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1
Esan, A. M., Z. Khan, R. Kiran, T. O. Omolekan, K. A. Aremu, and H. R. Y. Adeyemi. "Ameliorative Effects of Pseudomonas fluorescence Strains on Growth and Antioxidant Potential of Okra (Abelmoschus esculentus) Plant under Nematode Infection." European Journal of Biology and Biotechnology 2, no. 3 (June 5, 2021): 50–56. http://dx.doi.org/10.24018/ejbio.2021.2.3.168.
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Meloidogyne incognita is a plant pathogen causing root-knot nematodes disease in many crops worldwide. Due to the environmental threat on the use of chemical fumigants, there is a need for a biological control method using microbial antagonists on root-knot nematodes disease. Therefore, this study was conducted to screen and evaluate the biocontrol potential of P. fluorescens strains against root-knot nematodes. The effectiveness of six P. fluorescens strains viz., Pf1, Pf2, Pf3, Pf4, Pf5and Pf6 were tested in vitro and also in pots experiment for their inhibitory activities and biocontrol potential against root-knot nematodes disease caused by Meloidogyne incognita on okra plant. Treatments of the nematode with 1.0-6.0% concentrations of 108 CFU/mL of Pf4 and Pf5 strains caused 70.0-95.0% inhibition on nematode egg-hatch and 2nd stage juveniles activity. Pf3, Pf4 and Pf5showed a decrease in the number of roots galling with increased root and shoot dry weights of stressed okra plant. Moreover, there was 25.99-36.43%, 37.76-79.145% and 42.62-62.37%, 69.83-98.09% increase in shoot length and leaf areas after 15th and 30th day respectively of P. fluorescens inoculation. The inoculated okra plants exhibited higher photosynthetic pigments, higher antioxidant enzymes activity and mineral contents than the nematode treated groups. Higher mineral contents were observed in the roots than the leaves of the okra plant subjected to the nematode infection. The bacteria strains especially Pf4 and Pf3 have considerable potential to reduce the menace of the nematodes in the treated okra plant. Therefore, the strains can be used for crop management against root-knot nematodes disease.
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González-Serrano, Diego J., Milad Hadidi, Matin Varcheh, Aniseh Zarei Jelyani, Andres Moreno, and Jose M. Lorenzo. "Bioactive Peptide Fractions from Collagen Hydrolysate of Common Carp Fish Byproduct: Antioxidant and Functional Properties." Antioxidants 11, no. 3 (March 6, 2022): 509. http://dx.doi.org/10.3390/antiox11030509.
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Collagen isolated from byproducts of common carp was hydrolyzed with alcalase enzyme to obtain peptide fractions. The resulting >30 kDa (PF1), 10–30 kDa (PF2), 3–10 kDa (PF3) and <1 kDa (PF4) fractions were studied for their antioxidant and functional properties. All peptide fractions illustrated antioxidant activity at different concentrations (1, 5, and 10 mg/mL). Although PF4 indicated the highest DPPH radical-scavenging activity (87%) at a concentration of 1 mg/mL, the highest reducing power (0.34) and hydroxyl radical scavenging activity (95.4%) were also observed in PF4 at a concentration of 10 mg/mL. The solubility of the peptide fractions was influenced by pH. The lowest solubility of the peptide fractions was observed at pH 4. The highest emulsifying activity index (EAI) was observed for PF4 (121.1 m2/g), followed by PF3 (99.6 m2/g), PF2 (89.5 m2/g) and PF1 (78.2 m2/g). In contrast to what has been found in the case of EAI, the emulsion stability of the peptide fractions decreased at lower molecular weight, which ranged from 24.4 to 31.6 min. Furthermore, it was revealed that PF1 had the highest foam capacity (87.4%) and foam stability (28.4 min), followed by PF2 and PF3. Overall, the findings suggest that peptide fractions isolated from byproducts of common carp are a promising source of natural antioxidants for application in functional food and pharmaceutical products.
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Kakkar, Preeti Handa, R. M. Saxena, and Mamta Joshi. "Histopathological analysis of liver in Puntius ticto exposed to water soluble fraction (WSF) of petrol." Journal of Applied and Natural Science 2, no. 2 (December 1, 2010): 290–92. http://dx.doi.org/10.31018/jans.v2i2.136.
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The fresh water fish Puntius ticto were exposed to lethal concentration of water soluble fraction (WSF) of petrol (5%-PF1, 10%-PF2, 15%-PF3, 20%-PF4 and 25%-PF5) for 96 hours. The exposure of WSF produced some conspicuous histopathological changes in liver. The swelling of hepatocytes, degeneration, necrosis, hemolysis, dilation, congestion and fibrosis in blood sinusoids were the prominent changes observed. The histological analysis showed increasing damages dose-dependents and time-dependents.
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FELLOWES, R. A., A. G. MAULE, N. J. MARKS, T. G. GEARY, D. P. THOMPSON, and D. W. HALTON. "Nematode neuropeptide modulation of the vagina vera of Ascaris suum: in vitro effects of PF1, PF2, PF4, AF3 and AF4." Parasitology 120, no. 1 (January 2000): 79–89. http://dx.doi.org/10.1017/s0031182099005260.
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Ascaris suum possesses a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8/PF3) have been shown to modulate the intrinsic, rhythmic activity of the vagina vera of A. suum in vitro. In the present study, the effects of the nematode FaRPs, SDPNFLRFamide (PF1), SADPNFLREamide (PF2) and KPNFIRFamide (PF4) (from Panagrellus redivivus) and AVPGVLRFamide (AF3) and GDVPGVLRFamide (AF4) (from A. suum) on the in vitro activity of the vagina vera were examined. The effects of each of the peptides were qualitatively and quantitatively distinct. All 3 FaRPs from P. redivivus were inhibitory, causing a cessation of contractions. PF2 was 3 times more potent than PF1, with a threshold of 1 nM. Although PF4 was the least potent (threshold, 10 nM), its effects at [ges ]10 nM were quantitatively the greatest. Both AF3 and AF4 (1 μM) induced complex, multiphasic responses consisting of an initial contraction and spastic paralysis followed by a return of contractile activity of increased amplitude. AF3 was 3 times more potent than AF4. The effects of these peptides had some similarities to those observed on A. suum somatic body wall muscle in vitro, with PF1, PF2 and PF4 being inhibitory and AF3 and AF4 being excitatory.
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Lecomte-Raclet, Laurence, Mònica Alemany, Anabelle Sequeira-Le Grand, Jean Amiral, Gérard Quentin, Anne Marie Vissac, Jacques P. Caen, and Zhong Chao Han. "New Insights Into the Negative Regulation of Hematopoiesis by Chemokine Platelet Factor 4 and Related Peptides." Blood 91, no. 8 (April 15, 1998): 2772–80. http://dx.doi.org/10.1182/blood.v91.8.2772.2772_2772_2780.
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Platelet factor 4 (PF4) has been recognized as an inhibitor of myeloid progenitors. However, the mechanism of action of this chemokine remains poorly understood. The present study was designed to determine its structure/function relationship. A series of peptides overlapping the C-terminal and central regions of PF4 were analyzed in vitro for their action on murine hematopoietic progenitor growth to assess the minimal sequence length required for activity. The peptides p17-58 and p34-58 possessed an increased hematopoietic inhibitory activity when compared with PF4, whereas the shorter peptides p47-58 and p47-70 were equivalent to the native molecule and the peptide p58-70 was inactive. The PF4 functional motif DLQ located in 54-56 was required for the activity of these peptides. The peptide p34-58 impaired to a similar extent the growth of colony-forming unit-megakaryocyte (CFU-MK) as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit–granulocyte-macrophage (CFU-GM), whereas PF4 was more active on CFU-MK. In the experiments using purified murine CD34+ marrow cells, statistically significant inhibition induced by p34-58 was shown at concentrations of 2.2 nmol/L or greater for progenitors of the three lineages, whereas that induced by PF4 was seen at 130 nmol/L for CFU-MK and 650 nmol/L for CFU-GM and BFU-E, indicating that the p34-58 acts directly on hematopoietic progenitors and its activity is approximately 60- to 300-fold higher than PF4. The p34-58, unlike PF4, lacked affinity for heparin and its inhibitory activity could not be abrogated by the addition of heparin. In addition, an antibody recognizing p34-58 neutralized the activity of p34-58 but not whole PF4 molecule. These results demonstrate that PF4 contains a functional domain in its central region, which is independent of the heparin binding properties, and provide evidence for a model of heparin-dependent and independent pathways of PF4 in inhibiting hematopoiesis.
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Karuppaija, Sinthuja, Kapilan Ranganathan, and Vasantharuba Seevaratnam. "Characterization of best naringinase producing fungus strain isolated from palmyrah (Borrasus flabellifer) fruit pulp." International Journal of Biological Research 4, no. 2 (July 19, 2016): 97. http://dx.doi.org/10.14419/ijbr.v4i2.6289.
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Background: The Palmyrah (Borrasus flabellifer L.) fruit pulp has the bitter compound flabelliferin (a tetraglycoside) which can be hydrolyzed by naringinase enzyme. The diverse groups of filamentous fungi and bacteria that live in different substrates have the capacity of producing extracellular naringinase enzyme which is of tremendous industrial value.Objective: The objective of the study was to isolate the naringinase producing fungal strains from Palmyrah and to identify the best naringinase producer under liquid and solid state fermentation systems.Methods: Fungal strains isolated from Palmyrah fruit pulp and the soil where pulp is allowed to decay, were grown on naringin agar selective medium at pH 6.0 at room temperature and the production of extracellular naringinase was measured in the liquid fermentation media and solid state fermentation system using paddy husk as support.Results: Five fungal strains isolated from the palmyrah pulp and the pulp decaying in sand designated as PF1,PF2,PF3,PF4 & PF5 had the ability to produce extracellular naringinase enzyme in liquid fermentation media. Fungal strain PF4 that showed highest naringinase enzyme activity (1.769U/ml) was selected among the isolated five fungal strains and identified as Rhizophus stolonifer based on the morphological and biochemical characteristics. When this strain was grown in the solid state fermentation system using paddy husk as media, narininase production was higher (269.84 U/gram of dry substrate) in seven days.Conclusion: Rhizophus stolonifer could be used to produce large scale naringinase enzyme under solid state fermentation system using very cheap, easily available, agricultural waste paddy husk as support without the need of expensive and well equipped laboratories.
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PURCELL, J., A. P. ROBERTSON, D. P. THOMPSON, and R. J. MARTIN. "The time-course of the response to the FMRFamide-related peptide PF4 in Ascaris suum muscle cells indicates direct gating of a chloride ion-channel." Parasitology 124, no. 6 (June 2002): 649–56. http://dx.doi.org/10.1017/s0031182002001695.
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We investigated the effects of PF4 on Ascaris suum somatic muscle cells using a 2 electrode current-clamp technique. PF4 is a FaRP (FMRFamide-related peptide), originally isolated from the free-living nematode Panagrellus redivivus. PF4 caused hyperpolarization and an increase in chloride ion conductance when it was applied to the muscle cells of the Ascaris body wall. The delay between the application of the peptide and the appearance of the response was measured and compared with that of gamma-amino butyric acid (GABA), a compound that directly gates ion channels, and with PF1, a FaRP that acts via an intracellular signal transduction mechanism. The PF4 and GABA delay times were not significantly different; they were 1·51±0·11 sec and 1·22±0·10 sec respectively. The delay following application of PF1, 3·75±0·51 sec, was significantly longer. The rapid response to PF4 is consistent with direct gating of a chloride ion channel, which has not been described elsewhere in the literature.
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Bokka, Chandra Sekhar, Ganesh Kumar Veeramachaneni, V. B. S. C. Thunuguntla, Janakiram Bobbillapati, and Jayakumar Singh Bondili. "Peptide Mapping, In Silico and In Vivo Analysis of Allergenic Sorghum Profilin Peptides." Medicina 55, no. 5 (May 21, 2019): 178. http://dx.doi.org/10.3390/medicina55050178.
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Background and objectives: Nearly 20–30% of the world’s population suffers from allergic rhinitis, among them 15% are progressing to asthma conditions. Sorghum bicolor profilin (Sorb PF), one of the panallergens, was identified, but the allergen specificity is not yet characterized. Materials and Methods: To map the antigenic determinants responsible for IgE binding, the present study is focused on in silico modeling, simulation of Sorb PF and docking of the Sorb PF peptides (PF1-6) against IgG and IgE, followed by in vivo evaluation of the peptides for its allergenicity in mice. Results: Peptide PF3 and PF4 displayed high docking G-scores (−9.05) against IgE only. The mice sensitized with PF3 peptide showed increased levels of IL5, IL12, TNF-alpha, and GMCSF when compared to other peptides and controls, signifying a strong, Th2-based response. Concurrently, the Th1 pathway was inhibited by low levels of cytokine IL2, IFN-γ, and IL-10 justifying the role of PF3 in allergenic IgE response. Conclusions: Based on the results of overlapping peptides PF3 and PF4, the N-terminal part of the PF3 peptide (TGQALVI) plays a crucial role in allergenic response of Sorghum profilin.
