide. The presence of exogenous calcium during initial cell dissociation retains a functional CD aggregation mechanism. However, incubation of such cells with EGTA releases the CD component and converts the cells to a predominantly CI aggregation. Rabbits were immunized with limb bud cells exhibiting the recovered CD aggregation mechanism and the resulting immune sera were screened for their effect on cell aggregation. Relative to pre-immune sera, intact immune IgG agglutinated dissociated limb bud cells whilst immune Fab fragments inhibited their aggregation. The aggregation-inhibiting antiserum recognizes five major limb bud cell surface components with apparent molecular weights of 72K, 50K, 23K, 14.5K and 8.5K (K = 10(3) Mr), respectively. Limb bud cell surface plasma membranes were isolated by sucrose gradient density centrifugation and detergent-solubilized proteins coupled to Sepharose 4B with cyanogen bromide. Equivalent cell surface plasma membrane proteins were 125I-iodinated and applied to the affinity column. Limb bud cell surface protein affinity chromatography in the presence of exogenous calcium yields a single protein with an apparent molecular weight of approximately 8.5 K. This protein molecule elutes at 0.6 M NaCl, indicating a high affinity, is recognized by the aggregation-inhibiting antiserum, and is itself capable of inhibiting CD limb bud cell aggregation. Fab fragments prepared from rabbit antisera specifically directed against the affinity-purified material also inhibit CD limb bud cell aggregation and this inhibition is neutralized by the 8.5 K protein. Our data thus demonstrate that CD limb bud cell aggregation is not mediated by fibronectin and/or collagen type I and indicate that this process is governed by a novel 8.5 K cell adhesion molecule.
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Wang, Yiming, Reid C. Gallant, Miguel A. D. Neves, Xi Lei, Sahil Gupta, Rodrigo Coelho, Tatianna Wong, Ronald D. Cohn, Kevin P. Campbell, and Heyu Ni. "Alpha-Dystroglycan Supports Platelet Aggregation and Thrombus Formation." Blood 134, Supplement_1 (November 13, 2019): 11. http://dx.doi.org/10.1182/blood-2019-131521.
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Introduction: Fibrinogen (Fg) and von Willebrand factor (VWF) have been considered essential for platelet adhesion and aggregation. However, platelet aggregation still occurs in mice lacking Fg and/or VWF or plasma fibronectin but not β3 integrin (JCI, 2000; JTH, 2006; Blood, 2009; JCI, 2014). This suggests that other non-classical αIIbβ3 integrin ligand(s) mediate platelet aggregation. α-dystroglycan (α-DG) is a component of the dystrophin-glycoprotein complex that binds extracellular matrix proteins containing laminin-G like domains via unique heteropolysaccharide [-GlcA-β1,3-Xyl-α1,3-]n called matriglycan, which can be targeted specifically with monoclonal antibody IIH6C4. Although α-DG was identified in a recent proteomic study of platelet releasate, its membrane expression and function in platelets have never been investigated. Methods and Results: Using the anti-α-DG monoclonal antibody IIH6C4, we found expression of α-DG in mouse and human resting platelets in Western blots. α-DG expression was also identified on the non-permeabilized mouse and human resting platelets by flow cytometry, indicating that α-DG is constitutively expressed on the platelet surface. We next examined whether disruption of the integrity of the dystrophin-glycoprotein complex affects the platelet aggregation. In a dystrophin-deficient mouse model of Duchenne muscular dystrophy with reduced α-DG expression (mdx mice), we found that ADP induced platelet aggregation in platelet-rich plasma (PRP) decreased 50%, suggesting that the integrity of the dystrophin-glycoprotein complex is required for normal platelet aggregation. To test whether inhibition of platelet aggregation can be achieved by targeting α-DG, we applied the well-established polyclonal (H300) and monoclonal (IIH6C4 and VIA4) anti-α-DG antibodies. Mouse gel-filtered platelet aggregation induced by thrombin was significantly inhibited by all three antibodies. Mouse platelet aggregation in PRP was also inhibited by H300. For platelets from healthy human donors, the inhibitive effect was more profound. Using a lower concentration of H300 (1 µg/mL in human vs. 2 µg/mL in mouse), ADP induced human platelet aggregation in PRP was inhibited to less than 50% of control and quickly de-aggregated within 5 minutes, while no de-aggregation was observed in the controls. Human gel-filtered platelet aggregation was also inhibited in a dose-dependent manner by these antibodies. Our results thus revealed a vital role of α-DG in platelet aggregation. In an ex vivo perfusion chamber model, human platelet adhesion and thrombus formation on collagen were markedly decreased at an arterial shear rate of 1800/s by anti-α-DG antibodies. Interestingly, although α-DG was found to be a ligand of laminin, platelet adhesion on laminin was not significantly altered by these antibodies, suggesting that contribution of α-DG to thrombus formation is not through its classical ligand laminin but other previously unidentified mechanisms. Next, we tested the role of α-DG in thrombus formation in vivo. Using a mouse cremaster artery laser-injury intravital microscopy model, we found that the anti-α-DG antibodies significantly delayed and decreased thrombus formation. To investigate the underlying mechanism of the surprisingly profound impact of α-DG on platelet aggregation and thrombus formation, we performed the co-immunoprecipitation assay and found that α-DG interacts with both β3 integrin and fibronectin, even in the absence of Fg and VWF, suggesting that α-DG may directly or form an α-DG-fibronectin complex to bind αIIbβ3 integrin, contributing to platelet aggregation and thrombus formation. Conclusion: Our data demonstrated that α-DG and likely other components of the dystrophin-glycoprotein complex are expressed on the platelet surface, and play a vital role in platelet aggregation and thrombus formation. α-DG may contribute to platelet aggregation independent of VWF and Fg through direct or indirect interaction with αIIbβ3 integrin. It is likely that patients with muscular dystrophies, such as those with Duchenne muscular dystrophy, are protected from thrombosis. More importantly, our data established α-DG, and potentially other components of the dystrophin-glycoprotein complex, as novel targets for the treatment of thrombotic disorders. Disclosures No relevant conflicts of interest to declare.
