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Mortimer Poncz - One of the best experts on this subject based on the ideXlab platform.
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polyphosphate Platelet Factor 4 complexes can mediate heparin independent Platelet activation in heparin induced thrombocytopenia
Blood Advances, 2016Co-Authors: Douglas B Cines, Gowthami M Arepally, Mortimer Poncz, Lubica Rauova, Serge Yarovoi, Sanjay Khandelwal, Sergei Zaitsev, Tatiana Lebedeva, Victoria Stepanova, Ann H RuxAbstract:Heparin-induced thrombocytopenia (HIT) is a thrombotic disorder initiated by antibodies to complexes between Platelet Factor 4 (PF4) and heparin. The risk of recurrent thromboembolism persists after heparin is cleared and Platelet activation leading to release of PF4 has dissipated. We asked whether antigenic complexes between polyphosphates and PF4 released from activated Platelets might intensify or sustain the prothrombotic phenotype of HIT. PF4 forms stable, ultralarge complexes with polyphosphates of various sizes, including those released from Platelets, which are recognized by the HIT-like monoclonal KKO, an immunoglobulin G2bκ monoclonal heparin/PF4 binding antibody, and by human HIT antibodies. KKO helps to protect PF4/polyphosphate complexes from degradation by phosphatases. Complement is activated when HIT antibodies bind to PF4/polyphosphate complexes and PF4 reverses the inhibition of complement by polyphosphates. Polyphosphates and PF4 are stored primarily in separate granules in resting Platelets, but they colocalize when the cells are activated. Platelets activated by subaggregating doses of thrombin receptor activating peptide release polyphosphates and PF4, which form antigenic complexes that allow KKO to further activate Platelets in the absence of heparin and exogenous PF4. These studies suggest that thrombin- or immune complex-mediated release of endogenous antigenic PF4/polyphosphate complexes from Platelets may augment the prothrombotic risk of HIT and perpetuate the risk of thrombosis after heparin has been discontinued.
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heparin enhances uptake of Platelet Factor 4 heparin complexes by monocytes and macrophages
Journal of Thrombosis and Haemostasis, 2015Co-Authors: Manali Joglekar, Mortimer Poncz, Lubica Rauova, Douglas B Cines, Sanjay Khandelwal, Gowthami M ArepallyAbstract:Summary Background Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies to a self-antigen, Platelet Factor (4) and heparin. The reasons why antibodies form to PF4/heparin, but not to PF4 bound to other cellular glycosaminoglycans are poorly understood. Objective To investigate differences in cellular responses to cell-bound PF4 and PF4/heparin complexes, we studied the internalization of each by peripheral blood-derived monocytes, dendritic cells and neutrophils. Methods and results Using unlabeled and fluorescently-labeled antigen and/or labeled monoclonal antibody to PF4/heparin complexes (KKO), we show that PF4/heparin complexes are taken up by monocytes in a heparin-dependent manner and are internalized by human monocytes and dendritic cells, but not by neutrophils. Complexes of PF4/low-molecular-weight heparin and complexes composed of heparin and murine PF4, protamine or lysozyme are internalized similarly, suggesting a common endocytic pathway. Uptake of complexes is mediated by macropinocytosis, as shown by inhibition using cytochalasin D and amiloride. Internalized complexes are transported intact to late endosomes, as indicated by co-staining of vesicles with KKO and lysosomal associated membrane protein-2 (LAMP-2). Lastly, we show that cellular uptake is accompanied by expression of MHCII and CD83 co-stimulatory molecules. Conclusions Taken together, these studies establish a distinct role for heparin in enhancing antigen uptake and activation of the initial steps in the cellular immune response to PF4-containing complexes.
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Platelet Factor 4 limits th17 differentiation and cardiac allograft rejection
Journal of Clinical Investigation, 2014Co-Authors: Guanfang Shi, Anna M Kowalska, Mortimer Poncz, Kalyan Srivastava, David J Field, Sara Ture, Scott Levy, Deborah J Fowell, Craig N MorrellAbstract:Th cells are the major effector cells in transplant rejection and can be divided into Th1, Th2, Th17, and Treg subsets. Th differentiation is controlled by transcription Factor expression, which is driven by positive and negative cytokine and chemokine stimuli at the time of T cell activation. Here we discovered that chemokine Platelet Factor 4 (PF4) is a negative regulator of Th17 differentiation. PF4-deficient and Platelet-deficient mice had exaggerated immune responses to cardiac transplantation, including increased numbers of infiltrating Th17 cells and increased plasma IL-17. Although PF4 has been described as a Platelet-specific molecule, we found that activated T cells also express PF4. Furthermore, bone marrow transplantation experiments revealed that T cell–derived PF4 contributes to a restriction in Th17 differentiation. Taken together, the results of this study demonstrate that PF4 is a key regulator of Th cell development that is necessary to limit Th17 differentiation. These data likely will impact our understanding of Platelet-dependent regulation of T cell development, which is important in many diseases, in addition to transplantation.