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Ismail, Muhammad Hafiz, Katharine A. Michie, Yu Fen Goh, Parisa Noorian, Staffan Kjelleberg, Iain G. Duggin, Diane McDougald, and Scott A. Rice. "The Repressor C Protein, Pf4r, Controls Superinfection of Pseudomonas aeruginosa PAO1 by the Pf4 Filamentous Phage and Regulates Host Gene Expression." Viruses 13, no. 8 (August 15, 2021): 1614. http://dx.doi.org/10.3390/v13081614.
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It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in Pseudomonas aeruginosa PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the E.coli bacteriophage P2 RepC protein. A mutation, Pf4r*, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r* protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r’s immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in P. aeruginosa biofilm development.
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Hara, Francisco Adilson dos Santos, and Luiz Antonio de Oliveira. "Características fisiológicas e ecológicas de isolados de rizóbios oriundos de solos ácidos e álicos de Presidente Figueiredo, Amazonas." Acta Amazonica 34, no. 3 (September 2004): 343–57. http://dx.doi.org/10.1590/s0044-59672004000300002.
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Alguns isolados de rizóbio, além de fixarem o N2, são capazes de solubilizar fosfatos pouco solúveis, disponibilizando o P para as plantas e para si mesmos. No entanto, o Al e a acidez dos solos da Amazônia podem diminuir a população desses microrganismos. O presente trabalho avaliou a capacidade nodulífera, a tolerância à acidez e ao Al tóxico, bem como a capacidade de solubilizar fosfatos de Ca e de Al de 88 isolados de rizóbio de solos agrícolas, do município de Presidente Figueiredo, AM. Amostras de solo sob cultivos agrícolas foram coletadas e utilizadas como fontes de inóculo para plantas de feijão caupi. As amostras de solo continham isolados de rizóbio capazes de induzir a nodulação e incrementar a biomassa aérea do feijão caupi em condição ácida (pH 4,5) e álica (2cmol c Al. L-1). Os isolados de rizóbio presentes nas amostras de solo identificadas como INPA-PF2, INPA-PF3, INPA-PF4, INPA-PF5, INPA-PF13, INPA-PF15, INPA-PF22 e INPA-PF24 promoveram rendimentos de biomassa aérea superiores à testemunha. A tolerância à acidez foi apresentada por 25% dos isolados e apenas 23% apresentaram tolerância ao Al. O fosfato de Ca foi solubilizado por 39% dos isolados. No entanto, apenas um isolado apresentou alto índice de solubilização. A capacidade de solubilização de fosfato de Al foi identificada em 67% dos isolados. A maioria dos isolados de rizóbio que solubilizou fosfato de Ca (76,5% dos isolados) também solubilizou o fosfato de Al.
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Webb, Jeremy S., Mathew Lau, and Staffan Kjelleberg. "Bacteriophage and Phenotypic Variation in Pseudomonas aeruginosa Biofilm Development." Journal of Bacteriology 186, no. 23 (December 1, 2004): 8066–73. http://dx.doi.org/10.1128/jb.186.23.8066-8073.2004.
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ABSTRACT A current question in biofilm research is whether biofilm-specific genetic processes can lead to differentiation in physiology and function among biofilm cells. In Pseudomonas aeruginosa, phenotypic variants which exhibit a small-colony phenotype on agar media and a markedly accelerated pattern of biofilm development compared to that of the parental strain are often isolated from biofilms. We grew P. aeruginosa biofilms in glass flow cell reactors and observed that the emergence of small-colony variants (SCVs) in the effluent runoff from the biofilms correlated with the emergence of plaque-forming Pf1-like filamentous phage (designated Pf4) from the biofilm. Because several recent studies have shown that bacteriophage genes are among the most highly upregulated groups of genes during biofilm development, we investigated whether Pf4 plays a role in SCV formation during P. aeruginosa biofilm development. We carried out immunoelectron microscopy using anti-Pf4 antibodies and observed that SCV cells, but not parental-type cells, exhibited high densities of Pf4 filaments on the cell surface and that these filaments were often tightly interwoven into complex latticeworks surrounding the cells. Moreover, infection of P. aeruginosa planktonic cultures with Pf4 caused the emergence of SCVs within the culture. These SCVs exhibited enhanced attachment, accelerated biofilm development, and large regions of dead and lysed cells inside microcolonies in a manner identical to that of SCVs obtained from biofilms. We concluded that Pf4 can mediate phenotypic variation in P. aeruginosa biofilms. We also performed partial sequencing and analysis of the Pf4 replicative form and identified a number of open reading frames not previously recognized in the genome of P. aeruginosa, including a putative postsegregational killing operon.
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Rucinski, Boguslaw, Stefan Niewiarowski, Marian Strzyzewski, John C. Holt, and Kevin H. Mayo. "Human Platelet Factor 4 and Its C-Terminal Peptides: Heparin Binding and Clearance from the Circulation." Thrombosis and Haemostasis 63, no. 03 (1990): 493–98. http://dx.doi.org/10.1055/s-0038-1645072.
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SummaryHuman platelet factor 4 (PF4), a high affinity heparin binding protein, is released from stimulated platelets and stored at vascular sites, predominantly in liver, from where it can be brought back into circulation by heparin. We attempted to define structural requirements for PF4 binding to heparin and for the pattern of its clearance from the circulation. Intact PF4 bound strongly to heparin agarose and was eluted at 1.4 M NaCl, while reduced PF4 and PF4 C-terminal peptides PF4 (47-70) and PF4 (58-70) bound weakly and were eluted at 0.2-0.5 M NaCl. 125I-radiolabeled intact PF4, reduced PF4 and C-terminal PF4 peptides injected into rabbits were cleared from the circulation in a biphasic pattern with components having half-life time of 1-2 min and 20-140 min. Heparin eliminated the fast component of PF4 clearance, but it did not affect clearance of reduced PF4 or C-terminal PF4 peptides. In contrast to reduced PF4 and PF4 (47-70), intact PF4 that accumulated in the liver and spleen, was displaced by heparin into circulating blood. In conclusion, specific binding sites and native conformation of the molecule are critical for high affinity PF4 binding to insolubilized heparin and for a pattern of PF4 clearance from the circulation in the presence of heparin.
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Lambert, Michele P., Lubica Rauova, Matthew Bailey, Martha C. Sola-Visner, M. Anna Kowalska, and Mortimer Poncz. "Platelet factor 4 is a negative autocrine in vivo regulator of megakaryopoiesis: clinical and therapeutic implications." Blood 110, no. 4 (August 15, 2007): 1153–60. http://dx.doi.org/10.1182/blood-2007-01-067116.
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Abstract Platelet factor 4 (PF4) is a negative regulator of megakaryopoiesis in vitro. We have now examined whether PF4 regulates megakaryopoiesis in vivo by studying PF4 knockout mice and transgenic mice that overexpress human (h) PF4. Steady-state platelet count and thrombocrit in these animals was inversely related to platelet PF4 content. Growth of megakaryocyte colonies was also inversely related to platelet PF4 content. Function-blocking anti-PF4 antibody reversed this inhibition of megakaryocyte colony growth, indicating the importance of local PF4 released from developing megakaryocytes. The effect of megakaryocyte damage and release of PF4 on 5-fluorouracil–induced marrow failure was then examined. Severity of thrombocytopenia and time to recovery of platelet counts were inversely related to initial PF4 content. Recovery was faster and more extensive, especially in PF4-overexpressing mice, after treatment with anti-PF4 blocking antibodies, suggesting a means to limit the duration of such a chemotherapy-induced thrombocytopenia, especially in individuals with high endogenous levels of PF4. We found that approximately 8% of 250 healthy adults have elevated (> 2 times average) platelet PF4 content. These individuals with high levels of platelet PF4 may be especially sensitive to developing thrombocytopenia after bone marrow injury and may benefit from approaches that block the effects of released PF4.
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Greinacher, Andreas, Birte Holtfreter, Krystin Krauel, Daniela Gätke, Claudia Weber, Till Ittermann, Sven Hammerschmidt, and Thomas Kocher. "Association of natural anti-platelet factor 4/heparin antibodies with periodontal disease." Blood 118, no. 5 (August 4, 2011): 1395–401. http://dx.doi.org/10.1182/blood-2011-03-342857.
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Abstract Platelet factor 4 (PF4) and heparin (H) form PF4/H complexes, the target of the immune reaction in heparin-induced thrombocytopenia (HIT). HIT seems to be a secondary immune response as anti-PF4/H-IgG antibodies occur as early as day 4 of heparin treatment. This study investigated whether prevalent infections such as periodontitis may induce the PF4/H immune response as: (1) natural anti-PF4/H Abs are present in the normal population; (2) PF4 bound to bacteria exposes the same antigen(s) as PF4/H complexes; and (3) sepsis induces PF4/H Abs in mice. We found PF4 bound to periodontal pathogens (Aggregatibacter actinomycetemcomitans; Porphyromonas gingivalis) enabling subsequent binding of human anti-PF4/H Abs. The association of natural PF4/H Abs and periodontitis was assessed in a case-control study, enrolling individuals with natural anti-PF4/H Abs (n = 40 matched pairs), and in the cross-sectional population-based Study of Health in Pomerania (SHIP; n = 3500). Both studies showed a robust association between periodontitis and presence of anti-PF4/H Abs independent of inflammation markers (case-control study: lowest vs highest tertile, odds ratio, 7.12 [95% confidence interval, 1.73-46.13; P = .005]; SHIP study, ptrend ≤ 0.001). Thus, preimmunization to PF4/bacteria complexes by prevalent infections, for example, periodontitis, likely explains the presence of natural anti-PF4/heparin Abs and the early occurrence of anti-PF4/H-IgG in HIT.
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Krauel, Krystin, Christian Pötschke, Claudia Weber, Wolfram Kessler, Birgitt Fürll, Till Ittermann, Stefan Maier, Sven Hammerschmidt, Barbara M. Bröker, and Andreas Greinacher. "Platelet factor 4 binds to bacteria, inducing antibodies cross-reacting with the major antigen in heparin-induced thrombocytopenia." Blood 117, no. 4 (January 27, 2011): 1370–78. http://dx.doi.org/10.1182/blood-2010-08-301424.
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AbstractA clinically important adverse drug reaction, heparin-induced thrombocytopenia (HIT), is induced by antibodies specific for complexes of the chemokine platelet factor 4 (PF4) and the polyanion heparin. Even heparin-naive patients can generate anti-PF4/heparin IgG as early as day 4 of heparin treatment, suggesting preimmunization by antigens mimicking PF4/heparin complexes. These antibodies probably result from bacterial infections, as (1) PF4 bound charge-dependently to various bacteria, (2) human heparin-induced anti-PF4/heparin antibodies cross-reacted with PF4-coated Staphylococcus aureus and Escherichia coli, and (3) mice developed anti-PF4/heparin antibodies during polymicrobial sepsis without heparin application. Thus, after binding to bacteria, the endogenous protein PF4 induces antibodies with specificity for PF4/polyanion complexes. These can target a large variety of PF4-coated bacteria and enhance bacterial phagocytosis in vitro. The same antigenic epitopes are expressed when pharmacologic heparin binds to platelets augmenting formation of PF4 complexes. Boosting of preformed B cells by PF4/heparin complexes could explain the early occurrence of IgG antibodies in HIT. We also found a continuous, rather than dichotomous, distribution of anti-PF4/heparin IgM and IgG serum concentrations in a cross-sectional population study (n = 4029), indicating frequent preimmunization to modified PF4. PF4 may have a role in bacterial defense, and HIT is probably a misdirected antibacterial host defense mechanism.
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Turrentine, Tiffany, Jennine Dawicki McKenna, Ann Rux, Daniel Rader, Anna Kowalska, and Bruce Sachais. "Elimination of platelet factor 4 (PF4) from platelets reduces atherosclerosis in C57Bl/6 and apoE-/- mice." Thrombosis and Haemostasis 98, no. 11 (2007): 1108–13. http://dx.doi.org/10.1160/th07-04-0271.
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SummaryActivated platelets, which release platelet factor 4 (PF4) are present in patients with atherosclerosis. To date, no direct invivo evidence exists for the involvement of PF4 in atherogenesis. In the current study, we tested the hypothesis that PF4 is atherogenic, and that genetic elimination of PF4 would protect mice from atherosclerosis. We have bred PF4-/- mice onto two athero-susceptible backgrounds, WT-C57Bl/6(WT) and apoE-/- to examine the importance of PF4 in atherogenesis. In order to induce atherosclerosis, WT and PF4-/- mice were fed an atherogenic diet for 30 weeks, while apoE-/- and apoE-/- PF4-/- mice were fed a high-fat Western-style diet for 10 weeks. Examination of lesions in the aortic roots of atherogenic diet fed mice demonstrated reduced atherosclerosis in PF4-/- (20% compared to WT). Examination of apoE-/- mice demonstrated similar changes, with apoE-/- PF4-/- mice demonstrating 37% of the aortic atherosclerotic burden compared to apoE-/- mice. Although we found similar levels of total and non-HDL cholesterol inWT and PF4-/- mice, HDL-cholesterol levels were increased in PF4-/- on both backgrounds. These data demonstrate, for the first time, that the platelet specific chemokine PF4 promotes atherosclerotic lesion development in vivo.