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Kokubo, T., Y. Hiki, H. Iwase, A. Tanaka, K. Toma, K. Hotta, and Y. Kobayashi. "Protective role of IgA1 glycans against IgA1 self-aggregation and adhesion to extracellular matrix proteins." Journal of the American Society of Nephrology 9, no. 11 (November 1998): 2048–54. http://dx.doi.org/10.1681/asn.v9112048.
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The aim of this study was to investigate the role of carbohydrate moieties attached to IgA1 hinge region in IgA1 self-aggregation and adhesion to extracellular matrix (ECM) proteins previously reported in IgA nephropathy. Serum IgA1 samples isolated from healthy individuals were digested with neuraminidase (NA), NA + beta-galactosidase, and NA + beta-galactosidase + alpha-N-acetylgalactosaminidase to remove the carbohydrates from the hinge region and were named asialo, agalacto, and naked IgA1, respectively. First, polyacrylamide gel electrophoresis was performed under the native condition, and consequently, a broad band indicating IgA1 self-aggregation was clearly observed in asialo, agalacto, and naked IgA1, but not in native IgA1. However, the broad band disappeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the nonreducing condition. Second, it was shown that IgA1 adhesion activities to type IV collagen, fibronectin, and laminin were significantly higher in asialo, agalacto, and naked IgA1 than in native IgA1, using enzyme-linked immunosorbent assay (asialo, agalacto, and naked versus native: P < 0.01). In addition, agalacto IgA1 had the highest affinity for all of the ECM proteins among the deglycosylated IgA1 (agalacto versus asialo and naked, P < 0.05). These results indicated that the removal of carbohydrates from the IgA1 molecule resulted in noncovalent self-aggregation and a significant increase in adhesion to the ECM proteins. It was therefore suggested that the IgA1 glycans played a protective role against aggregation and adhesion and that the underglycosylation of the IgA1 molecule found in IgA nephropathy could be involved in the nonimmunologic glomerular accumulation of IgA1.
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Reheman, Adili, Yang Hong, Christopher M. Spring, Pingguo Chen, Denisa D. Wagner, Reinhard Fassler, John Freedman, and Heyu Ni. "Fibronectin Is Not the Only Important Molecule Required for Fibrinogen/VWF-Independent Platelet Aggregation: Study of Thrombosis in a New Strain of Triple Deficient Mice." Blood 108, no. 11 (November 16, 2006): 1515. http://dx.doi.org/10.1182/blood.v108.11.1515.1515.
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Abstract Fibrinogen (Fg) has been considered essential for platelet aggregation. We demonstrated, however, that thrombi do form in Fg-deficient mice and in mice doubly deficient for both fibrinogen and von Willebrand factor (Fg/VWF−/−). We further reported that β3 integrin and thrombin are critical for this Fg/VWF-independent platelet aggregation. In Fg−/− or Fg/VWF−/− mice, platelet fibronectin (Fn) content is increased 3–5 fold. Furthermore, thrombus growth and stability are impaired in plasma Fn conditional deficient (M×-Cre, Fnflox/flox) mice. These data are consistent with the most recent studies of Fn assembly and suggest that Fn may support platelet thrombus formation. To examine whether Fn is the alternative key molecule which mediates platelet aggregation and thrombus formation in Fg/VWF−/− mice, we developed a novel strain of triple knockout (TKO) mice by breeding Fg/VWF−/− mice with M×-Cre+/− Fnflox/flox conditional knockout mice. Cre- littermates delivered from the same parents were used as a control. Fn depletion was induced by i.p. injections of polyinonic-polycytidylic acid. We found that TKO mice are viable with dramatically decreased levels of Fn in both the plasma (<2% of control) and platelets (<20% of control) as determined by immunoblot. No significant difference was found in peripheral blood cell counts or platelet surface adhesion proteins (GPIbα, P-selectin, β3 and β1 integrins) as compared with control mice. Unexpectedly, TKO platelet aggregation induced by thrombin and thrombin receptor activation peptide (TRAP) was enhanced in both platelet rich plasma and PIPES buffer (P<0.05). This phenomenon was also observed in a parallel-plate flow chamber. Significantly more TKO aggregates formed when fluorescently labelled whole blood was perfused over a collagen surface at 500s−1 (P<0.001). We also studied thrombus formation in TKO and Cre- control mice using intravital microscopy. Our preliminary data showed no obvious decrease in thrombus formation in TKO mice and injured arterioles occluded in one of two experimental mice. Our data suggest that Fn is not the only important molecule required for platelet aggregation and thrombus formation in Fg/VWF−/− mice. Further investigation of how Fn may inhibit Fg/VWF-independent platelet aggregation and the identification of what ligand(s) support this novel aggregation pathway may unveil an alternative haemostatic pathway in the absence of Fg and VWF.