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atherosclerosis is not a risk Factor for anti Platelet Factor 4 heparin antibody formation after cardiopulmonary bypass surgery
Thrombosis and Haemostasis, 2014Co-Authors: Adam Cuker, Mortimer Poncz, Lubica Rauova, Douglas Bolgiano, William H Matthai, Barbara A KonkleAbstract:Atherosclerosis is not a risk Factor for anti-Platelet Factor 4/heparin antibody formation after cardiopulmonary bypass surgery -
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modulation of protein c activation by histones Platelet Factor 4 and heparinoids new insights into activated protein c formation
Arteriosclerosis Thrombosis and Vascular Biology, 2014Co-Authors: Anna M Kowalska, Guohua Zhao, Li Zhai, George David, Stephen Marcus, Sriram Krishnaswamy, Mortimer PonczAbstract:Objective— Histones are detrimental in late sepsis. Both activated protein C (aPC) and heparin can reverse their effect. Here, we investigated whether histones can modulate aPC generation in a manner similar to another positively charged molecule, Platelet Factor 4, and how heparinoids (unfractionated heparin or oxygen-desulfated unfractionated heparin with marked decrease anticoagulant activity) may modulate this effect. Approach and Results— We measured in vitro and in vivo effects of histones, Platelet Factor 4, and heparinoids on aPC formation, activated partial thromboplastin time, and murine survival. In vitro, histones and Platelet Factor 4 both affect thrombin/thrombomodulin aPC generation following a bell-shaped curve, with a peak of >5-fold enhancement. Heparinoids shift these curves rightward. Murine aPC generation studies after infusions of histones, Platelet Factor 4, and heparinoids supported the in vitro data. Importantly, although unfractionated heparin and 2-O, 3-O desulfated heparin both reversed the lethality of high-dose histone infusions, only mice treated with 2-O, 3-O desulfated heparin demonstrated corrected activated partial thromboplastin times and had significant levels of aPC. Conclusions— Our data provide a new contextual model of how histones affect aPC generation, and how heparinoid therapy may be beneficial in sepsis. These studies provide new insights into the complex interactions controlling aPC formation and suggest a novel therapeutic interventional strategy.
Andreas Greinacher - One of the best experts on this subject based on the ideXlab platform.
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characterization of the interaction between Platelet Factor 4 and homogeneous synthetic low molecular weight heparins
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Thihuong Nguyen, Sven Brandt, Martin Mandelkow, Ricarda Raschke, Ulrike Strobel, Mihaela Delcea, Wen Zhou, Jian Liu, Andreas GreinacherAbstract:BACKGROUND Heparins are usually produced from animal tissues. It is now possible to synthesize heparins. This provides the abilities to overcome shortages of heparin, to optimize biological effects, and to reduce adverse drug effects. Heparins interact with Platelet Factor 4 (PF4), which can induce an immune response causing thrombocytopenia. This side effect is called heparin-induced thrombocytopenia (HIT). We characterized the interaction of PF4 and HIT antibodies with oligosaccharides of 6-, 8-, 10-, and 12-mer size and a hypersulfated 12-mer (S12-mer). METHODS We utilized multiple methodologies including isothermal calorimetry, circular dichroism spectroscopy, single molecule force spectroscopy (SMFS), enzyme immunosorbent assay (EIA), and Platelet aggregation test to characterize the interaction of synthetic heparin analogs with PF4 and anti-PF4/heparin antibodies. RESULTS The synthetic heparin-like compounds display stronger binding characteristics to PF4 than animal-derived heparins of corresponding lengths. Upon complexation with PF4, 6-mer and S12-mer heparins showed much lower enthalpy, induced less conformational changes in PF4, and interacted with weaker forces than 8-, 10-, and 12-mer heparins. Anti-PF4/heparin antibodies bind more weakly to complexes formed between PF4 and heparins ≤ 8-mer than with complexes formed between PF4 and heparins ≥ 10-mer. Addition of one sulfate group to the 12-mer resulted in a S12-mer, which showed substantial changes in its binding characteristics to PF4. CONCLUSIONS We provide a template for characterizing interactions of newly developed heparin-based anticoagulant drugs with proteins, especially PF4 and the resulting potential antigenicity.