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Hong, Shu, Zhongji Gu, Ling Chen, Ping Zhu, and Hailan Lian. "Synthesis of phenol formaldehyde (PF) resin for fast manufacturing laminated veneer lumber (LVL)." Holzforschung 72, no. 9 (September 25, 2018): 745–52. http://dx.doi.org/10.1515/hf-2017-0184.
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AbstractPhenol formaldehyde (PF) resin is a well-tried adhesive for manufacturing laminated veneer lumber (LVL). PF has a high bonding strength, good cold pressing property and contributes a lot to the high production efficiency of LVL. In the present paper, PFs were synthesized at three different alkaline condition levels with a molar formaldehyde to phenol (F/P) ratio of 2.25. The bonding strength of PFs was not influenced by the alkalinity. Compared with PFs synthesized under alkalinity of 1 and 4%, PF with 8% alkalinity formed a resin with a high mole mass (MM), uniform mole mass distribution (MMD) and a high cross-linking density. With PF8%, the cold pressing property could be shortened from 30 to 12 min in the winter time. Cured PF8%had a higher cross-linking density than PF1%and PF4%. PF8%has a high potential for industrial production of LVL.
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Lambert, Michele P., Ronghua Meng, Dawn Harper, Liqing Xiao, Michael S. Marks, and Mortimer Poncz. "Megakaryocytes Exchange Significant Levels of Their Alpha-Granular PF4 with Their Environment." Blood 124, no. 21 (December 6, 2014): 1432. http://dx.doi.org/10.1182/blood.v124.21.1432.1432.
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Abstract Platelet factor 4 (PF4, CXCL4) is a major chemokine in megakaryocytes (megs). It is synthesized almost exclusively by megs during their development and may have important roles in regulating both hematopoietic stem cell and megakaryocyte proliferation. We now show that megs both release significant amounts of PF4 into their environment as well as take up PF4 into alpha granules. This PF4 is then available for release by thrombin activation. We examined PF4 recycling during megakaryopoiesis based on the observation that in vitro-cultured human meg hematopoietic precursors release significant amounts of PF4 into the media beginning after approximately 7 days of culture, when definitive megs begin to emerge. Using immunohistochemistry, we find that in vivo in murine bone marrow, human PF4 (hPF4) is released by hPF4 transgenic (hPF4+) megs during the steady-state, and this release is markedly accentuated 48 hours after sub-lethal 660 cGy whole body irradiation from an X-ray source to induce bone marrow injury. By comparison, animals without endogenous PF4 expression (Pf4-/-) showed only background staining. After irradiation, the levels of PF4 staining within the hPF4+ megs decreased with a concomitant increase in background staining suggesting that the stored PF4 was released into the bone marrow milieu. The increase in the PF4 staining in the intramedullary space was not due to released PF4 from entrapped platelets as similar changes were seen in untreated hPF4+ mice and in mice made thrombocytopenic by injection of antiCD41 antibody. We then asked whether the released PF4 could be taken back up by the megs and whether internalized PF4 could reach significant levels compared to endogenously synthesized PF4. We show that murine megs can take up significant levels of hPF4 so that peak hPF4 uptake at 24 hours (19±2 ng/10e6 cells) is equivalent to the amount of mouse (m) PF4 (30±1 ng/106 cells) natively present within the megs. Blocking antibodies to either PF4 itself or to lipoprotein receptor related protein 1 (LRP1) prevented PF4 uptake (53±17 IU/10e6 cells and 32±9 IU/10e6, respectively, vs 95±9 IU/10e6 cells, p <0.01, for either vs. no treatment), consistent with our previous report that LRP1 was necessary for PF4’s negative paracrine effect on megakaryopoiesis. The PF4 that was taken up by megs localizes at least in part to alpha granules, as evidenced by co-localization with P-selectin by immunofluorescence microscopy. Quantification showed a higher degree of colocalization between endogenous mPF4 and internalized hPF4 than between other alpha-granule markers, including vWF, P-selectin and internalized fibrinogen. Moreover like endogenous mPF4, the internalized PF4 can be re-released upon thrombin-induced meg activation. Finally, we asked whether the PF4 uptake was unusual and began by studying uptake of the related chemokine, platelet basic protein (PBP, CXCL7), another protein synthesized by megs and stored in alpha-granules. Unlike PF4, PBP was not internalized by megs as judged by immunohistochemistry or ELISA, indicating that the ability to be internalized and re-released is a relatively unique property of PF4. In summary, we demonstrate that PF4 - an important regulator of megakaryopoiesis and hematopoiesis - is released by megs in the intramedullary space at steady-state and even more so when stressed. Moreover, the released PF4 can be taken up into alpha-granules and stored for potential rerelease. Whether this complex cycle of PF4 in megs is unique to PF4 or applies to other alpha-granular proteins and whether it is necessary for the PF4 effect on hematopoiesis/ megakaryopoiesis needs further investigation Disclosures Xiao: ECRI Institute: Employment.
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Krauel, Krystin, Claudia Weber, Sven Brandt, Ulrich Zähringer, Uwe Mamat, Andreas Greinacher, and Sven Hammerschmidt. "Platelet factor 4 binding to lipid A of Gram-negative bacteria exposes PF4/heparin-like epitopes." Blood 120, no. 16 (October 18, 2012): 3345–52. http://dx.doi.org/10.1182/blood-2012-06-434985.
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AbstractThe positively charged chemokine platelet factor 4 (PF4) forms immunogenic complexes with heparin and other polyanions. Resulting antibodies can induce the adverse drug effect heparin-induced thrombocytopenia. PF4 also binds to bacteria, thereby exposing the same neoantigen(s) as with heparin. In this study, we identified the negatively charged lipopolysaccharide (LPS) as the PF4 binding structure on Gram-negative bacteria. We demonstrate by flow cytometry that mutant bacteria with progressively truncated LPS structures show increasingly enhanced PF4 binding activity. PF4 bound strongest to mutants lacking the O-antigen and core structure of LPS, but still exposing lipid A on their surfaces. Strikingly, PF4 bound more efficiently to bisphosphorylated lipid A than to monophosphorylated lipid A, suggesting that phosphate residues of lipid A mediate PF4 binding. Interactions of PF4 with Gram-negative bacteria, where only the lipid A part of LPS is exposed, induce epitopes on PF4 resembling those on PF4/heparin complexes as shown by binding of human anti-PF4/heparin antibodies. As both the lipid A on the surface of Gram-negative bacteria and the amino acids of PF4 contributing to polyanion binding are highly conserved, our results further support the hypothesis that neoepitope formation on PF4 after binding to bacteria is an ancient host defense mechanism.
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Braga, Diego De Ávila Martins, Juarez Lopes Donzele, Rita Flávia Miranda de Oliveira Donzele, Matheus Faria de Sousa, Evandro Ferreira Cardoso, Igor De Freitas Donzeles, João Paulo de Oliveira, and Jessica Mansur Siqueira Furtado. "Evaluation of the standardized ileal digestible lysine requirement of nursery pigs from 28 to 63 d of age in a three-phase feeding program." Semina: Ciências Agrárias 39, no. 2 (March 15, 2018): 719. http://dx.doi.org/10.5433/1679-0359.2018v39n2p719.
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The objective of this study was to determine the standardized ileal digestible lysine (SID Lys) requirement of nursery pigs from 28 to 63 d of age fed a multi-phase feeding (PF) program and its possible adaptation to SID Lys-deficient diets. Ninety-six commercial hybrid piglets (Topigs Norsvin, 46 castrated males and 50 females) that had been weaned at 28 d of age with an initial body weight of 8.82 ± 0.28 kg were distributed in a randomized block design composed of four treatments, with eight replicates per treatment and three animals per replicate. The treatments were as follows: PF1, SID Lys levels of 1.05, 0.95, and 0.85%; PF 2, SID Lys levels of 1.15, 1.05, and 0.95%; PF 3, SID Lys levels of 1.25, 1.15, and 1.05%; and PF 4, SID Lys levels of 1.35, 1.25, and 1.15% from 28 to 35, 36 to 49, and 50 to 63 d of age, respectively. From 28 to 63 d of age, the average daily feed intake (ADFI) and average daily gain (ADG) were not affected by the SID Lys levels tested; however, final body weight (fBW) was affected, with PF1 having the lowest fBW. The SID Lys levels tested had a significant effect on the feed conversion ratio (FCR), which varied linearly from 28 to 35 d of age. In the period from 28 to 63 d of age, pigs fed PF4 had the highest FCR results. The protein deposition ratio (PDR) was also affected by the SID Lys levels tested, with PF3 and PF4 having the highest PDR results. Therefore, the optimal SID Lys requirement for nursery pigs from 28 to 35 d of age that provided better performance results was 1.25%, corresponding to a daily Lys intake of 4.13 g/d. PF3 provided the best performance and PDR results for piglets from 28 to 63 days of age.
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Brandt, Sven, Krystin Krauel, Miriam Jaax, Thomas Renné, Christiane A. Helm, Sven Hammerschmidt, Mihaela Delcea, and Andreas Greinacher. "Polyphosphates form antigenic complexes with platelet factor 4 (PF4) and enhance PF4-binding to bacteria." Thrombosis and Haemostasis 114, no. 12 (2015): 1189–98. http://dx.doi.org/10.1160/th15-01-0062.
Full textAbstract:
SummaryShort chain polyphosphates (polyP) are pro-coagulant and pro-inflammatory platelet released inorganic polymers. The platelet chemokine platelet factor 4 (PF4) binds to lipid A on bacteria, inducing an antibody mediated host defense mechanism, which can be misdirected against PF4/heparin complexes leading to the adverse drug reaction heparin-induced thrombocytopenia (HIT). Here, we demonstrate that PF4 complex formation with soluble short chain polyP contributes to host defense mechanisms. Circular dichroism spectroscopy and isothermal titration calorimetry revealed that PF4 changed its structure upon binding to polyP in a similar way as seen in PF4/heparin complexes. Consequently, PF4/polyP complexes exposed neoepitopes to which human anti-PF4/heparin antibodies bound. PolyP enhanced binding of PF4 to Escherichia coli, hereby facilitating bacterial opsonisation and, in the presence of human anti-PF4/polyanion antibodies, phagocytosis. Our study indicates a role of polyP in enhancing PF4-mediated defense mechanisms of innate immunity.
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Fiore, Martine Marie, and Ian J. Mackie. "Dual Effect of Platelet Factor Four on the Activities of Factor Xa." Blood 112, no. 11 (November 16, 2008): 1034. http://dx.doi.org/10.1182/blood.v112.11.1034.1034.
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Abstract Platelet Factor 4 (PF4) is a cationic molecule that binds to heparin with high affinity and neutralises the activity of the latter. Our recent studies indicate that heparin can promote an interaction of fXa with PF4 since neutralization of heparin activity by PF4 was dependent on the concentration of protease. To examine the contribution of PF4 in protease function, fXa activity was determined in chromogenic assays. Upon preincubation with fXa and heparin, PF4 (at a concentration of 100 nM) decreased the kcat of S2765 peptide hydrolysis 4-fold and that of prothrombin activation about 2-fold. These results suggested an effect of PF4 on the primary specificity of the protease. In fact, PF4 exerted a mild effect (30 % decrease) on the Na+ dependence of fXa, consistent with linkage between Na+ and S1. PF4 preincubation with fXa also prevented the binding of the S1 probe p-aminobenzamidine (pAB) while simultaneous addition of PF4 and pAB diminished the contribution of PF4. In the presence of excess fVa (relative to fXa), kinetic parameters measuring fXa amidolytic activity in the presence of PF4 were restored to control values in the absence of PF4. Interestingly, high concentrations of PF4 (> 1 μM) totally restored fXa activity toward peptidyl substrate and strongly enhanced prothrombin activation, indicating a dual effect of PF4 on fXa activities. The inhibitory contribution of PF4 during prothrombin activation was due to a three-fold decreased affinity of fXa for fVa while enhancement of prothrombin activation was accompanied by a three-fold increase in fVa-dependent cofactor activity. Thus, the effects of PF4 possibly involved a region of the heparin/fVabinding exosite that is linked to the S1 and Na+ sites. These findings suggest that PF4 is a probe of fVa-dependent changes occurring in the active site of fXa and provide an explanation for the in vivo paradoxical effects of PF4 reported in the literature.
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Kowalska, M. Anna, Karine Amirikian, Laurent O. Mosnier, Hartmut Weiler, Sriram Krishnaswamy, and Mortimer Poncz. "Structural and Functional Studies to Define the Molecular Basis by Which Platelet Factor 4 (PF4) Increases Survival of Mice in Lipopolysaccharide (LPS)-Induced Endotoxicity." Blood 112, no. 11 (November 16, 2008): 19. http://dx.doi.org/10.1182/blood.v112.11.19.19.