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Hussain, Muzaffar, Axana Haggar, Georg Peters, Gursharan S. Chhatwal, Mathias Herrmann, Jan-Ingmar Flock, and Bhanu Sinha. "More than One Tandem Repeat Domain of the Extracellular Adherence Protein of Staphylococcus aureus Is Required for Aggregation, Adherence, and Host Cell Invasion but Not for Leukocyte Activation." Infection and Immunity 76, no. 12 (September 15, 2008): 5615–23. http://dx.doi.org/10.1128/iai.00480-08.
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ABSTRACT The extracellular adherence protein (Eap) is a multifunctional Staphylococcus aureus protein and broad-spectrum adhesin for several host matrix and plasma proteins. We investigated the interactions of full-length Eap and five recombinant tandem repeat domains with host proteins by use of surface plasmon resonance (BIAcore) and ligand overlay assays. In addition, agglutination and host cell interaction, namely, adherence, invasion, and stimulation of proliferation, were determined. With plasmon resonance, the interaction of full-length Eap isoforms (from strains Newman and Wood 46) with fibrinogen, fibronectin, vitronectin, and thrombospondin-1 was found to be specific but with different affinities for the ligands tested. In the ligand overlay assay, the interactions of five single tandem repeat domains (D1 to D5) of Eap-7 (from strain CI-7) with fibronectin, fibrinogen, vitronectin, thrombospondin-1, and collagen I differed substantially. Most prominently, D3 bound most strongly to fibronectin and fibrinogen. Full-length Eap, but none of the single tandem repeat domains, agglutinated S. aureus and enhanced adherence to and invasion of host cells by S. aureus. Constructs D3-4 and D1-3 (in cis) increased adherence and invasiveness compared to what was seen for single Eap tandem repeat domains. By contrast, single Eap tandem repeat domains and full-length Eap similarly modulated the proliferation of peripheral blood mononuclear cells (PBMCs): low concentrations stimulated, whereas high concentrations inhibited, proliferation. Taken together, the data indicate that Eap tandem repeat domains appear to have distinct characteristics for the binding of soluble ligands, despite a high degree of sequence similarity. In addition, more than one Eap tandem repeat domain is required for S. aureus agglutination, adherence, and cellular invasion but not for the stimulation of PBMC proliferation.
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Cox, Dermot, Yukio Motoyama, Jiro Seki, Toshiaki Aoki, Miwako Dohi, and Keizo Yoshida. "Pentamidine: A Non-Peptide GPIIb/IIIa Antagonist – In Vitro Studies on Platelets from Humans and Other Species." Thrombosis and Haemostasis 68, no. 06 (1992): 731–36. http://dx.doi.org/10.1055/s-0038-1646352.
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SummaryIn this paper we show that the non-peptide anti-parasite agent pentamidine is a broad spectrum anti-platelet agent with an IC50 of 1.1 µM in ADP-induced platelet aggregation in human platelet rich plasma (PRP). It had similar activity when collagen, arachidonic acid, platelet activating factor, thrombin and epinephrine were used. It had no effect on platelet intracellular cAMP levels. It inhibited 125I-fibrinogen, 125I-fibronectin and 125I-von Willebrand factor binding to ADP-activated fixed platelets with IC50 values of 160, 160 and 60 nM respectively. Pentamidine showed a high degree of species selectivity with slightly less activity in monkey and dog PRP and little activity in guinea pig, rabbit, rat and mouse PRP compared with human. This was similar to the other RGD analogues tested. This species specificity was shown to be dependent on the species of platelets and independent of the species of fibrinogen. Thus, pentamidine is a potent non-peptide inhibitor of fibrinogen binding to GPIIb/IIIa.