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Platelets kill bacteria by bridging innate and adaptive immunity via Platelet Factor 4 and fcγriia
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Raghavendra Palankar, Krystin Krauel, Thomas P Kohler, Jan Wesche, Sven Hammerschmidt, Andreas GreinacherAbstract:Essentials Human Platelets specifically interact with IgG opsonized bacteria through FcγRIIA. Platelet Factor 4 (PF4) binds to polyanions (P) and undergoes a conformational change. Anti-PF4/P IgG opsonizes PF4-coated Gram-positive and Gram-negative bacteria. Platelets specifically kill E.coli opsonized with PF4 and human anti-PF4/P IgG. SUMMARY Background Activated Platelets release the chemokine Platelet Factor 4 (PF4) stored in their granules. PF4 binds to polyanions (P) on bacteria, undergoes a conformational change and exposes neoepitopes. These neoepitopes induce production of anti-PF4/P antibodies. As PF4 binds to a variety of bacteria, anti-PF4/P IgG can bind and opsonize several bacterial species. Objective Here we investigated whether Platelets are able to kill bacteria directly after recognizing anti-PF4/P IgG opsonized bacteria in the presence of PF4 via their FcγRIIA. Methods Using Platelet-bacteria suspension co-culture experiments and micropatterns with immobilized viable bacteria, in combination with pharmacological inhibitors and human anti- PF4/P IgG we analyzed the role of Platelet-mediated killing of bacteria. Results In the presence of PF4, human anti-PF4/P IgG and Platelets, E. coli killing (> 50%) with colony forming units (CFU mL-1 ) 0.71 × 104 ± 0.19 was observed compared with controls incubated only with anti-PF4/P IgG (CFU mL-1 3.4 × 104 ± 0.38). Blocking of Platelet FcγRIIA using mAb IV.3 (CFU mL-1 2.5 × 104 ± 0.45), or integrin αIIbβ3 (CFU mL-1 2.26 × 104 ± 0.31), or disruption of cytoskeletal functions (CFU mL-1 2.7 × 104 ± 0.4) markedly reduced E. coli killing by this mechanism. Our observation of E. coli killing by Platelets on micropatterned arrays is compatible with the model that Platelets kill bacteria by covering them, actively concentrating them into the area under their granulomere and then releasing antimicrobial substances of Platelet α-granules site directed towards bacteria. Conclusion These findings collectively indicate that by bridging of innate and adaptive immune mechanisms, Platelets and anti-PF4/polyanion antibodies cooperate in an antibacterial host response.
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anti Platelet Factor 4 polyanion antibodies mediate a new mechanism of autoimmunity
Nature Communications, 2017Co-Authors: Thihuong Nguyen, Mihaela Delcea, Nikolay Medvedev, Andreas GreinacherAbstract:Antibodies recognizing complexes of the chemokine Platelet Factor 4 (PF4/CXCL4) and polyanions (P) opsonize PF4-coated bacteria hereby mediating bacterial host defense. A subset of these antibodies may activate Platelets after binding to PF4/heparin complexes, causing the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). In autoimmune-HIT, anti-PF4/P-antibodies activate Platelets in the absence of heparin. Here we show that antibodies with binding forces of approximately 60-100 pN activate Platelets in the presence of polyanions, while a subset of antibodies from autoimmune-HIT patients with binding forces ≥100 pN binds to PF4 alone in the absence of polyanions. These antibodies with high binding forces cluster PF4-molecules forming antigenic complexes which allow binding of polyanion-dependent anti-PF4/P-antibodies. The resulting immunocomplexes induce massive Platelet activation in the absence of heparin. Antibody-mediated changes in endogenous proteins that trigger binding of otherwise non-pathogenic (or coFactor-dependent) antibodies may also be relevant in other antibody-mediated autoimmune disorders.
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quantitative description of thermodynamic and kinetic properties of the Platelet Factor 4 heparin bonds
Nanoscale, 2015Co-Authors: Thihuong Nguyen, Andreas Greinacher, Mihaela DelceaAbstract:Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein Platelet Factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly understood. In this study, single molecule force spectroscopy (SMFS) is utilized to characterize the interaction of PF4 with heparins of defined length (5-, 6-, 8-, 12-, and 16-mers). Analysis of the force–distance curves shows that PF4/heparin binding strength rises with increasing heparin length. In addition, two binding pathways in the PF4/short heparins (≤8-mers) and three binding pathways in the PF4/long heparins (≥8-mers) are identified. We provide a model for the PF4/heparin complexes in which short heparins bind to one PF4 tetramer, while long heparins bind to two PF4 tetramers. We propose that the interaction between long heparins and PF4s is not only due to charge differences as generally assumed, but also due to hydrophobic interaction between two PF4s which are brought close to each other by long heparin. This complicated interaction induces PF4/heparin complexes more stable than other ligand–receptor interactions. Our results also reveal that the boundary between antigenic and non-antigenic heparins is between 8- and 12-mers. These observations are particularly important to understand processes in which PF4–heparin interactions are involved and to develop new heparin-derived drugs.
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binding of anti Platelet Factor 4 heparin antibodies depends on the thermodynamics of conformational changes in Platelet Factor 4
Blood, 2014Co-Authors: Martin Kreimann, Sven Brandt, Andreas Greinacher, Krystin Krauel, Stephan Block, Christiane A Helm, Werner Weitschies, Mihaela DelceaAbstract:The chemokine Platelet Factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia (HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 antiparallel β-sheets exceeding ∼30% (achieved by UFH, LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic.