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Abstract Our previous studies have demonstrated that platelet-released PF4 (chemokine CXCL4) promotes survival in a murine LPS-induced endotoxicity model, although the molecular basis for PF4’s protective effects was not fully defined. We hypothesized that enhanced generation of cytoprotective activated protein C (APC) by PF4 might contribute to the molecular mechanism of PF4’s beneficial effects in vivo, based on the observation that PF4 stimulates protein C (PC) activation by the thrombin/thrombomodulin complex both in vitro and in vivo. Here we show that PF4 in vitro affects human (h) PC activation in the presence of human thrombomodulin (hTM) in a bell-shaped concentration curve, i.e. stimulation at low, but inhibition at high PF4 concentrations with a peak around 3 μM. This curve is similar to that seen with PF4 for surface-bound heparin-induced thrombocytopenia (HIT) antigenicity, suggesting that similar complexes of PF4 with glycosaminoglycans (GAGs) that occur in HIT (termed ultralarge complexes (ULC)) are relevant to PF4’s interaction with the hPC/hTM. Addition of heparin blocks PF4 increase of APC generation in a similar fashion as it does for surface-bound ULC. A PF4 variant PF4K50E that poorly forms PF4 tetramers requires 8-fold higher concentrations to enhance APC generation, supporting that PF4 tetramers are central for APC generation as they are for formation of ULC. Neither PF4 nor PF4K50E accelerated in vitro generation of APC in the presence of hTM that was depleted of its chondroitin sulfate chain, suggesting that PF4 binds to this domain on hTM. In vivo studies involving simultaneous infusions of PF4 and thrombin into PF4 knock out (mPF4−/−) mice showed that PF4 leads to enhanced mouse (m) APC generation not seen with infused PF4K50E, consistent with our in vitro studies. We then asked if surface heparan sulfate on the endothelial lining was necessary for the observed PF4 effect on in vivo mAPC formation. We studied mice with a Tie2-Cre conditional knock out of N-deacetylase-N-sulfotransferase-1 activity (NDST-1−/−) that have only 15% of normal endothelial cell surface heparan sulfate content using a similar thrombin/PF4 infusion model. We found that mAPC generation was accelerated by PF4 to the same extent both in NDST1−/−/mPF4−/− and mPF4−/− mice, suggesting that surface GAGs are not involved in the PF4 effect. We have also tested the in vivo effect of PF4 on mAPC formation in TM mutant (TMpro/pro) mice that have impaired capacity for APC formation to further demonstrate that PF4’s positive effect in LPS endotoxic shock survival involves enhanced mAPC generation. Upon injection of high doses of thrombin (40 U/kg), mAPC levels are increased to the same extent in WT and TMpro/pro mice. After injection of low amounts of thrombin (8 U/kg), generation of mAPC was impaired in TMpro/pro as compared to WT mice. Concurrent infusion of PF4 increased mAPC formation in TMpro/pro mice after injection of low doses of thrombin, approximately equal to that seen in WT mice with no PF4 injected. As previously described, TMpro/pro mice had increased mortality after injection of LPS as compared to WT mice; however, with concurrent platelet PF4 overexpression, mortality decreased to that seen in WT mice, suggesting that the biological value of PF4 in LPS endotoxicity is related to its effect on the generation of APC. Thus, these studies support enhanced APC generation as the basis for the positive effect of PF4 on LPS endotoxicity and further define the molecular basis for increased APC generation by PF4 by forming ULC with the chondroitin sulfate domain of TM, but not with heparan sulfate on the vascular surface.
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Mosnier, Laurent O., Rachel L. Ochoa, and John H. Griffin. "Platelet Factor 4 (PF4) Modulation of Endothelial Protein C Receptor and Thrombomodulin Enhancements of Protein C Activation and TAFI Activation by Thrombin." Blood 108, no. 11 (November 16, 2006): 1791. http://dx.doi.org/10.1182/blood.v108.11.1791.1791.
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Abstract Protein C (PC) activation by thrombin on endothelial cells normally involves thrombomodulin (TM) and the endothelial cell protein C receptor (EPCR), and dysfunction of this mechanism is implicated in a variety of pathologies. Platelet factor 4 (PF4), a releasable platelet alpha-granule protein, stimulates PC activation in vitro and in vivo; PF4 stimulates PC activation in the presence but not in the absence of TM and this requires the PC Gla-domain (Slungaard et al). In contrast to PC activation, we found that thrombin/TM-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is inhibited by PF4. In assays where TAFI-dependent inhibition of clot lysis was promoted by thrombin/TM-mediated TAFI activation, PF4 dose-dependently inhibited TAFI’s anti-fibrinolytic effects. In the absence of TM, PF4 did not affect TAFI activation by thrombin alone and did not alter the clot-lysis times in normal plasma. Thus, the effects of PF4 on thrombin/TM actions are enhancing for PC activation and inhibitory for TAFI activation such that as PF4 concentration increases, the ratio for relative rates of PC and TAFI activation changes more than 40-fold to favor selective PC activation. The current model to explain PF4 stimulation of PC activation involves electrostatic bridging of the negatively charged Gla-domain of PC and the negatively charged glycosaminoglycan moiety of TM by the highly positively charged PF4. In assays using purified soluble reactants components, EPCR abrogated PF4 stimulation of PC activation by thrombin/TM. On 293 cells expressing TM but not EPCR, PF4 enhanced PC activation by thrombin, and this effect was inhibited by soluble EPCR (IC50 ~ 1.0 μM). Based on these and previous observations, we hypothesized that the binding sites for EPCR and PF4 on the Gla-domain of PC may overlap and, if so, that PF4 could interfere with EPCR-dependent APC cytoprotective activities involving protease activated recpotor-1 activation. Indeed, studies showed that PC and APC binding to 293 cells expressing EPCR was inhibited by PF4 (IC50 ~ 1.0 μM) and that PF4 concentrations which inhibited APC binding to EPCR significantly impaired EPCR-dependent anti-apoptotic activity of APC in assays of staurosporine-induced endothelial cell apoptosis. PF4 enhanced EPCR-dependent PC activation on endothelial cells at PF4 concentrations which did not significant inhibit PC binding to EPCR. On K293 cells transfected with both TM and EPCR, PF4 at low concentrations enhanced EPCR-dependent PC activation. Anti-EPCR antibodies abrogated both EPCR-dependent and PF4-dependent stimulation of PC activation on cells containing TM and EPCR, further suggesting a direct interaction between PF4 and EPCR. Proof for this interaction came from experiments showing that PF4 bound to immobilized soluble EPCR. Thus, these results suggest that on endothelial cells, PF4 enhances APC generation by enhancement of the ability of both TM and EPCR to promote PC activation while inhibiting TAFI activation. Therefore, the current model for stimulation of PC activation by PF4 involving thrombin/TM-PF4-PC quaternary ternary complex interactions does not adequately predict the effects of PF4 on PC activation on cells in the presence of EPCR. We propose a new model to explain the effects of PF4 on the generation of APC on endothelial cells in which the tetrameric character of PF4 promotes multiple simultaneous interactions of PF4 in a macromolecular complex with thrombin, TM, PC and EPCR.
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Warkentin, Theodore E., Richard J. Cook, Victor J. Marder, Jo-Ann I. Sheppard, Jane C. Moore, Bengt I. Eriksson, Andreas Greinacher, and John G. Kelton. "Anti–platelet factor 4/heparin antibodies in orthopedic surgery patients receiving antithrombotic prophylaxis with fondaparinux or enoxaparin." Blood 106, no. 12 (December 1, 2005): 3791–96. http://dx.doi.org/10.1182/blood-2005-05-1938.
Full textAbstract:
Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating IgG antibodies that recognize platelet factor 4 (PF4) bound to heparin. Immunogenicity of heparins differs in that unfractionated heparin (UFH) induces more anti–PF4/heparin antibodies than low-molecular-weight heparin (LMWH) and UFH also causes more HIT. Fondaparinux, a synthetic anticoagulant modeled after the antithrombin-binding pentasaccharide, is believed to be nonimmunogenic. We tested 2726 patients for anti–PF4/heparin antibodies after they were randomized to receive antithrombotic prophylaxis with fondaparinux or LMWH (enoxaparin) following hip or knee surgery. We also evaluated in vitro cross-reactivity of the IgG antibodies generated against PF4 in the presence of UFH, LMWH, danaparoid, or fondaparinux. We found that anti–PF4/heparin antibodies were generated at similar frequencies in patients treated with fondaparinux or enoxaparin. Although antibodies reacted equally well in vitro against PF4/UFH and PF4/LMWH, and sometimes weakly against PF4/danaparoid, none reacted against PF4/fondaparinux, including even those sera obtained from patients who formed antibodies during fondaparinux treatment. At high concentrations, however, fondaparinux inhibited binding of HIT antibodies to PF4/polysaccharide, indicating that PF4/fondaparinux interactions occur. No patient developed HIT. We conclude that despite similar immunogenicity of fondaparinux and LMWH, PF4/fondaparinux, but not PF4/LMWH, is recognized poorly by the antibodies generated, suggesting that the risk of HIT with fondaparinux likely is very low.
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Krauel, Krystin, Christine Hackbarth, Birgitt Fürll, and Andreas Greinacher. "Heparin-induced thrombocytopenia: in vitro studies on the interaction of dabigatran, rivaroxaban, and low-sulfated heparin, with platelet factor 4 and anti-PF4/heparin antibodies." Blood 119, no. 5 (February 2, 2012): 1248–55. http://dx.doi.org/10.1182/blood-2011-05-353391.
Full textAbstract:
Abstract Heparin is a widely used anticoagulant. Because of its negative charge, it forms complexes with positively charged platelet factor 4 (PF4). This can induce anti-PF4/heparin IgG Abs. Resulting immune complexes activate platelets, leading to the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). HIT requires treatment with alternative anticoagulants. Approved for HIT are 2 direct thrombin inhibitors (DTI; lepirudin, argatroban) and danaparoid. They are niche products with limitations. We assessed the effects of the DTI dabigatran, the direct factor Xa-inhibitor rivaroxaban, and of 2-O, 3-O desulfated heparin (ODSH; a partially desulfated heparin with minimal anticoagulant effects) on PF4/heparin complexes and the interaction of anti-PF4/heparin Abs with platelets. Neither dabigatran nor rivaroxaban had any effect on the interaction of PF4 or anti-PF4/heparin Abs with platelets. In contrast, ODSH inhibited PF4 binding to gel-filtered platelets, displaced PF4 from a PF4-transfected cell line, displaced PF4/heparin complexes from platelet surfaces, and inhibited anti-PF4/heparin Ab binding to PF4/heparin complexes and subsequent platelet activation. Dabigatran and rivaroxaban seem to be options for alternative anticoagulation in patients with a history of HIT. ODSH prevents formation of immunogenic PF4/heparin complexes, and, when given together with heparin, may have the potential to reduce the risk for HIT during treatment with heparin.
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K. Ahmed, Dua'a, and Balkis A. Kamal. "Formulation and Optimization of Oral Fast Dissolving Prochloperazine Maleate Tablets." Iraqi Journal of Pharmaceutical Sciences ( P-ISSN 1683 - 3597 E-ISSN 2521 - 3512) 21, no. 1 (March 28, 2017): 46–55. http://dx.doi.org/10.31351/vol21iss1pp46-55.
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Prochloperazine maleate (PCM) is one of the most prescribed phenothiazine. The purpose of the present research was to develop fast dissolving tablets of PCM with β-cyclodextrin inclusion complex. Tablets prepared by wet granulation with sublimation and by using different superdisintegrants type [ low-hydroxypropylcellulose LH21 (L-HPC LH21), carboxymethylcellulose calcium (ECG505), crospovidone (CP)], and different type of subliming agents (urea and ammonium bicarbonate (AB)). Tablets evaluated for its % friability, disintegration time, wetting time, hardness, content uniformity, weight variation, in vitro dissolution studies. For further enhancement of disintegration and dissolution, PCM orodispersible tablet were formulated as (PF2,PF3 and PF4) using inclusion complex of drug in β-cyclodextrin at different ratios (1:1, 1:2 and 1:3) respectively. These formulation showed disintegration times between (22s and 14.6s), and drug release showed t80% between (2.9% and 0.9%). Among all the formulations PF4 was considered as the best, containing PCM: β-cyclodextrin 1:3, 10% crospovidone and 25% ammonium bicarbonate which shows the shortest DT, good dissolution study and stability. The overall results suggest that the orodispersible tablet of PCM enhance the absorbable part than that of corresponding conventional tablet by using superdisintegrants and β-cyclodextrin inclusion complex of drug.
Key words: Prochloperazine maleate, Orodispersible tablet, Crospovidone, Ammonium Bicarbonate, Sublimation.
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Singh, Bandana, Adam Kanack, Antonios Bayas, Gemlyn George, Mouhamed Yazan Abou Abou-Ismail, Mindy Kohlhagen, Monika Christ, et al. "Monoclonal and Oligoclonal Anti-PF4 Antibodies Mediate VITT." Blood 138, Supplement 1 (November 5, 2021): 3220. http://dx.doi.org/10.1182/blood-2021-153307.