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Liu, Huan, Xuening Yang, John Andersen, Yipeng Wang, Fuyuki Tokumasu, José Ribeiro, Dongying Ma, et al. "A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbβ3 or αVβ3 and inhibits platelet aggregation and angiogenesis." Thrombosis and Haemostasis 105, no. 06 (2011): 1032–45. http://dx.doi.org/10.1160/th11-01-0029.
Full textAbstract:
SummaryA novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet αIIbβ3 and endothelial cell αVβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 μM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.
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Montealegre-Sánchez, Leonel, Sarah N. C. Gimenes, Daiana S. Lopes, Samuel C. Teixeira, Luis Solano-Redondo, Veridiana de Melo Rodrigues, and Eliécer Jiménez-Charris. "Antitumoral Potential of Lansbermin-I, a Novel Disintegrin from Porthidium lansbergii lansbergii Venom on Breast Cancer Cells." Current Topics in Medicinal Chemistry 19, no. 22 (October 24, 2019): 2069–78. http://dx.doi.org/10.2174/1568026619666190806151401.
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Background: Disintegrins from snake venoms bind with high specificity cell surface integrins, which are important pharmacological targets associated with cancer development and progression. Objective: In this study, we isolated a disintegrin from the Porthidium lansbergii lansbergii venom and evaluated its antitumoral effects on breast cancer cells. Methods: The isolation of the disintegrin was performed on RP-HPLC and the inhibition of platelet aggregation was evaluated on human platelet-rich plasma. The inhibition of cell adhesion was also evaluated in vitro on cultures of cell lines by the MTT method as well as the inhibition of breast cancer cell migration by the wound healing assay. The binding of the disintegrin to integrin subunits was verified by flow cytometry and confocal microscopy. Finally, inhibition of angiogenesis was assessed in vitro on HUVEC cells and the concentration of VEGF was measured in the cellular supernatants. Results: The disintegrin, named Lansbermin-I, is a low molecular weight protein (< 10 kDa) that includes an RGD on its sequence identified previously. Lansbermin-I showed potent inhibition of ADP and collagen-induced platelet aggregation on human plasma and also displayed inhibitory effects on the adhesion and migration of breast cancer MCF7 and MDA-MB 231cell lines, without affecting nontumorigenic breast MCF-10A and lung BEAS cells. Additionally, Lansbermin-I prevented MCF7 cells to adhere to fibronectin and collagen, and also inhibited in vitro angiogenesis on human endothelial HUVEC cells. Conclusion: Our results display the first report on the antitumor and anti-metastatic effects of an RGDdisintegrin isolated from a Porthidium snake venom by possibly interfering with α2 and/or β1-containing integrins. Thus, Lansbermin-I could be an attractive model to elucidate the role of disintegrins against breast cancer development.
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Zaidi, TN, LV McIntire, DH Farrell, and P. Thiagarajan. "Adhesion of platelets to surface-bound fibrinogen under flow." Blood 88, no. 8 (October 15, 1996): 2967–72. http://dx.doi.org/10.1182/blood.v88.8.2967.bloodjournal8882967.
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After platelet activation, fibrinogen mediates platelet-platelet interactions leading to platelet aggregation. In addition, fibrinogen can also function as a cell adhesion molecule, providing a substratum for adhesion of platelets and endothelial cells. In this report, we studied the adhesion of platelets to surface-immobilized fibrinogen under flow in different shear rates. Heparinized whole blood containing mepacrine-labeled platelets was perfused for two minutes at various wall shear rates from 250 to 2,000 s-1 in a parallel plate flow chamber. The number of adherent fluorescent platelets was quantitated every 15 seconds with an epifluorescent videomicroscope and digital image processing system. When compared with platelet adhesion and aggregation seen on glass surfaces coated with type I bovine collagen, a significant increase in platelet adhesion was observed on immobilized fibrinogen up to wall shear rates of 800 s-1. The adherent platelets formed a single layer on fibrinogen-coated surfaces. Under identical conditions, no significant adhesion was observed on fibronectin- or vitronectin-coated surfaces. Although platelet adhesion to collagen was substantially inhibited by the platelet inhibitors prostaglandin E1 and theophylline, these inhibitors had no effect on platelet adhesion to fibrinogen. Platelets adhered to recombinant homodimeric wild-type (gamma 400–411) fibrinogen, but not to the recombinant homodimeric gamma' variant of fibrinogen. Platelet adhesion to recombinant fibrinogen with RGD to RGE mutations at positions alpha 95–97 and alpha 572–574 was similar to that with plasma-derived fibrinogen. These results show that platelets adhere to fibrinogen-coated surfaces under moderate wall shear rates, that the interaction is mediated by the fibrinogen 400–411 sequence at the carboxy-terminus of the gamma chain, and that the interaction is independent of platelet activation and the RGD sequences in the alpha chain.