Douglas B Cines - One of the best experts on this subject based on the ideXlab platform.
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polyphosphate Platelet Factor 4 complexes can mediate heparin independent Platelet activation in heparin induced thrombocytopenia
Blood Advances, 2016Co-Authors: Douglas B Cines, Gowthami M Arepally, Mortimer Poncz, Lubica Rauova, Serge Yarovoi, Sanjay Khandelwal, Sergei Zaitsev, Tatiana Lebedeva, Victoria Stepanova, Ann H RuxAbstract:Heparin-induced thrombocytopenia (HIT) is a thrombotic disorder initiated by antibodies to complexes between Platelet Factor 4 (PF4) and heparin. The risk of recurrent thromboembolism persists after heparin is cleared and Platelet activation leading to release of PF4 has dissipated. We asked whether antigenic complexes between polyphosphates and PF4 released from activated Platelets might intensify or sustain the prothrombotic phenotype of HIT. PF4 forms stable, ultralarge complexes with polyphosphates of various sizes, including those released from Platelets, which are recognized by the HIT-like monoclonal KKO, an immunoglobulin G2bκ monoclonal heparin/PF4 binding antibody, and by human HIT antibodies. KKO helps to protect PF4/polyphosphate complexes from degradation by phosphatases. Complement is activated when HIT antibodies bind to PF4/polyphosphate complexes and PF4 reverses the inhibition of complement by polyphosphates. Polyphosphates and PF4 are stored primarily in separate granules in resting Platelets, but they colocalize when the cells are activated. Platelets activated by subaggregating doses of thrombin receptor activating peptide release polyphosphates and PF4, which form antigenic complexes that allow KKO to further activate Platelets in the absence of heparin and exogenous PF4. These studies suggest that thrombin- or immune complex-mediated release of endogenous antigenic PF4/polyphosphate complexes from Platelets may augment the prothrombotic risk of HIT and perpetuate the risk of thrombosis after heparin has been discontinued.
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heparin enhances uptake of Platelet Factor 4 heparin complexes by monocytes and macrophages
Journal of Thrombosis and Haemostasis, 2015Co-Authors: Manali Joglekar, Mortimer Poncz, Lubica Rauova, Douglas B Cines, Sanjay Khandelwal, Gowthami M ArepallyAbstract:Summary Background Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies to a self-antigen, Platelet Factor (4) and heparin. The reasons why antibodies form to PF4/heparin, but not to PF4 bound to other cellular glycosaminoglycans are poorly understood. Objective To investigate differences in cellular responses to cell-bound PF4 and PF4/heparin complexes, we studied the internalization of each by peripheral blood-derived monocytes, dendritic cells and neutrophils. Methods and results Using unlabeled and fluorescently-labeled antigen and/or labeled monoclonal antibody to PF4/heparin complexes (KKO), we show that PF4/heparin complexes are taken up by monocytes in a heparin-dependent manner and are internalized by human monocytes and dendritic cells, but not by neutrophils. Complexes of PF4/low-molecular-weight heparin and complexes composed of heparin and murine PF4, protamine or lysozyme are internalized similarly, suggesting a common endocytic pathway. Uptake of complexes is mediated by macropinocytosis, as shown by inhibition using cytochalasin D and amiloride. Internalized complexes are transported intact to late endosomes, as indicated by co-staining of vesicles with KKO and lysosomal associated membrane protein-2 (LAMP-2). Lastly, we show that cellular uptake is accompanied by expression of MHCII and CD83 co-stimulatory molecules. Conclusions Taken together, these studies establish a distinct role for heparin in enhancing antigen uptake and activation of the initial steps in the cellular immune response to PF4-containing complexes.
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distinct specificity and single molecule kinetics characterize the interaction of pathogenic and non pathogenic antibodies against Platelet Factor 4 heparin complexes with Platelet Factor 4
Journal of Biological Chemistry, 2013Co-Authors: Rustem I Litvinov, Gowthami M Arepally, Lubica Rauova, Serge Yarovoi, Valeri Barsegov, Bruce S Sachais, Ann H Rux, Jillian L Hinds, Douglas B Cines, John W WeiselAbstract:Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy mediated by antibodies to complexes between Platelet Factor 4 (PF4) and heparin or cellular glycosaminoglycans. However, only a fraction of patients with anti-PF4-heparin antibodies develop HIT, implying that only a subset of these antibodies is pathogenic. The basis for the pathogenic potential of anti-PF4-heparin antibodies remains unclear. To elucidate the intrinsic PF4-binding properties of HIT-like monoclonal antibody (KKO) versus non-pathogenic antibody (RTO) at the single-molecule level, we utilized optical trap-based force spectroscopy to measure the strength and probability of binding of surface-attached antibodies with oligomeric PF4 to simulate interactions on cells. To mimic the effect of heparin in bringing PF4 complexes into proximity, we chemically cross-linked PF4 tetramers using glutaraldehyde. Analysis of the force histograms revealed that KKO-PF4 interactions had ∼10-fold faster on-rates than RTO-PF4, and apparent equilibrium dissociation constants differed ∼10-fold with similar force-free off-rates (koff = 0.0031 and 0.0029 s−1). Qualitatively similar results were obtained for KKO and RTO interacting with PF4-heparin complexes. In contrast to WT PF4, KKO and RTO showed lower and similar binding probabilities to cross-linked PF4K50E, which forms few if any oligomers. Thus, formation of stable PF4 polymers results in much stronger interactions with the pathogenic antibody without a significant effect on the binding of the non-pathogenic antibody. These results suggest a fundamental difference in the antigen-binding mechanisms between model pathogenic and non-pathogenic anti-PF4 antibodies that might underlie their distinct pathophysiological behaviors.