Full textAbstract:
Abstract Background: ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen Johnson & Johnson) vaccines against COVID-19 have been associated with thrombotic thrombocytopenic reactions referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT) characterized by the presence of platelet-activating, anti-PF4 antibodies. While VITT shares key clinical features with a similar but separate entity, Heparin-induced thrombocytopenia (HIT), there appear to be important differences: 1) VITT patients have extremely high thrombosis rates and are very strongly positive in PF4-polyanion ELISAs, and 2) Many patients with VITT frequently present with refractoriness to therapy or have disease recurrence that suggests distinct antibody characteristics due to a strong autoimmune anti-PF4 response. Aims: The goal of this study was to characterize anti-PF4 antibodies in VITT. Methods: Five VITT patients were studied, one after ChAdOx1 nCoV-19 vaccination and four after Ad26.COV2. Reactivity of VITT anti-PF4 antibodies to uncomplexed PF4, PF4-Polyvinyl sulfonate (PVS), and PF4-heparin targets was evaluated, and the platelet-activating ability of these antibodies was examined in the PF4-dependent P-selectin Expression assay (PEA). Anti-PF4 antibodies were isolated from patient blood samples using PF4-treated heparin sepharose beads, and isolated antibodies were subject to mass spectrometric evaluation (Liquid Chromatography Electrospray Ionization Quadrupole time-of-flight mass spectrometry [LC-ESI-QTOF MS]). Results: Antibodies from all VITT patients recognized both uncomplexed and complexed PF4 (Fig. 1A). Interestingly, recognition of PF4 by VITT antibodies was lower if PF4 targets were complexed with polyanions, PVS, or heparin (Fig. 1A). These results contrasted with those obtained in a "classical" HIT patient which showed reactivity to PF4/polyanion complexes, but not to uncomplexed PF4 (Fig 1A). All samples activated platelets in the PEA (data not shown). Mass spectrometric evaluation of anti-PF4 antibodies isolated from VITT patients demonstrated monoclonal anti-PF4 antibodies in three patients, and bi- and tri-clonal antibodies in one patient each (a representative monoclonal antibody anti-PF4 antibody is shown in Fig 1B). Consistent with current dogma, polyclonal anti-PF4/polyanion antibodies were seen in "classical" HIT (Fig 1C). Evaluation of anti-PF4 antibodies in spontaneous HIT, a type of autoimmune HIT seen in pro-inflammatory milieus such as orthopedic surgery and infectious prodromes also demonstrated monoclonal anti-PF4 antibodies (Fig 1D). Eluates from control heparin-sepharose beads did not reveal any immunoglobulins (data not shown). Conclusion: Although development of platelet-activating anti-PF4 antibodies and the thrombotic thrombocytopenia syndrome seen after ChAdOx1 nCoV-19 and Ad26.COV2.S vaccination resembles HIT, these findings demonstrate that clonally restricted anti-PF4 antibodies mediate VITT while polyclonal anti-PF4 antibodies mediate HIT. In addition, we noted clonally-restricted anti-PF4 antibodies in another condition that does not require proximate heparin exposure, spontaneous ("autoimmune") HIT. In VITT, the strong immune response after vaccine administration may result in the activation of a single or few pre-existing anti-PF4 reactive clones, and development of clonally restricted anti-PF4 antibodies with a similar pathophysiology to Spontaneous HIT. It is also likely that high levels of monoclonal/oligoclonal anti-PF4 antibodies cause the severe thrombotic phenotypes seen in VITT and Spontaneous HIT. The high mortality rate and reports of disease refractoriness to therapy in VITT may warrant consideration of additional therapeutic modalities like rituximab and therapeutic plasma exchange in select cases. Figure Legends: (A): VITT (Patient 1-ChAdOx1 nCoV-19; Patients 2-5, Ad26.COV2.S) patient samples were tested in ELISA against uncomplexed PF4 (white), and PF4 in complex with polyvinyl sulfonate (light grey), or unfractionated heparin (dark gray). (B-D) Mass spectrometric evaluation of anti-PF4 antibodies isolated from VITT (B), HIT (C) and spontaneous HIT patient sera (D). "Relative Intensity" refers to abundance of the Ig light chain relative to the polyclonal background. Numbers above Ig light chain peaks depict mass/charge ratios. NC- Normal control. Figure 1 Figure 1. Disclosures Murray: Mayo Clinic: Other: Has received patents for the Mass-Fix technology which has been licensed to the Binding Site with potential royalties.. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.
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Meyer, Sara C., Eva Steinmann, Thomas Lehmann, Patricia Muesser, Jakob R. Passweg, Radek C. Skoda, and Dimitrios A. Tsakiris. "Anti-Platelet Factor 4/Heparin Antibody Formation Occurs Endogenously and at Unexpected High Frequency in Polycythemia Vera." BioMed Research International 2017 (2017): 1–13. http://dx.doi.org/10.1155/2017/9876819.
Full textAbstract:
Background.Myeloproliferative neoplasms (MPN) encounter thromboses due to multiple known risk factors. Heparin-induced thrombocytopenia (HIT) is a thrombotic syndrome mediated by anti-platelet factor 4 (PF4)/heparin antibodies with undetermined significance for thrombosis in MPN. We hypothesized that anti-PF4/heparin Ab might occur in MPN and promote thrombosis.Methods.Anti-PF4/heparin antibodies were analyzed in 127 MPN patients including 76 PV and 51 ET. Screening, validation testing, and isotype testing of anti-PF4/heparin Ab were correlated with disease characteristics.Results.Anti-PF4/heparin antibodies were detected in 21% of PV and 12% of ET versus 0.3–3% in heparin-exposed patients. Validation testing confirmed anti-PF4/heparin immunoglobulins in 15% of PV and 10% of ET. Isotype testing detected 9.2% IgG and 5.3% IgM in PV and exclusively IgM in ET. IgG-positive PV patients encountered thromboses in 57.1% suggesting anti-PF4/heparin IgG may contribute to higher risk for thrombosis in MPN. Overall, 45% of PV patients experienced thromboses with 11.8% positive for anti-PF4/heparin IgG versus 7.1% in PV without thrombosis.Conclusion.Anti-PF4/heparin antibodies occur endogenously and more frequently in MPN than upon heparin exposure. Thrombotic risk increases in anti-PF4/heparin IgG-positive PV reflecting potential implications and calling for larger, confirmatory cohorts. Anti-PF4/heparin IgG should be assessed upon thrombosis in PV to facilitate avoidance of heparin in anti-PF4/heparin IgG-positive PV.
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Pongas, Georgios, Swapan Dasgupta, and Perumal Thiagarajan. "Anti-Platelet Factor 4/Heparin Antibodies in Patients with Gram Negative Bacteremia." Blood 120, no. 21 (November 16, 2012): 3391. http://dx.doi.org/10.1182/blood.v120.21.3391.3391.
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Abstract Abstract 3391 Introduction The anti-platelet factor 4(PF4)/heparin antibodies, arising as a result of previous heparin exposure, are causally related to the procoagulant state due to platelet and monocyte activation. Formation of these antibodies with subsequent thrombocytopenia or thrombosis has also been described in patients, who have not been previously exposed to heparin. The presence of anti-PF4/heparin antibodies in individuals correlates with the severity of periodontal disease, implying that their occurrence may be triggered by periodontal pathogens. In this study, we determined the presence of anti-PF4/heparin antibodies in gram-negative bacteremic patients in a hospital setting and propose a pathophysiologic mechanism of their presence. Method We developed an in house ELISA for quantifying anti-PF4/heparin antibodies using therapeutic heparin and PF4 isolated from platelets. We used serum from a patient with high optical density as a standard and assigned an unit of 100 arbitrarily to construct a standard curve. We tested the sera from gram negative bacteremic patients (n= 34) in the quantitative ELISA along with normal controls (n=10). We also developed an in house ELISA for studying cross reactivity between anti-PF4/heparin antibodies and lipopolysaccharide (LPS)/PF4. We tested the sera from patients (n=5) with heparin induced thrombocytopenia in this cross reactivity ELISA. To test the interaction of LPS with PF4, we labeled PF4 with Alexa488 and measured its binding to LPS by monitoring the changes in fluorescence emission spectrum following excitation at λ480. Results Patients with bacteremia had higher titers of antiPF4/heparin antibodies compared to normal controls (26.4 ± SD 33 units, N=34 versus 6.3 ± SD 2.38 units, N=10, P=0.032). Bacterial LPS interacted with alexa488-labeled PF4 in a concentration-dependent manner, as measured by the quenching of the excitation spectrum. Patients with ant-PF4/heparin antibodies also reacted with LPS/PF4 complex in ELISA. Prior absorption of serum with PF4/heparin complex coated on ELISA plates decreased the reactivity of the serum towards PF4/LPS complex (19–46%) in two out of the five patients tested suggesting some were cross-reaction between PF4/Heparin and PF4/LPS complex. Conclusions PF4 forms a complex with lipopolysaccharide and this complex is immunogenic. Antibodies to PF4/LPS complex can cross-react with PF4/heparin complex raising the possibility that these antibodies may be responsible for the detection of PF4/heparin in individuals never been exposed to heparin previously. These antibodies may also be at least partly responsible for increased thrombosis associated with infection. Disclosures: No relevant conflicts of interest to declare.
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von Hundelshausen, Philipp, Rory R. Koenen, Markus Sack, Sebastian F. Mause, Wencke Adriaens, Amanda E. I. Proudfoot, Tilman M. Hackeng, and Christian Weber. "Heterophilic interactions of platelet factor 4 and RANTES promote monocyte arrest on endothelium." Blood 105, no. 3 (February 1, 2005): 924–30. http://dx.doi.org/10.1182/blood-2004-06-2475.
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AbstractThe chemokines platelet factor 4 (PF4) and RANTES (regulated on activation normal T cell expressed and secreted) are secreted by activated platelets and influence multiple cell types and biologic processes. For instance, PF4 inhibits progenitor cell proliferation and angiogenesis, while platelet-derived RANTES is involved in vascular recruitment of monocytes. However, little is known about functional interactions of PF4 and RANTES. Here we show that the presence of PF4 enhanced the arrest of RANTES-stimulated monocytes and monocytic cells on activated endothelial cells under flow conditions, while binding of PF4 to the monocyte surface was increased by RANTES. Both RANTES-triggered arrest and PF4 binding involved monocytic chondroitin sulfate. Ligand blots and surface plasmon resonance revealed a robust heterophilic interaction of PF4 with RANTES but not with RANTES variants defective in higher order oligomerization. The tetrameric mutant E26A bound to the monocyte surface without increasing PF4 binding, and monocyte arrest induced by E26A-RANTES was not enhanced by PF4. Stimulation of monocytes with supernatants of activated platelets triggered arrest involving RANTES and PF4, as shown by inhibition studies. Our results suggest that heterophilic interactions with PF4 require structural motifs important in RANTES oligomerization and amplify RANTES-triggered effects on monocyte adhesion. This may have implications for the modulation of inflammatory recruitment by platelet-derived chemokines.
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Liu, Chao Yan, Rhonda A. Geoffrey, Qi-Hong Sun, and Gian Paolo Visentin. "Identification of Critical Amino Acids in Human Platelet Factor 4 (PF4) Required for Its Modulatory Effects on CD4+CD25+ Regulatory T Cells." Blood 104, no. 11 (November 16, 2004): 1339. http://dx.doi.org/10.1182/blood.v104.11.1339.1339.