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Bruggeman, L. A., E. A. Horigan, S. Horikoshi, P. E. Ray, and P. E. Klotman. "Thromboxane stimulates synthesis of extracellular matrix proteins in vitro." American Journal of Physiology-Renal Physiology 261, no. 3 (September 1, 1991): F488—F494. http://dx.doi.org/10.1152/ajprenal.1991.261.3.f488.
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The vasoconstrictor eicosanoid thromboxane plays an important role in the pathogenesis of several renal diseases. As an autacoid, its local release alters blood flow and induces platelet aggregation. We report a direct stimulatory effect of thromboxane on extracellular matrix protein production and gene expression in vitro. Treatment of two cell types, differentiated mouse teratocarcinoma cells (F9+) and human glomerular mesangial cells, with two different thromboxane analogues resulted in increased production of components of the extracellular matrix including fibronectin and the basement membrane proteins laminin and type IV collagen. These responses to thromboxane were not the result of a mitogenic effect of thromboxane nor the result of an increase in total cellular protein. The increased production of extracellular matrix proteins was, at least in part, due to an increase in the steady-state level of mRNA for these genes. Furthermore, the effect of thromboxane was markedly inhibited by cotreatment with a thromboxane-receptor antagonist. These results suggest a new potential role for thromboxane as a mediator of the sclerotic and fibrotic responses to injury.
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Berfield, A. K., G. J. Raugi, and C. K. Abrass. "Insulin induces rapid and specific rearrangement of the cytoskeleton of rat mesangial cells in vitro." Journal of Histochemistry & Cytochemistry 44, no. 2 (February 1996): 91–101. http://dx.doi.org/10.1177/44.2.8609378.
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Mesangial cells (MCs) grown without supplemental insulin (SI-MCs) express a quiescent phenotype and extracellular matrix (ECM) composition similar to MCs in vivo. In contrast, MCs routinely propagated in insulin (SI+MCs) are stimulated to proliferate, change their phenotype, and produce large amounts of collagens I and III. These effects of insulin may in part be mediated through cytoskeletal rearrangement. Differences in cytoskeletal arrangement were compared between SI-MCs and SI+MCs and 1 hr after addition of insulin (1 nM) or IGF-1 (100 nM) to SI-MCs. Cells were examined by light microscopy, electron microscopy, and immunostaining for specific cytoskeletal proteins and fibronectin. Insulin induced rapid rearrangement of stress fibers. Surface ruffling, actin aggregation, vimentin retraction, rearrangement of vinculin in focal adhesions, and fibronectin extraction were apparent. These direct effects of insulin on the SI-MC cytoskeleton occurred before insulin-induced changes in ECM composition. IGF-I induced cytoskeletal reorganization distinct from insulin. These observations demonstrate that insulin and IGF-I have unique effects on the MC cytoskeleton, which is turn may mediate secondary ligand effects on MCs.
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Lavergne, Marion, Emily Janus-Bell, Mathieu Schaff, Christian Gachet, and Pierre Mangin. "Platelet Integrins in Tumor Metastasis: Do They Represent a Therapeutic Target?" Cancers 9, no. 10 (September 28, 2017): 133. http://dx.doi.org/10.3390/cancers9100133.
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Platelets are small anucleated cell fragments that ensure the arrest of bleeding after a vessel wall injury. They are also involved in non-hemostatic function such as development, immunity, inflammation, and in the hematogeneous phase of metastasis. While the role of platelets in tumor metastasis has been recognized for 60 years, the molecular mechanism underlying this process remains largely unclear. Platelets physically and functionally interact with various tumor cells through surface receptors including integrins. Platelets express five integrins at their surface, namely α2β1, α5β1, α6β1, αvβ3, and αIIbβ3, which bind preferentially to collagen, fibronectin, laminin, vitronectin, and fibrinogen, respectively. The main role of platelet integrins is to ensure platelet adhesion and aggregation at sites of vascular injury. Two of these, α6β1 and αIIbβ3, were proposed to participate in platelet–tumor cell interaction and in tumor metastasis. It has also been reported that pharmacological agents targeting both integrins efficiently reduce experimental metastasis, suggesting that platelet integrins may represent new anti-metastatic targets. This review focuses on the role of platelet integrins in tumor metastasis and discusses whether these receptors may represent new potential targets for novel anti-metastatic approaches.
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Ekman, S., and D. Heinegård. "Immunohistochemical Localization of Matrix Proteins in the Femoral Joint Cartilage of Growing Commercial Pigs." Veterinary Pathology 29, no. 6 (November 1992): 514–20. http://dx.doi.org/10.1177/030098589202900605.