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rational design and characterization of Platelet Factor 4 antagonists for the study of heparin induced thrombocytopenia
Blood, 2012Co-Authors: Bruce S Sachais, Serge Yarovoi, Ann H Rux, Jillian L Hinds, Douglas B Cines, Lee I Garner, Stephen P Watson, John J RuxAbstract:Patients with heparin-induced thrombocytopenia (HIT) remain at risk for recurrent thromboembolic complications despite improvements in management. HIT is caused by antibodies that preferentially recognize ultralarge complexes (ULCs) of heparin and Platelet Factor 4 (PF4) tetramers. We demonstrated previously that a variant PF4K50E forms dimers but does not tetramerize or form ULCs. Here, we identified small molecules predicted to bind PF4 near the dimer-dimer interface and that interfere with PF4 tetramerization. Screening a library of small molecules in silico for binding at this site, we identified 4 compounds that inhibited tetramerization at micromolar concentrations, designated PF4 antagonists (PF4As). PF4As also inhibited formation of pathogenic ULCs, and 3 of these PF4As promoted the breakdown of preformed ULCs. To characterize the ability of PF4As to inhibit cellular activation, we developed a robust and reproducible assay that measures cellular activation by HIT antibodies via FcγRIIA using DT40 cells. PF4As inhibit FcγRIIA-dependent activation of DT40 cells by HIT antibodies as well as Platelet activation, as measured by serotonin release. PF4As provide new tools to probe the pathophysiology of HIT. They also may provide insight into the development of novel, disease-specific therapeutics for the treatment of thromboembolic complications in HIT.
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Platelet Factor 4 enhances the binding of oxidized low density lipoprotein to vascular wall cells
Journal of Biological Chemistry, 2003Co-Authors: Taher Nassar, Bruce S Sachais, Saed Akkawi, Maria Anna Kowalska, Khalil Bdeir, Eran Leitersdorf, Edna Hiss, Leah Ziporen, Michael Aviram, Douglas B CinesAbstract:Accumulation of low-density lipoprotein (LDL)-derived cholesterol by macrophages in vessel walls is a pathogenomic feature of atherosclerotic lesions. Platelets contribute to lipid uptake by macrophages through mechanisms that are only partially understood. We have previously shown that Platelet Factor 4 (PF4) inhibits the binding and degradation of LDL through its receptor, a process that could promote the formation of oxidized LDL (ox-LDL). We have now characterized the effect of PF4 on the binding of ox-LDL to vascular cells and macrophages and on the accumulation of cholesterol esters. PF4 bound to ox-LDL directly and also increased ox-LDL binding to vascular cells and macrophages. PF4 did not stimulate ox-LDL binding to cells that do not synthesize glycosaminoglycans or after enzymatic cleavage of cell surface heparan and chondroitin sulfates. The effect of PF4 on binding ox-LDL was dependent on specific lysine residues in its C terminus. Addition of PF4 also caused an ∼10-fold increase in the amount of ox-LDL esterified by macrophages. Furthermore, PF4 and ox-LDL co-localize in atherosclerotic lesion, especially in macrophage-derived foam cells. These observations offer a potential mechanism by which Platelet activation at sites of vascular injury may promote the accumulation of deleterious lipoproteins and offer a new focus for pharmacological intervention in the development of atherosclerosis.
Gowthami M Arepally - One of the best experts on this subject based on the ideXlab platform.