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Abstract Human platelet factor 4 (PF4; CXCL4), a heparin-binding CXC chemokine contained in platelet α-granules, is secreted upon activation of platelets. Several reports have identified PF4 as an inhibitor of hematopoietic progenitor and endothelial cell proliferation and angiogenesis. Furthermore, PF4 has been shown to strongly inhibit T cell proliferation as well as IFN-γ and IL-2 release by activated T cells. We have recently reported that human PF4 inhibits the proliferative response of human CD4+CD25− T cells, while inducing expansion of CD4+CD25+ T regulatory (Tr) cells stimulated by anti-CD3 or anti-CD3 and anti CD-28 monoclonal antibodies (mAbs), and that PF4-induced CD4+CD25+ Tr cells lose their potent suppressor function in vitro. The antithetic effects of PF4 on CD4+CD25− T cells and CD4+CD25+ Tr cells in response to anti-CD3 and anti-CD3/28 mAbs appear to be specific since these effects were not mimicked by equivalent quantities of protamine, a positively charged heparin-binding protein used as a control for any putative interaction mediated only by positive charges, or heparin alone. We hypothesize that PF4 acts not only as the target for the antibody, in patients experiencing heparin-induced thrombocytopenia (HIT), but also as a modulator of T regulatory lymphocytes. How PF4 modulates T cell proliferation and CD4+CD25+ Tr cell-mediated suppression of CD4+CD25− T cells remains to be determined. To delineate which domain(s) of PF4 is(are) critical for its antithetical activity on the two T cell subsets, we compared PF4 from rat and cattle, which are about 74% homologous to human PF4, for their ability to affect proliferation of CD4+CD25+ and CD4+CD25− T cells stimulated by anti-CD3 mAb. Both rat and bovine PF4 were recognized by a rabbit polyclonal Ab against human PF4, but neither one, in contrast with human PF4, was capable to induce proliferation of CD4+CD25+ Tr cells. This suggests that the relatively few amino acid (AA) residues at which rat and human differ are critical for the modulatory activity of PF4 on T cells. Therefore, we introduced specific modifications into human PF4 cDNA using site-directed mutagenesis to determine the importance of individual AA residues for PF4 effects on T cell proliferation. Our strategy involved converting selected amino acids in human PF4 (reactive) to the corresponding residues in rat PF4 (non-reactive) and determining the effect of each change on the proliferation of CD4+CD25+ and CD4+CD25− T cells stimulated by anti-CD3 mAb. We created seven mutant forms of PF4. Four of the mutants involved one or more of six residues in the 47 C-terminal AAs, known to comprise the most important heparin binding region. The other three mutants were created at the N-terminus. All the PF4 constructs, expressed in E. coli, assembled properly into tetramers as determined by their HPLC profile and electrophoretic mobility in SDS-PAGE and were comparable to wild-type PF4 in their avidity for heparin. PF4 constructs bearing mutations at the N-terminus (E4S, L11V, and T16S) and three C-terminal mutants (the combined P37A/T38V/A39P, R49S, and L55R) were comparable to wild type recombinant human PF4 for their proliferative effects on the two T cell subpopulations. In contrast, another C-terminal mutant (A57V) completely abrogated PF4 proliferative effects on CD4+CD25+ Tr cells, suggesting the importance of this determinant in the interaction with a specific receptor on T cells.
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Kowalska, M. Anna, Lubica Rauova, Vincent Hayes, Douglas B. Cines, Daniel W. Bougie, Richard H. Aster, Sriram Krishnaswamy, and Mortimer Poncz. "Heparin-Induced Thrombocytopenia Antibodies Inhibit PF4-Dependent Enhancement of Activated Protein C Formation by Binding to Antigenic Complexes Formed with the Chondroitin Sulfate Side-Chain of Thrombomodulin." Blood 116, no. 21 (November 19, 2010): 721. http://dx.doi.org/10.1182/blood.v116.21.721.721.
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Abstract Abstract 721 Previous studies have shown that platelet factor 4 (PF4) increases activated protein C (aPC) generation both in vitro and in vivo. PF4 increase of aPC generation by thrombin (IIa) and thrombomodulin (TM) complex followed a bell-shaped curve when tested in solution, on human TM expressing HEK293 and on endothelial cells. PF4 failed to enhance aPC in the presence of chondroitin sulfate (CS)-free TM. These results were consistent with PF4 binding to the CS on the TM glycosaminoglycan (GAG) domain and forming complexes that are similar to PF4/GAG antigenic complexes seen in heparin-induced thrombocytopenia (HIT). We tested the hypothesis that PF4 forms a HIT-like antigenic complex with the TM-CS using the HIT-like monoclonal antibody KKO. KKO abolished the potentiating effects of PF4 on aPC formation measured with TM in solution or with a TM-expressing cell line. To further address the nature of complexes formed between PF4 and TM, we used a mutant of PF4, PF4T38Q, which forms complexes with GAGs that are not recognized by KKO and a subgroup of HIT antibodies. Similar to PF4, PF4T38Q potentiated TM-dependent aPC generation in a bell-shaped manner, but this potentiation was not blocked by KKO. Moreover, KKO did not have any effect when PF4 was replaced with protamine sulfate (PS), which can also form macromolecular complexes with heparin/GAGs and can also enhance aPC generation. We also tested HIT antibodies isolated from patients that developed HIT with thrombocytopenia and thromboembolism developing >4 days after the last exposure to heparin. Patient IgGs specific for PF4/GAG complex were purified using PF4 bound to heparin columns. Specific binding of antibodies to PF4/heparin complexes was checked by ELISA. Complex-specific antibodies were then tested in an aPC generation assay in the presence IIa and TM and near peak concentration of PF4 and compared to a control human IgG. Three of four patient‘s antibodies significantly inhibited the increase in aPC generation in the presence of PF4. These studies provide evidence that HIT-like PF4/GAG complexes develop naturally in vivo. In this case, the ability of HIT or HIT-like antibodies to specifically inhibit the PF4-dependent increase in aPC formation may contribute to the prothrombotic state in HIT. Disclosures: No relevant conflicts of interest to declare.
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Krauel, Krystin, Patricia Preuße, Theodore E. Warkentin, Catja Trabhardt, Sven Brandt, Inga Jensch, Martin Mandelkow, Elke Hammer, Sven Hammerschmidt, and Andreas Greinacher. "Fibronectin modulates formation of PF4/heparin complexes and is a potential factor for reducing risk of developing HIT." Blood 133, no. 9 (February 28, 2019): 978–89. http://dx.doi.org/10.1182/blood-2018-05-850370.
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Abstract Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating anti–platelet factor 4 (PF4)/heparin antibodies. Platelet activation assays that use “washed” platelets are more sensitive for detecting HIT antibodies than platelet-rich plasma (PRP)–based assays. Moreover, heparin-exposed patients vary considerably with respect to the risk of PF4/heparin immunization and, among antibody-positive patients, the risk of subsequent “breakthrough” of clinical HIT with manifestation of thrombocytopenia. We used washed platelets and PRP, standard laboratory HIT tests, and physicochemical methods to identify a plasma factor interfering with PF4/heparin complexes and anti-PF4/heparin antibody–platelet interaction, thus explaining differences in functional assays. To investigate a modulating risk for PF4/heparin immunization and breakthrough of HIT, we also tested 89 plasmas from 2 serosurveillance trials. Fibronectin levels were measured in 4 patient groups exhibiting different degrees of heparin-dependent immunization and expression of HIT. The heat-labile plasma protein, fibronectin, inhibited PF4 binding to platelets in a dose-dependent fashion, particularly in washed (vs PRP) systems. Fibronectin also inhibited PF4/heparin binding to platelets, anti-PF4/heparin antibody binding to PF4/heparin complexes, and anti-PF4/heparin antibody–induced platelet activation as a result of PF4/heparin complex disruption. In addition, plasma fibronectin levels increased progressively among the following 4 patient groups: enzyme-linked immunosorbent assay (ELISA)+/serotonin-release assay (SRA)+/HIT+ < ELISA+/SRA+/HIT− ∼ ELISA+/SRA−/HIT− < ELISA−/SRA−/HIT−. Altogether, these findings suggest that fibronectin interferes with PF4/heparin complex formation and anti-PF4/heparin antibody–induced platelet activation. Reduced fibronectin levels in washed platelet assays help to explain the greater sensitivity of washed platelet (vs PRP) assays for HIT. More importantly, lower plasma fibronectin levels could represent a risk factor for PF4/heparin immunization and clinical breakthrough of HIT.
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Sartori, Maria Teresa, Chiara Zurlo, Maria Bon, Antonella Bertomoro, Raffaele Bendo, Irene Bertozzi, Claudia Maria Radu, Elena Campello, Paolo Simioni, and Fabrizio Fabris. "Platelet-Derived Microparticles Bearing PF4 and Anti-GAGS Immunoglobulins in Patients with Sepsis." Diagnostics 10, no. 9 (August 24, 2020): 627. http://dx.doi.org/10.3390/diagnostics10090627.
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PF4 is a megakaryocyte-derived cationic chemokine that plays a part in innate immunity through its activity on the macrophages. In bacterial sepsis, PF4 binds to glycosaminoglycans (GAGs) on the surface of aerobic bacteria, giving rise to an antigenic complex that induces the early formation of anti-PF4 IgG-IgA-IgM. This triggers the immune response in patients receiving heparin therapy who develop heparin-induced thrombocytopenia (HIT). These antibodies have also been identified in patients with chronic Gram-negative infections. Given the complexity of this innate immune response network, our study on 45 patients with sepsis focused on the immune response mediated by platelet PF4. We analyzed the role of IgG-IgA-IgM against PF4-GAGs, and the presence of specific PF4-bearing platelet microparticles (PMPs). Anti-GAGs/PF4 IgG-IgA-IgM levels were significantly higher in septic patients than in control groups (healthy controls or acute patients without sepsis, p < 0.001). PF4-bearing PMP levels were only significantly higher in septic patients (p < 0.001). The occurrence of IgG-IgA-IgM against PF4-GAGs and PF4+ PMPs correlated with an improvement in patients’ sepsis. In conclusion, we demonstrated that, in the course of bacterial sepsis, platelet activation leads to the formation of specific PF4-bearing PMPs. These specific microparticles bind to polyanionic sequences on the surface of aerobic bacteria, giving rise to an antigenic complex that induces the early formation of IgG-IgA-IgM against PF4-GAGs as an innate immune response to infection.
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Sachais, Bruce S., Tiffany Turrentine, Jeanine M. Dawicki-McKenna, Daniel J. Rader, and M. Anna Kowalska. "Elimination of Platelet Factor 4 (PF4) from Platelets Reduces Atherosclerosis and Increases HDL Cholesterol in C57Bl/6 and ApoE−/− Mice." Blood 108, no. 11 (November 16, 2006): 418. http://dx.doi.org/10.1182/blood.v108.11.418.418.
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Abstract There is a presence of circulating, activated platelets in blood of patients with atherosclerosis, coronary disease and hypercholesterolemia. Upon activation, platelets release a large amount of platelet factor 4 (PF4), a platelet specific chemokine. Our laboratory has previously demonstrated several potentially proatherogenic properties of PF4 including alteration of LDL metabolism and cellular trafficking, and activation of NFkB, a proinflammatory transcription factor involved in atherosclerosis. We have also localized PF4 to human atherosclerotic lesions. However, to date, no direct in vivo evidence for the involvement of PF4 in atherogenesis. In the current study, we have bred PF4−/− mice onto two athero-susceptible backgrounds, WT-C57Bl/6(WT) and apoE−/−, to examine the importance of PF4 in atherogenesis. PF4−/− and PF4−/−apoE−/− (DKO) mice are viable and healthy, with no spontaneous bleeding disorders. In order to induce atherosclerosis, WT and PF4−/− mice were fed an atherogenic Paigen diet for 30 weeks (Study 1), while apoE−/− and DKO mice were fed a high fat Western style diet for 10 weeks (Study 2). Examination of lesions in the aortic roots of Study 1 animals demonstrated a 5-fold reduction in PF4−/− compared to WT mice (p = 0.008). Measurement of cholesterol levels demonstrated similar total and non-HDL cholesterol levels in WT and PF4−/− mice. However, HDL cholesterol was significantly increased in PF4−/− mice compared to WT (2.5-fold, p = 0.001). Examination of apoE−/− mice (Study 2) demonstrated similar changes, with DKO mice demonstrating a 2.7-fold reduction in aortic atherosclerosis (measured by the en face method; p = 0.03) and a 1.7-fold increase in HDL cholesterol (p = 0.02) compared to apoE−/− mice. Although platelet counts were increased by ~30% in mice lacking PF4, the activation state of the platelets in our mice at sacrifice (WT vs PF4−/− and apoE−/− vs DKO) were similar as measured by both p-selectin expression and annexin V binding. These data demonstrate, for the first time, that the platelet specific chemokine PF4 promotes atherosclerotic lesion development in vivo.
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Podolnikova, Nataly P., Valentin P. Yakubenko, and Tatiana P. Ugarova. "Platelet Factor 4 Induces Leukocyte Responses through Integrin Mac-1 (CD11b/CD18)." Blood 128, no. 22 (December 2, 2016): 2529. http://dx.doi.org/10.1182/blood.v128.22.2529.2529.
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Abstract The proteins/peptides secreted from α-granules of activated platelets not only aid in thrombus formation and blood coagulation, but also exert various immune-modulating effects. Among many secreted products, Platelet Factor 4 (PF4), a chemokine that belongs to the CXC family, is one of the most abundant platelet proteins. While PF4 assignment to the chemokine family is based on its structural similarity with other CXC chemokines and chemotactic activity, to date no receptor for PF4 on leukocytes has been identified. Our recent elucidation of the recognition specificity of a major leukocyte integrin αMβ2 (Mac-1) allowed the prediction that PF4 contains several putative Mac-1 recognition motifs and thus could potentially interact with this receptor. Using a peptide library spanning the sequence of PF4, we showed that the αMI-domain of Mac-1 bound several overlapping PF4-derived peptides. The biolayer interferometry analyses demonstrated that PF4 bound recombinant active αMI-domain of Mac-1 in a concentration-dependent manner with a KD of 1.3 ± 0.2x10-6 M. No interaction of PF4 with the inactive αMI-domain (α7 helix extended) or the αLI-domain of a homologous integrin αLβ2 was detected. The full-length recombinant PF4 and the αMI-domain-binding peptide (residues 58-70) identified in the peptide library supported strong adhesion and spreading of Mac-1-expressing cells, including neutrophils, U937 monocytic and Mac-1-transfected HEK293 cells. The cell adhesion to PF4 was partially inhibited by anti-Mac-1 mAbs and completely blocked when anti-Mac-1 antibodies were combined with heparin, suggesting that cell surface proteoglycans act cooperatively with integrin Mac-1. PF4 induced a potent migratory response of wild-type, but not Mac-1-deficient, macrophages in a Transwell system. PF4 also enhanced phagocytosis: coating of E. coli bacteria or latex beads with PF4 enhanced ~ 4-fold their phagocytosis by macrophages, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by wild-type, but not Mac-1-deficient, macrophages. These results identify PF4 as a ligand for integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated via interaction with Mac-1. Disclosures No relevant conflicts of interest to declare.