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The immunocytochemical localization of several matrix macromolecules, including collagen type II and proteoglycans, in the distal femoral articular-epiphyseal cartilage complex of 15 commercial pigs between the age of 6 and 18 weeks was studied. Early osteochondrotic lesions, i.e., chondronecrosis in the resting region of the growth cartilage, as well as extensions of necrotic cartilage into the subchondral bone, were present in all animals, except those 6 weeks old. A battery of antibodies were used for identification of macromolecules in the matrix at different stages of the disease. Chondrocyte involvement in the process could be studied by identifying the sequence of alterations in matrix macromolecules as the lesion developed. The immunostaining for aggrecan (large aggregating proteoglycans), cartilage oligomeric matrix protein, fibronectin, collagen type II, fibromodulin, and biglycan was more prominent in the areas of chondronecrosis, extending into the subchondral bone, than in the normal resting region. This altered pattern of matrix macromolecules resembled that of the matrix of the proliferative chondrocytes and suggests that the chondrocyte maturation had stopped in the proliferative zone. The matrix in the areas of chondronecrosis in the resting region resembled that in the normal resting region. Thus the chondronecrosis appears to have preceded alterations of the matrix composition. The antibody reactivity pattern was, however, altered in the matrix of the clustered chondrocytes in areas of chondronecrosis. Staining in these regions suggested a more prominent appearance of fibronectin and collagen type II than in the normal matrix of the resting region. These changes are suggestive of attempt to repair. The chondronecrotic areas restricted to the resting region have a matrix that is different from the matrix of the abnormal cartilage extending into the subchondral bone, which resembled the matrix of the proliferative region. Hence the osteochondrotic lesion may not start in the resting region, instead the maturation of chondrocytes seems to stop in the proliferative zone, which would result in impaired bone formation.
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Scharf, Rüdiger. "Platelet Signaling in Primary Haemostasis and Arterial Thrombus Formation: Part 1." Hämostaseologie 38, no. 04 (October 23, 2018): 203–10. http://dx.doi.org/10.1055/s-0038-1675144.
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AbstractPlatelets react immediately in response to traumatic vascular injury by adhesion, activation, aggregation and subsequent haemostatic plug formation. While this reaction pattern is essential for haemostasis, platelet responses can also cause occlusive thrombi in diseased arteries, leading to myocardial infarction or stroke. Initially, flowing platelets are captured from the circulation to vascular lesions. This step is mediated by glycoprotein (GP) Ib-IX-V interacting with immobilized von Willebrand factor (VWF) on exposed subendothelial components. Tethered platelets can now bind to collagen through GPVI and integrin α2β1. Outside-in signals from the adhesion receptors act synergistically with inside-out signals from soluble stimuli and induce platelet activation. These mediators operate through G protein–coupled receptors and reinforce adhesion and activation. Typical manifestations of activated platelets include calcium mobilization, procoagulant activity, cytoskeletal reorganization, granule secretion and aggregation. This requires activation of integrin αIIbβ3 with shifting into a high-affinity state and is indispensable to bind soluble fibrinogen, VWF and fibronectin. The multiple interactions and the impact of thrombin result in firm adhesion and recruitment of circulating platelets into growing aggregates. A fibrin meshwork supports stabilization of haemostatic thrombi and prevents detachment by the flowing blood. This two-part review provides an overview of platelet activation and signal transduction mechanisms with a focus on αIIbβ3-mediated outside-in signaling in integrin variants. In the first part, a three-stage model of platelet recruitment and activation in vivo is presented. Along with that, platelet responses upon exposure to thrombogenic surfaces followed by platelet-to-platelet interactions and formation of haemostatic thrombi are discussed. Moreover, several determinants involved in pathological thrombosis will be reviewed.
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van de Wiel-van Kemenade, E., Y. van Kooyk, AJ de Boer, RJ Huijbens, P. Weder, W. van de Kasteele, CJ Melief, and CG Figdor. "Adhesion of T and B lymphocytes to extracellular matrix and endothelial cells can be regulated through the beta subunit of VLA." Journal of Cell Biology 117, no. 2 (April 15, 1992): 461–70. http://dx.doi.org/10.1083/jcb.117.2.461.
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Investigating the regulation of very late antigen (VLA)-mediated functions, we found that TS2/16, a mAb directed against the beta chain of the VLA group of integrins, can induce binding of resting peripheral blood lymphocytes, cloned T lymphocytes, and Epstein Barr virus-transformed B cells to extracellular matrix components, fibronectin, laminin, and collagen, but not to fibrinogen. The antibody stimulates VLA-4-, VLA-5-, and VLA-6-mediated binding. Furthermore, it induces VLA-4-mediated binding to vascular cell adhesion molecule-1 expressed by rTNF-alpha-stimulated endothelial cells, but it does not stimulate homotypic aggregation of cells as described for a number of anti-VLA-4 alpha antibodies (Bednarczyk, J.L., and B. W. McIntyre. 1990. J. Immunol. 144: 777-784; Campanero, M. R., R. Pulido, M. A. Ursa, M. Rodríguez-Moya, M. O. de Landázuri, and F. Sánchez-Madrid. 1990. J. Cell Biol. 110:2157-2165). Therefore, the stimulating activity of this anti-beta 1 antibody clearly contrasts with that of the anti-VLA-4 alpha antibodies, which induce homotypic cell aggregation, but not binding of cells to extracellular matrix components or endothelial cells, indicating that TS2/16 may generate different signals. The observation that also F(ab')2 or Fab fragments of this anti-beta 1 antibody stimulate binding to extracellular matrix components and endothelial cells excludes the possibility that binding requires receptor crosslinking, or is Fc receptor mediated. Induction of this adhesion is cation and energy dependent and requires an intact cytoskeleton. Although changes in the conformation of VLA integrins induced by this antibody may regulate their functional activity, the dependence on metabolic energy indicates that intracellular processes may also play a role.