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polyphosphate Platelet Factor 4 complexes can mediate heparin independent Platelet activation in heparin induced thrombocytopenia
Blood Advances, 2016Co-Authors: Douglas B Cines, Gowthami M Arepally, Mortimer Poncz, Lubica Rauova, Serge Yarovoi, Sanjay Khandelwal, Sergei Zaitsev, Tatiana Lebedeva, Victoria Stepanova, Ann H RuxAbstract:Heparin-induced thrombocytopenia (HIT) is a thrombotic disorder initiated by antibodies to complexes between Platelet Factor 4 (PF4) and heparin. The risk of recurrent thromboembolism persists after heparin is cleared and Platelet activation leading to release of PF4 has dissipated. We asked whether antigenic complexes between polyphosphates and PF4 released from activated Platelets might intensify or sustain the prothrombotic phenotype of HIT. PF4 forms stable, ultralarge complexes with polyphosphates of various sizes, including those released from Platelets, which are recognized by the HIT-like monoclonal KKO, an immunoglobulin G2bκ monoclonal heparin/PF4 binding antibody, and by human HIT antibodies. KKO helps to protect PF4/polyphosphate complexes from degradation by phosphatases. Complement is activated when HIT antibodies bind to PF4/polyphosphate complexes and PF4 reverses the inhibition of complement by polyphosphates. Polyphosphates and PF4 are stored primarily in separate granules in resting Platelets, but they colocalize when the cells are activated. Platelets activated by subaggregating doses of thrombin receptor activating peptide release polyphosphates and PF4, which form antigenic complexes that allow KKO to further activate Platelets in the absence of heparin and exogenous PF4. These studies suggest that thrombin- or immune complex-mediated release of endogenous antigenic PF4/polyphosphate complexes from Platelets may augment the prothrombotic risk of HIT and perpetuate the risk of thrombosis after heparin has been discontinued.
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heparin enhances uptake of Platelet Factor 4 heparin complexes by monocytes and macrophages
Journal of Thrombosis and Haemostasis, 2015Co-Authors: Manali Joglekar, Mortimer Poncz, Lubica Rauova, Douglas B Cines, Sanjay Khandelwal, Gowthami M ArepallyAbstract:Summary Background Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies to a self-antigen, Platelet Factor (4) and heparin. The reasons why antibodies form to PF4/heparin, but not to PF4 bound to other cellular glycosaminoglycans are poorly understood. Objective To investigate differences in cellular responses to cell-bound PF4 and PF4/heparin complexes, we studied the internalization of each by peripheral blood-derived monocytes, dendritic cells and neutrophils. Methods and results Using unlabeled and fluorescently-labeled antigen and/or labeled monoclonal antibody to PF4/heparin complexes (KKO), we show that PF4/heparin complexes are taken up by monocytes in a heparin-dependent manner and are internalized by human monocytes and dendritic cells, but not by neutrophils. Complexes of PF4/low-molecular-weight heparin and complexes composed of heparin and murine PF4, protamine or lysozyme are internalized similarly, suggesting a common endocytic pathway. Uptake of complexes is mediated by macropinocytosis, as shown by inhibition using cytochalasin D and amiloride. Internalized complexes are transported intact to late endosomes, as indicated by co-staining of vesicles with KKO and lysosomal associated membrane protein-2 (LAMP-2). Lastly, we show that cellular uptake is accompanied by expression of MHCII and CD83 co-stimulatory molecules. Conclusions Taken together, these studies establish a distinct role for heparin in enhancing antigen uptake and activation of the initial steps in the cellular immune response to PF4-containing complexes.
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distinct specificity and single molecule kinetics characterize the interaction of pathogenic and non pathogenic antibodies against Platelet Factor 4 heparin complexes with Platelet Factor 4
Journal of Biological Chemistry, 2013Co-Authors: Rustem I Litvinov, Gowthami M Arepally, Lubica Rauova, Serge Yarovoi, Valeri Barsegov, Bruce S Sachais, Ann H Rux, Jillian L Hinds, Douglas B Cines, John W WeiselAbstract:Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy mediated by antibodies to complexes between Platelet Factor 4 (PF4) and heparin or cellular glycosaminoglycans. However, only a fraction of patients with anti-PF4-heparin antibodies develop HIT, implying that only a subset of these antibodies is pathogenic. The basis for the pathogenic potential of anti-PF4-heparin antibodies remains unclear. To elucidate the intrinsic PF4-binding properties of HIT-like monoclonal antibody (KKO) versus non-pathogenic antibody (RTO) at the single-molecule level, we utilized optical trap-based force spectroscopy to measure the strength and probability of binding of surface-attached antibodies with oligomeric PF4 to simulate interactions on cells. To mimic the effect of heparin in bringing PF4 complexes into proximity, we chemically cross-linked PF4 tetramers using glutaraldehyde. Analysis of the force histograms revealed that KKO-PF4 interactions had ∼10-fold faster on-rates than RTO-PF4, and apparent equilibrium dissociation constants differed ∼10-fold with similar force-free off-rates (koff = 0.0031 and 0.0029 s−1). Qualitatively similar results were obtained for KKO and RTO interacting with PF4-heparin complexes. In contrast to WT PF4, KKO and RTO showed lower and similar binding probabilities to cross-linked PF4K50E, which forms few if any oligomers. Thus, formation of stable PF4 polymers results in much stronger interactions with the pathogenic antibody without a significant effect on the binding of the non-pathogenic antibody. These results suggest a fundamental difference in the antigen-binding mechanisms between model pathogenic and non-pathogenic anti-PF4 antibodies that might underlie their distinct pathophysiological behaviors.