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Green, C. J., R. S. Charles, B. F. Edwards, and P. H. Johnson. "Identification and characterization of PF4varl, a human gene variant of platelet factor 4." Molecular and Cellular Biology 9, no. 4 (April 1989): 1445–51. http://dx.doi.org/10.1128/mcb.9.4.1445-1451.1989.
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A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.
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Green, C. J., R. S. Charles, B. F. Edwards, and P. H. Johnson. "Identification and characterization of PF4varl, a human gene variant of platelet factor 4." Molecular and Cellular Biology 9, no. 4 (April 1989): 1445–51. http://dx.doi.org/10.1128/mcb.9.4.1445.
Full textAbstract:
A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.
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Joglekar, Manali V., Sanjay Khandelwal, Mortimer Poncz, Lubica Rauova, and Gowthami M. Arepally. "Understanding the Underlying Immune Response in HIT: Uptake of PF4/Heparin Complexes By Monocytes & Dendritic Cells." Blood 124, no. 21 (December 6, 2014): 4197. http://dx.doi.org/10.1182/blood.v124.21.4197.4197.
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Abstract Platelet Factor 4 (PF4), a strongly positive-charged small protein, and the negatively-charged polymer heparin (Hep) form ultra-large complexes (ULCs) that have several unusual features including their remarkable stability and their ability to elicit a robust antibody response in vivo (Rauova L; Blood 2005 and Suvarna S, Blood 2005). Recently, we and others have shown that similar complexes of another positively charged protein, protamine sulfate with heparin can also lead to a clinically relevant immune response. To better understand the cellular basis for PF4/Hep antibody formation, we investigated mechanisms of cellular interactions and uptake of PF4 and PF4/Hep ULCs. For these studies, we examined cellular uptake of unlabeled or labeled PF4, heparin, or PF4/Hep using monocytes (PBMCs), dendritic cells (DCs) and/or neutrophils derived from peripheral blood. For these studies, cells were incubated with varying concentrations of unlabeled or fluorescently-labeled antigen (PF4, Hep or PF4/Hep ULCs). In cellular studies using unlabeled antigen, uptake was detected by fluorescently-labeled KKO, a monoclonal antibody to PF4/Hep complexes. Cellular uptake was visualized by confocal microscopy or flow cytometry. In initial studies, we defined the time course of uptake. As shown in Figure 1, we demonstrate that PF4/Hep-FITC ULCs are taken up by PBMCs in a time-dependent manner, with maximal uptake occurring between 12-24 hours (Figure 1, only 24 hour time point shown). This uptake is independent of the fluorescent label, as labeled or unlabeled intracellular PF4/Hep ULCs were readily visualized by KKO-AF647. To examine the effect of Hep on PF4 uptake, PBMCs were incubated with unlabeled PF4 alone or in the presence of increasing concentrations of Hep-FITC (0.1-2.5 U/mL). As shown in Figure 2A, Hep markedly enhances the efficiency of cellular uptake of PF4 in a Hep-dependent manner. Increased number of intracellular vesicles containing labeled PF4/Hep-FITC was noted at Hep-FITC concentration of 0.25-1 U/mL (Figure 2B; fluorescent vesicles/cell: 0.6 ± 0.22 for 0.35 U/mL and 0.5 ± 0.26 for 1U/mL) as compared to PF4 alone (25 µg/mL; number of fluorescent vesicles/cell: 0.35 ± 0.07). On examining the uptake of ULCs by other phagocytic cells, we could not demonstrate PF4/Hep uptake by neutrophils, suggesting that only monocytes/DCs provide clearance of complexes. Cellular uptake of Hep-containing ULCs was not limited to complexes of PF4 and heparin but also other positively-charged proteins, as intracellular complexes could be demonstrated when Hep-FITC was incubated with murine PF4, protamine, or lysozyme to form corresponding protein/Hep-FITC ULCs. This uptake was an active process of monocytes as PF4/Hep ULC endocytosis was inhibited by 4C, and cytochalasin D, an actin polymerization inhibitor and was associated with cellular activation and expression of MHC II and CD83 co-stimulatory molecules as shown by flow cytometry (Figure 3). Finally, co-staining with KKO and lysosomal associated membrane protein-2 (LAMP-2) localized intact PF4/Hep ULCs into late endosomes. Taken together, these studies demonstrate that PF4/Hep and other protein/heparin ULCs are taken up actively by monocytes and/or DCs, intact ULCs can be detected in late endosomes and uptake is accompanied by cellular activation. These studies establish a distinct role for heparin in increasing the uptake and cellular activation of PF4 and other positively charged complexes. These studies additionally provide insights into why the majority of clinical cases of HIT occur in the wake of heparin exposure. Disclosures Arepally: TEVA Pharma: Consultancy.
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Xiao, Liqing, Mortimer Poncz, and Michele Lambert. "Platelet Factor 4 (PF4) Causes Cell Cycle Arrest in Megakaryocytes (Megs) by Inactivating CDC2 (CDK1) and CDK2." Blood 120, no. 21 (November 16, 2012): 1238. http://dx.doi.org/10.1182/blood.v120.21.1238.1238.
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Abstract Abstract 1238 PF4 (CXCL4), a platelet specific chemokine released in large amounts from activated platelet α -granules, is a negative regulator of megakaryopoiesis. In mouse studies, we have shown that PF4 levels regulate steady-state platelet count and impact chemotherapy and radiation-induced thrombocytopenia. In a clinical study in leukemia patients, we found that PF4 levels were inversely related to steady-state platelet count and to recovery after chemotherapy. The molecular basis for the effect of PF4 in megakaryopoiesis is largely unknown. Our studies in cell models suggested that PF4 might act through the cell surface receptor low-density lipoprotein related protein-1 (LRP1). Using an early megakaryoblastic cell line, which expresses LRP1, Meg-like cell line (Meg01), we show that PF4 exerts an anti-proliferative effect on the cells through inactivation of cell cycle regulators CDC2 (CDK1) and CDK2. PF4 treatment (200 μg/ml for 48 hrs) of Meg01 cells induced a decrease in cells in G1 (from 68% of cells to 51%, p=0.001) with a concurrent increase in the percentage of cells in S (12% of cells to 21%, p = 0.02 for no PF4 vs. PF4 treatment) and G2 (from 20% to 28% of cells) phase, without significant bromodeoxyuridine (BrdU) incorporation by the cells in the S phase, suggesting that PF4 causes a cell cycle arrest resulting in decreased cell proliferation. The cell cycle arrest and lack of BrDU incorporation was confirmed in primary murine Megs. No apoptosis was detected in PF4 treated Meg01 or primary cells. To determine the molecular mechanisms by which PF4 causes cell cycle arrest, we used Western blots interrogating cell cycle proteins. We detected a transient increase in the inhibitory phosphorylation (at Tyr15) of CDC2 after PF4 treatment, as well as a decrease in phosphorylation of the activating site (Thr160) on CDK2. In addition, we found PF4 treatment resulted in the degradation of Cdc25c, the upstream phosphatase of Tyr15 of CDC2. In primary murine Megs, we detected a significant decrease of total CDC2, biologically equivalent to the CDC2 inactivation seen in Meg01 cells. The CDK inhibitor Roscovitine inhibited Meg01 cell proliferation and had minimum additive effect with PF4. Overexpression of the constitutively active CDC2 mutant CDC2AF with the inhibitory phosphorylation sites Thr14 and Tyr15 replaced by Ala and Phe, respectively, desensitized the cells to PF4 treatment. These results suggested that PF4 inhibits megakaryopoiesis by decreasing the proliferation of megakaryocytes in their early developmental stage by inactivating cell cycle regulators CDC2 and CDK2. Unraveling the mechanisms by which PF4 inhibits megakaryopoiesis may lead to the development of novel therapeutics to regulate platelet counts. Disclosures: No relevant conflicts of interest to declare.
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Rauova, Lubica, Li Zhai, M. Anna Kowalska, Gowthami M. Arepally, Douglas B. Cines, and Mortimer Poncz. "Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications." Blood 107, no. 6 (March 15, 2006): 2346–53. http://dx.doi.org/10.1182/blood-2005-08-3122.
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AbstractHeparin-induced thrombocytopenia (HIT) antibodies recognize complexes between heparin and platelet factor 4 (PF4). Heparin and PF4 bind HIT antibodies only over a narrow molar ratio. We explored the involvement of platelet surface–bound PF4 as an antigen in the pathogenesis of experimental HIT. We show that cell-surface PF4 complexes are also antigenic only over a restricted concentration range of PF4. Heparin is not required for HIT antibody binding but shifts the concentration of PF4 needed for optimal surface antigenicity to higher levels. These data are supported by in vitro studies involving both human and murine platelets with exogenous recombinant human (h) PF4 and either an anti–PF4-heparin monoclonal antibody (KKO) or HIT immunoglobulin. Injection of KKO into transgenic mice expressing different levels of hPF4 demonstrates a correlation between the severity of the thrombocytopenia and platelet hPF4 expression. Therapeutic interventions in this model using high-dose heparin or protamine sulfate support the pathogenic role of surface PF4 antigenic complexes in the etiology of HIT. We believe that this focus on surface PF4 advances our understanding of the pathogenesis of HIT, suggests ways to identify patients at high risk to develop HIT upon heparin exposure, and offers new therapeutic strategies.
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Amiral, J., F. Bridey, M. Wolf, C. Boyer-Neumann, E. Fressinaud, A. M. Vissac, E. Peynaud-Debayle, M. Dreyfus, and D. Meyer. "Antibodies to Macromolecular Platelet Factor 4-Heparin Complexes in Heparin-induced Thrombocytopenia: a Study of 44 Cases." Thrombosis and Haemostasis 73, no. 01 (1995): 021–28. http://dx.doi.org/10.1055/s-0038-1651670.
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SummaryAs heparin-PF4 (H-PF4) complexes are the target for antibodies associated to heparin-induced thrombocytopenia (HIT), an ELISA has been developed and optimised for testing antibodies binding to H-PF4. This test was consistently negative in 50 healthy subjects (A492 <0.3) and 35 patients with other causes of thrombocytopenia (A492 <0.5). In contrast, 43 out of 44 HIT patients showed antibodies to H-PF4 (A492 = 1.70 ± 0.81) including 5 patients with a negative platelet aggregation test. In one patient with HIT, antibodies to H-PF4 were already present at day 7, whereas platelet counts dropped ≤ 100 × 109/l only at days 11–12. Surprisingly, among 41 patients under heparin for >7 days, 5 showed antibodies to H-PF4, without HIT. These findings underline the major interest of this ELISA for the early diagnosis of HIT. We also showed that LMWH as well as other sulphated polysaccharides can bind to HIT antibodies in the presence of PF4 and that their reactivity is dependent on the molecular weight and the sulphation grade. The mechanism for HIT involves platelet PF4 receptors which bind the macromolecular H-PF4 complexes formed in the presence of a well defined heparin/PF4 ratio.
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Dudek, Arkadiusz Z., Irina Nesmelova, Kevin Mayo, Catherine M. Verfaillie, Simon Pitchford, and Arne Slungaard. "Platelet factor 4 promotes adhesion of hematopoietic progenitor cells and binds IL-8: novel mechanisms for modulation of hematopoiesis." Blood 101, no. 12 (June 15, 2003): 4687–94. http://dx.doi.org/10.1182/blood-2002-08-2363.
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AbstractPlatelet factor 4 (PF4) is an abundant platelet α-granule C-X-C chemokine that has weak chemotactic potency but strongly inhibits hematopoiesis through an unknown mechanism. We find that PF4 binds to human CD34+ hematopoietic progenitor cells (HPCs) with a median effective concentration of 1 μg/mL but not after exposure to chondroitinase ABC. PF4 enhances adhesion of HPCs to intact stroma. Committed progenitors also adhere avidly to immobilized PF4. This adhesion is time-dependent, requires metabolic activity, causes cytoskeletal rearrangement, and induces cell-cycle inhibition. Using extracellular acidification rate to indicate transmembrane signaling, we find that interleukin-8 (IL-8), but not PF4, activates CD34+ progenitors, and PF4 blocks IL-8–mediated activation. Surface plasmon resonance analysis shows that PF4 binds IL-8 with high (dissociation constant [Kd] = 42 nM) affinity. Nuclear magnetic resonance analysis of IL-8 and PF4 in solution confirms this interaction. We conclude that PF4 has the capacity to influence hematopoiesis through mechanisms not mediated by a classical high-affinity, 7-transmembrane domain chemokine receptor. Instead, PF4 may modulate the hematopoietic milieu both directly, by promoting progenitor adhesion and quiescence through interaction with an HPC chondroitin sulfate–containing moiety, and indirectly, by binding to or interfering with signaling caused by other, hematopoietically active chemokines, such as IL-8.