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WATTAM, Beth, Dazhuang SHANG, Salman RAHMAN, Sofia EGGLEZOU, Mike SCULLY, Vijay KAKKAR, and Xinjie LU. "Arg-Tyr-Asp (RYD) and Arg-Cys-Asp (RCD) motifs in dendroaspin promote selective inhibition of β1 and β3 integrins." Biochemical Journal 356, no. 1 (May 8, 2001): 11–17. http://dx.doi.org/10.1042/bj3560011.
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Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence that is found within several glycoprotein ligands. This sequence has also been found in snake-venom anti-platelet proteins, including the disintegrins and dendroaspin, a natural variant of short-chain neurotoxins isolated from the venom of Dendroaspis jamesonii. In the present study, the motifs RYD and RCD were introduced into the dendroaspin scaffold to replace RGD. Both motifs in dendroaspin caused inhibition of ADP-induced platelet aggregation with IC50 values of 200 and 300nM respectively, similar to that of the wild-type RGD motif (170nM). In comparison with wild-type dendroaspin, both RYD- and RCD-containing dendroaspins were more selective in the inhibition of the adhesion of K562 cells to laminin rather than to fibrinogen and fibronectin, even though they were 10–30-fold less potent at inhibiting K562 cell (containing α5β1 integrin) adhesion to laminin compared with wild-type. Interestingly, the RYD motif produced a similar IC50 value to the RGD motif at inhibiting A375-SM cell (β3 integrin) adhesion to collagen, whereas the RCD motif was approx. 2–6-fold less potent compared with either RGD or RYD. These findings show that the selectivity of dendroaspin binding to β1 and β3 integrins can be modulated by the introduction of alternative cell recognition sequences.
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Callaghan, Michael U., Meera B. Chitlur, Jeanne M. Lusher, Madhvi Rajpurkar, and Patricia Fleming. "Thromboelastogram Platelet Mapping Accurately Predicts Bleeding Phenotype in Glanzmann Thrombasthenia." Blood 112, no. 11 (November 16, 2008): 4560. http://dx.doi.org/10.1182/blood.v112.11.4560.4560.
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Abstract Glanzmann thrombasthenia (GT) is caused by genetic defects in the ITGA2B and ITGB3 genes that encode the proteins of the αIIbβ3 complex on the surface of the platelet. The αIIbβ3 complex on activated platelets acts a receptor principally for fibrinogen but can also bind fibronectin, vonWillebrand factor, vitronectin and CD40 ligand under conditions of high flow. The binding of these proteins results in the formation of protein bridges and aggregation of platelets. Patients with GT have deficiency of platelet surface αIIbβ3 resulting in insufficient platelet spreading and aggregation and resultant bleeding. Conventional platelet aggregation studies demonstrate aggregation with ristocetin but absence of aggregation with other platelet agonists including thrombin, collagen, arachadonic acid and epinephrine. Platelet immunophenotyping demonstrates absent (Type I) or diminished (Type II) surface expression of CD41 (Gp IIb) and CD61 (Gp IIIa). Bleeding phenotype in individual patients can be quite variable and is not well predicted by surface expression or platelet aggregation studies. Thromboelastogram platelet mapping (TEG-PM) is a newer technology that employs antagonists of coagulation cascade meditated thrombosis in concert with platelet agonists to determine the level of platelet inhibition. Here we have examined the bleeding phenotype (Table I) of 5 GT patients, from three separate families, followed at our center in relation to conventional platelet aggregation studies, platelet immunophenotype and TEG-PM (Figure I). Three of the patients are from a single kindred and have had frequent severe bleeding episodes requiring treatment with activated FVII, transfused platelets and transfusion support for blood loss. The fourth patient has had an intermediate bleeding phenotype. The fifth patient has had only a single severe bleeding episode after nasal surgery and an otherwise mild bleeding phenotype including a caesarian section with a single platelet transfusion and no bleeding. The fifth patient displayed reduced CD41 and CD61 platelet expression (type II GT) while the other 4 patients have no CD41 and CD61 platelet expression (type I GT) by flow cytometry. While all 5 patients have similar, classic platelet aggregation studies the TEG-PM accurately predicted the bleeding phenotype. We believe TEG-PM may be a useful tool for the management of patients with GT. Table I Patient I Patient II Patient III Patient IV Patient V Age 4 years 10 years 20 years 2 years 29 years Sex Female Female Male Female Female # of PRBC Transfusion 0 2 0 3 0 Hospitilizations 2 4 7 4 1 FVIIa days 69 47 27 17 0 Platelet transfusions 9 3 13 0 1 PFA Epi >262 s - - >196 s 259 s PFA ADP 272 s - - >210 s 173 s TEG K - - - 7.8 min 2.2 min TEG MA 13.6 13.4 16.4 22.8 65.9 Platelet Count 83–439K 91–364K 76–449K TEG ADP Inh 89.3% 100% 83.2% 100% 6.8% TEG AA Inh 98.7% 93.2% 89.7% 100% 68.6% Figure I Figure I.