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Platelet Factor 4 heparin antibodies in blood bank donors
American Journal of Clinical Pathology, 2010Co-Authors: Marcie J Hursting, Poulomi J Pai, Julianna E Mccracken, Fred Hwang, Shayela Suvarna, Yuliya Lokhnygina, Nicholas Bandarenko, Gowthami M ArepallyAbstract:Platelet Factor 4 (PF4)/heparin antibody, typically associated with heparin therapy, is reported in some heparin-naive people. Seroprevalence in the general population, however, remains unclear. We prospectively evaluated PF4/heparin antibody in approximately 4,000 blood bank donors using a commercial enzyme-linked immunosorbent assay for initial and then repeated (confirmatory) testing. Antibody was detected initially in 249 (6.6%; 95% confidence interval [CI], 5.8%-7.4%) of 3,795 donors and repeatedly in 163 (4.3%; 95% CI, 3.7%-5.0%) of 3,789 evaluable donors. “Unconfirmed” positives were mostly (93%) low positives (optical density [OD] = 0.40-0.59). Of 163 repeatedly positive samples, 116 (71.2%) were low positives, and 124 (76.1%) exhibited heparin-dependent binding. Predominant isotypes of intermediate to high seropositive samples (OD >0.6) were IgG (20/39 [51%]), IgM (9/39 [23%]), and indeterminate (10/39 [26%]). The marked background seroprevalence of PF4/heparin antibody (4.3%-6.6%) with the preponderance of low (and frequently nonreproducible) positives in blood donors suggests the need for further assay calibration, categorization of antibody level, and studies evaluating clinical relevance of “naturally occurring” PF4/heparin antibodies.
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Platelet Factor 4 heparin antibody igg m a in healthy subjects a literature analysis of commercial immunoassay results
Journal of Thrombosis and Thrombolysis, 2008Co-Authors: Gowthami M Arepally, Marcie J HurstingAbstract:Purpose We determined the seroprevalence of Platelet Factor 4 (PF4)/heparin antibodies in healthy subjects. Methods A literature search identified studies in which healthy subjects were evaluated using commercial immunoassays for PF4/heparin antibody (IgG/M/A). Proportions of test-positive subjects were calculated, by assay. Results Across 11 eligible studies, 860 healthy subjects were tested using the Stago enzyme-linked immunosorbent assay (ELISA) (nine studies), GTI ELISA (three studies), and/or DiaMed particle gel immunoassay (PGIA) (three studies). Seropositivity occurred in 17 of 790 (2.2%, 95% CI, 1.1–3.2%) subjects by Stago ELISA, one of 100 (1.0%, 95% CI, 0–3.0%) subjects by GTI ELISA, and three of 70 (4.3%, 95% CI, 0–9.0%) subjects by PGIA (P > 0.20). Of seven seropositive subjects tested further, none had Platelet-activating antibodies. Conclusion Commercial immunoassays detect PF4/heparin antibody in 1.0–4.3% of healthy subjects. Because this “background” prevalence overlaps seropositivity rates in heparin-treated patients in various clinical settings, normality cut-offs may require refinement.
Bruce S Sachais - One of the best experts on this subject based on the ideXlab platform.
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distinct specificity and single molecule kinetics characterize the interaction of pathogenic and non pathogenic antibodies against Platelet Factor 4 heparin complexes with Platelet Factor 4
Journal of Biological Chemistry, 2013Co-Authors: Rustem I Litvinov, Gowthami M Arepally, Lubica Rauova, Serge Yarovoi, Valeri Barsegov, Bruce S Sachais, Ann H Rux, Jillian L Hinds, Douglas B Cines, John W WeiselAbstract:Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy mediated by antibodies to complexes between Platelet Factor 4 (PF4) and heparin or cellular glycosaminoglycans. However, only a fraction of patients with anti-PF4-heparin antibodies develop HIT, implying that only a subset of these antibodies is pathogenic. The basis for the pathogenic potential of anti-PF4-heparin antibodies remains unclear. To elucidate the intrinsic PF4-binding properties of HIT-like monoclonal antibody (KKO) versus non-pathogenic antibody (RTO) at the single-molecule level, we utilized optical trap-based force spectroscopy to measure the strength and probability of binding of surface-attached antibodies with oligomeric PF4 to simulate interactions on cells. To mimic the effect of heparin in bringing PF4 complexes into proximity, we chemically cross-linked PF4 tetramers using glutaraldehyde. Analysis of the force histograms revealed that KKO-PF4 interactions had ∼10-fold faster on-rates than RTO-PF4, and apparent equilibrium dissociation constants differed ∼10-fold with similar force-free off-rates (koff = 0.0031 and 0.0029 s−1). Qualitatively similar results were obtained for KKO and RTO interacting with PF4-heparin complexes. In contrast to WT PF4, KKO and RTO showed lower and similar binding probabilities to cross-linked PF4K50E, which forms few if any oligomers. Thus, formation of stable PF4 polymers results in much stronger interactions with the pathogenic antibody without a significant effect on the binding of the non-pathogenic antibody. These results suggest a fundamental difference in the antigen-binding mechanisms between model pathogenic and non-pathogenic anti-PF4 antibodies that might underlie their distinct pathophysiological behaviors.