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Brandt, Sven, Krystin Krauel, Kay E. Gottschalk, Thomas Renné, Christiane A. Helm, Andreas Greinacher, and Stephan Block. "Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity." Thrombosis and Haemostasis 112, no. 07 (2014): 53–64. http://dx.doi.org/10.1160/th13-08-0634.
Full textAbstract:
SummaryHeparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32–38, which form together with C-terminal AA (66–70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express “HIT antigens”. The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.
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Zheng, Yongwei, Wen Zhu, Dipica Haribhai, Calvin B. Williams, Richard H. Aster, Renren Wen, and Demin Wang. "Regulatory T Cells Control PF4/Heparin Antibody Production in Mice." Blood 132, Supplement 1 (November 29, 2018): 2542. http://dx.doi.org/10.1182/blood-2018-99-113049.
Full textAbstract:
Abstract Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. Regulatory T (Treg) cells are a subpopulation of CD4 T cells that modulate immune system by suppressing or down-regulating immune response and play an important role in immune homeostasis. However, the role of Treg cells in controlling PF4/heparin-specific antibody production is not known. Here we found that FoxP3-deficient mice, which have no functional Treg cells, spontaneously generated PF4/heparin-specific antibodies as early as three weeks after birth. Similarly, following transplantation with bone marrow cells from FoxP3-deficient mice, Rag1-deficient mice that have no endogenous B cells and T cells spontaneously produced PF4/heparin-specific antibodies. In contrast, Rag1-deficient mice that received wild-type bone marrow cells failed to produce PF4/heparin-specific antibodies. In addition, adoptively transferred Treg cells prevented spontaneous production of PF4/heparin-specific antibodies in FoxP3-deficient mice. Adoptively transferred Treg cells, not conventional CD4 T cells, also suppressed PF4/heparin complex-induced production of PF4/heparin-specific IgGs in wild-type mice. Treg cells suppress immune response mainly through releasing anti-inflammatory cytokines, such as interleukin-10 (IL-10). Deficiency of IL-10 led to spontaneous production of PF4/heparin-specific antibodies in mice. Moreover, BM chimeric mice with CD4 T cell-specific deletion of IL-10 increased PF4/heparin-specific IgG production following PF4/heparin complex challenge. Taken together, these findings demonstrate that Treg cells play an important role in suppressing PF4/heparin-specific antibody production in mice. Disclosures Haribhai: AbbVie Inc.: Employment. Aster:BloodCenter of Wisconsin: Patents & Royalties.
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Cini, Michela, Caterina Pili, Ottavio Boggian, Mirella Frascaro, Gualtiero Palareti, and Cristina Legnani. "Evaluation of a new automated panel of assays for the detection of anti-PF4/heparin antibodies in patients suspected of having heparin-induced thrombocytopenia." Thrombosis and Haemostasis 104, no. 08 (2010): 402–9. http://dx.doi.org/10.1160/th10-01-0002.
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SummaryHeparin-induced thrombocytopenia (HIT) is a life-threatening complication of heparin treatment; the prognosis depends on early and accurate diagnosis, and prompt start of alternative anticoagulants. Because of high sensitivity, the commercially available immunologic assays are widely used, though not suited to be run on single samples and with a turnaround time of 2–3 hours. We evaluated two new, rapid, automated, semi-quantitative chemiluminescent immunoassays in HIT suspected patients: HemosIL® AcuStar HIT-IgG(PF4-H) (specific for IgG anti- PF4/heparin antibodies) and HemosIL® AcuStar HIT-Ab(PF4-H) (detecting IgG, IgM and IgA anti-PF4/heparin antibodies) (both from Instrumentation Laboratory). A total of 102 patients with suspected HIT were included; HIT was diagnosed in 17 (16.7%). No false negative cases were observed using either the HemosIL AcuStar HIT-IgG(PF4-H) or the HITAb(PF4-H) assay (sensitivity and negative predictive values = 100%; negative likelihood ratios <0.01). The specificity was higher for the He-mosIL AcuStar HIT-IgG(PF4-H) in comparison with that of the HemosIL AcuStar HIT-Ab(PF4-H) (96.5% vs. 81.2%). Higher values of the HemosIL AcuStar HIT-IgG(PF4-H) were associated with increased probability of HIT. Patients with confirmed HIT and thrombotic complications had significantly higher levels of HemosIL AcuStar HIT-IgG(PF4-H) than those without thrombotic complications. The HemosIL AcuStar HIT-IgG(PF4-H) and HIT-Ab(PF4-H) assays showed a very high sensitivity, and therefore they can reliably be used to rule out HIT in suspected patients. The diagnostic specificity was greatly increased by using the HemosIL AcuStar HITIgG(PF4-H). Both the assays are reproducible (CVs <6%), rapid (turnaround time 30 minutes), automated, and semi-quantitative, and they can be run for single sample testing.
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Lambert, Michele, Samriddhi S. Sharma, Liqing Xiao, Stephen Marcus, and Mortimer Poncz. "2-O, 3-O-Desulfated Heparin (ODSH) Mitigates Chemotherapy-Induced Thrombocytopenia (CIT) by Blocking the Negative Paracrine Effect of Platelet Factor 4 (PF4) On Megakaryopoiesis." Blood 120, no. 21 (November 16, 2012): 386. http://dx.doi.org/10.1182/blood.v120.21.386.386.
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Abstract Abstract 386 Thrombocytopenia is a significant complication of myelosuppressive chemotherapy treatments, which are a mainstay in cancer therapy. We and others have previously shown that PF4 is a negative paracrine affecting megakaryocyte number in culture. In murine studies we showed that platelet PF4 levels are inversely related to steady-state platelet counts and is a major contributor to the duration and severity of CIT. Recently, we have shown that PF4 influences both steady-state platelet count and platelet count recovery in pediatric patients treated for acute lymphoblastic leukemia. Pre-clinical studies in murine models suggest that blocking the effect of PF4 (using polyclonal anti-PF4 antibodies) can mitigate the effect of PF4 on megakaryopoiesis and results in a shortened time to platelet count recovery and higher nadir platelet count. Heparin is known to bind PF4 tightly and can clear PF4 from the vascular endothelium. Pre-clinical studies using heparin to mitigate CIT failed to show an effect, but were limited by dose considerations due to the anti-coagulant effect of unfractionated heparin (UFH) and likely by the inability of this highly negative polysaccharide to reach the intramedullary space in high enough amounts to alter available PF4. ODSH does not exhibit the same effect as unfractionated heparin (UFH) in enhancing antithrombin effects and may allow studies of non-anticoagulant pharmacologic effects of heparin. For example, ODSH retains UFH's anti-inflammatory effects and its ability to bind tightly to PF4. In preliminary results of a clinical trial using ODSH in conjunction with myelosuppressive chemotherapy in patients with metastatic pancreatic cancer, there was a striking lack of clinically significant CIT as will be reported separately at this meeting. This prompted our evaluation of whether ODSH affects CIT and modulates platelet count recovery and whether this is through its interaction with PF4. We first examined the ability of ODSH to prevent the PF4 effect on megakaryopoiesis in vitro. Using a megakaryocyte colony assay, we show that PF4 treatment of PF4null murine megakaryocytes decreases megakaryocyte colony numbers (54±3% control vs. 23±6% PF4-treated, p<0.001) and treatment with ODSH completely blocks the PF4 effect (55±9% ODSH/PF4-treated, p<0.001 vs. PF4-treated). This suggested that the major mechanism by which ODSH prevents CIT is through inhibition of the PF4 effect. We then examined the effect of ODSH on liquid murine bone marrow culture and showed that ODSH treatment (50 μg/mL) was able to improve cell counts in the presence of added recombinant human PF4 (50 μg/mL) (9.2±3.3 × 104 cells/mL in PF4-treated cells vs. 19.3±4.2 × 104 cells/mL in ODSH+PF4-treated cells, p<0.01). Finally, we examined the in vivo effect of ODSH on CIT in a murine model of chemotherapy in transgenic mice that overexpress human (h) PF4. In these hPF4 mice, endogenous PF4 levels significantly affect enhances the degree and duration of 5FU-induced CIT. We previously reported that treatment of animals with anti-hPF4 antibodies was able to completely abolish the PF4 effect. Using the same model, injecting 180 mg/kg 5-fluourouracil (5-FU) intraperitoneally on day 0, we examined the effect on platelet counts of treatment with 2 clinically relevant doses of ODSH (25 mg/kg/dose) given subcutaneously 30 minutes and 24 hours after injection of 5-FU. hPF4 mice treated with 5-FU and ODSH had a higher platelet count nadir (70±14% versus vs. 44±1% of baseline). The nadir platelet count in the ODSH-treated mice was similar to that in mPF4null mice (57±20% of baseline). In addition, animals treated with ODSH recovered approximately 2 days earlier. In summary, ODSH mitigates CIT resulting in decreased severity and duration of thrombocytopenia. These studies suggest that this effect is mediated in large part by PF4 as in vitro experiments show that ODSH completely blocks the effect of PF4 on megakaryopoiesis. The in vivo studies support that sufficient ODSH reaches the marrow to block intramedullary-released PF4 and prevents its inhibition of megakaryopoiesis. This drug is already in clinical trials in humans and may be the first clinically relevant inhibitor of CIT. Further studies will examine its effect in other thrombocytopenic settings. Disclosures: Marcus: Paringenix: Employment, Equity Ownership.
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Levine, SP, LK Knieriem, and MA Rager. "Platelet factor 4 and the platelet secreted proteoglycan: immunologic characterization by crossed immunoelectrophoresis." Blood 75, no. 4 (February 15, 1990): 902–10. http://dx.doi.org/10.1182/blood.v75.4.902.902.
Full textAbstract:
Abstract Platelet factor 4 (PF4) is a hydrophobic, alpha-granule protein with potent antiheparin activity. It also binds to a chondroitin sulfate- containing proteoglycan (PG) isolated from platelets. In order to evaluate further the relationship between PF4 and the chondroitin sulfate-containing proteoglycan in resting platelets, the PF4-binding proteoglycan from human platelets has been purified using purified PF4 as an affinity ligand and used to prepare polyclonal antiserum. Two antisera have been characterized: one reacts primarily with chondroitin sulfate (CS), the other reacts with the protein core of the platelet proteoglycan after chondroitinase AC digestion. PF4 and PG core protein antigen are present in separate, dissimilar precipitin arcs when triton- solubilized platelets are analyzed by crossed immunoelectrophoresis using polyclonal antisera to purified PF4 and PG. PF4 was demonstrated in a complex with a separate chondroitin sulfate antigen by crossed immunoelectrophoresis (CIE) experiments in which either anti-PF4 or anti-CS antisera was incorporated in the intermediate gel. Both the PF4- chondroitin sulfate complex and the proteoglycan are secreted from platelets when fresh, washed human platelets are stimulated by human alpha-thrombin. This second antigen may represent the PG after posttranslational modification of a precursor form of the proteoglycan.
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Levine, SP, LK Knieriem, and MA Rager. "Platelet factor 4 and the platelet secreted proteoglycan: immunologic characterization by crossed immunoelectrophoresis." Blood 75, no. 4 (February 15, 1990): 902–10. http://dx.doi.org/10.1182/blood.v75.4.902.bloodjournal754902.
Full textAbstract:
Platelet factor 4 (PF4) is a hydrophobic, alpha-granule protein with potent antiheparin activity. It also binds to a chondroitin sulfate- containing proteoglycan (PG) isolated from platelets. In order to evaluate further the relationship between PF4 and the chondroitin sulfate-containing proteoglycan in resting platelets, the PF4-binding proteoglycan from human platelets has been purified using purified PF4 as an affinity ligand and used to prepare polyclonal antiserum. Two antisera have been characterized: one reacts primarily with chondroitin sulfate (CS), the other reacts with the protein core of the platelet proteoglycan after chondroitinase AC digestion. PF4 and PG core protein antigen are present in separate, dissimilar precipitin arcs when triton- solubilized platelets are analyzed by crossed immunoelectrophoresis using polyclonal antisera to purified PF4 and PG. PF4 was demonstrated in a complex with a separate chondroitin sulfate antigen by crossed immunoelectrophoresis (CIE) experiments in which either anti-PF4 or anti-CS antisera was incorporated in the intermediate gel. Both the PF4- chondroitin sulfate complex and the proteoglycan are secreted from platelets when fresh, washed human platelets are stimulated by human alpha-thrombin. This second antigen may represent the PG after posttranslational modification of a precursor form of the proteoglycan.