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Tiwari, Kalpana, Farendra Bhardwaj, Shagun Gupta, Umang Juneja, Tushar Prabha, and Harsh Patel. "CASE REPORT: VAGINAL LEIOMYOMA." International Journal Of Advanced Research 10, no. 04 (April 30, 2022): 199–203. http://dx.doi.org/10.21474/ijar01/14533.
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Leiomyoma are benign, monoclonal tumors of the smooth muscle cells of the myometrium and contain large aggregations of extracellular matrix composed of collagen, elastin, fibronectin, and proteoglycan. [1] They are the most common benign tumors in females and are most frequently seen in uterus. Vaginal leiomyomas are very rare and often confused with a variety of vaginal tumors. Since the first detected case back in 1733 by Denys de Leyden approximately 300 cases have been reported in the literature so far. [2] They may present with varied symptoms depending on site, size and vascularity including lower abdominal pain, low back pain, vaginal bleeding, dyspareunia and urinary symptoms like frequency, dysuria or other features of urinary obstruction. Here we discuss a case of a 35 year old female who presented to us with complains consistent with prolapse and was provisionally diagnosed as paraurethral vaginal mass which turned out to be vaginal fibroid.
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Snyder, Christina, Monica Rohde, and Ravi N. Singh. "Abstract 1745: Exploiting vulnerabilities to nanoparticle induced lipid peroxidation and proteotoxicity to treat drug resistant lung cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1745. http://dx.doi.org/10.1158/1538-7445.am2022-1745.
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Abstract Protein quality control plays an essential role in cell survival. Clinically translated strategies to target protein quality control in cancer show some efficacy, but off-target effects are severe. We are investigating a new approach using nanomaterials to induce lipid peroxidation to trigger a cascade of protein oxidation, protein misfolding, and decreased protein degradative capacity in cancer cells without affecting normal cells. Our studies show that silver nanoparticles (AgNPs) cause dramatic increases in lipid peroxidation, 4-hydroxynonenal adducts (4-HNE), protein oxidation, protein aggregation, and proteotoxic stress signaling in a mesenchymal-like subset of non-small cell lung (NSCLC) and other cancers at doses that do not affect normal cells in vitro or vivo. Notably, AgNP sensitive NSCLCs are inherently resistant to clinically relevant therapies including epidermal growth factor receptor tyrosine kinase inhibitors ((EGFR-TKIs) e.g. erlotinib, gefitinib, olfatinib) and cisplatin. We identified two defining characteristics causing some cancers to be sensitive to AgNPs. The first is enrichment of cell membranes in polyunsaturated fatty acids (PUFAs), especially arachidonic (AA) and adrenic acid (AdA), which are prone to oxidation. The second is high-level synthesis and secretion of extracellular matrix (ECM), especially fibronectin and collagen, which places AgNP sensitive cancers under substantial baseline endoplasmic reticulum stress, making them susceptible to agents that increase proteotoxicity. Mechanistically, we find that in PUFA-enriched cancers, signaling from ECM sustains the AgNP sensitive phenotype by stimulating Akt activity and stabilizing cytoplasmic β-catenin. Transcriptionally active β-catenin increases expression of fibronectin and collagen, resulting in a positive feedback loop locking cells into the AgNP sensitive phenotype. Use of AgNPs to targeting vulnerabilities associated with ECM synthesis and signaling downstream of β-catenin is a novel approach to overcoming multiple EGFR-TKI resistance mechanisms. Ultimately, our goal is to elucidate the precise mechanism by which AgNPs overcome resistance to clinically available EGFR-TKIs. Identification of the specific drivers of sensitivity to AgNPs (and potentially other metal nanoparticles) will guide development of therapeutic metal nanoparticles, enable selection of cancer patients who will benefit most or are at greatest risk to nanomaterial exposure, and will spur innovation of other treatments that exploit the vulnerabilities we identify. Citation Format: Christina Snyder, Monica Rohde, Ravi N. Singh. Exploiting vulnerabilities to nanoparticle induced lipid peroxidation and proteotoxicity to treat drug resistant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1745.