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rational design and characterization of Platelet Factor 4 antagonists for the study of heparin induced thrombocytopenia
Blood, 2012Co-Authors: Bruce S Sachais, Serge Yarovoi, Ann H Rux, Jillian L Hinds, Douglas B Cines, Lee I Garner, Stephen P Watson, John J RuxAbstract:Patients with heparin-induced thrombocytopenia (HIT) remain at risk for recurrent thromboembolic complications despite improvements in management. HIT is caused by antibodies that preferentially recognize ultralarge complexes (ULCs) of heparin and Platelet Factor 4 (PF4) tetramers. We demonstrated previously that a variant PF4K50E forms dimers but does not tetramerize or form ULCs. Here, we identified small molecules predicted to bind PF4 near the dimer-dimer interface and that interfere with PF4 tetramerization. Screening a library of small molecules in silico for binding at this site, we identified 4 compounds that inhibited tetramerization at micromolar concentrations, designated PF4 antagonists (PF4As). PF4As also inhibited formation of pathogenic ULCs, and 3 of these PF4As promoted the breakdown of preformed ULCs. To characterize the ability of PF4As to inhibit cellular activation, we developed a robust and reproducible assay that measures cellular activation by HIT antibodies via FcγRIIA using DT40 cells. PF4As inhibit FcγRIIA-dependent activation of DT40 cells by HIT antibodies as well as Platelet activation, as measured by serotonin release. PF4As provide new tools to probe the pathophysiology of HIT. They also may provide insight into the development of novel, disease-specific therapeutics for the treatment of thromboembolic complications in HIT.
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elimination of Platelet Factor 4 pf4 from Platelets reduces atherosclerosis in c57bl 6 and apoe mice
Thrombosis and Haemostasis, 2007Co-Authors: Bruce S Sachais, T Turrentine, A H Rux, D Rader, M A KowalskaAbstract:Activated 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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Platelet Factor 4 enhances the binding of oxidized low density lipoprotein to vascular wall cells
Journal of Biological Chemistry, 2003Co-Authors: Taher Nassar, Bruce S Sachais, Saed Akkawi, Maria Anna Kowalska, Khalil Bdeir, Eran Leitersdorf, Edna Hiss, Leah Ziporen, Michael Aviram, Douglas B CinesAbstract:Accumulation of low-density lipoprotein (LDL)-derived cholesterol by macrophages in vessel walls is a pathogenomic feature of atherosclerotic lesions. Platelets contribute to lipid uptake by macrophages through mechanisms that are only partially understood. We have previously shown that Platelet Factor 4 (PF4) inhibits the binding and degradation of LDL through its receptor, a process that could promote the formation of oxidized LDL (ox-LDL). We have now characterized the effect of PF4 on the binding of ox-LDL to vascular cells and macrophages and on the accumulation of cholesterol esters. PF4 bound to ox-LDL directly and also increased ox-LDL binding to vascular cells and macrophages. PF4 did not stimulate ox-LDL binding to cells that do not synthesize glycosaminoglycans or after enzymatic cleavage of cell surface heparan and chondroitin sulfates. The effect of PF4 on binding ox-LDL was dependent on specific lysine residues in its C terminus. Addition of PF4 also caused an ∼10-fold increase in the amount of ox-LDL esterified by macrophages. Furthermore, PF4 and ox-LDL co-localize in atherosclerotic lesion, especially in macrophage-derived foam cells. These observations offer a potential mechanism by which Platelet activation at sites of vascular injury may promote the accumulation of deleterious lipoproteins and offer a new focus for pharmacological intervention in the development of atherosclerosis.
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heparin induced thrombocytopenia thrombosis in a transgenic mouse model requires human Platelet Factor 4 and Platelet activation through fcγriia
Blood, 2001Co-Authors: Michael P Reilly, Gowthami M Arepally, Bruce S Sachais, Scott M Taylor, Nealie K Hartman, Douglas B CinesAbstract:Heparin-induced thrombocytopenia/thrombosis (HIT/HITT) is a severe, life-threatening complication that occurs in 1% to 3% of patients exposed to heparin. Interactions between heparin, human Platelet Factor 4 (hPF4), antibodies to the hPF4/heparin complex, and the Platelet Fc receptor (FcR) for immunoglobulin G, FcγRIIA, are the proposed primary determinants of the disease on the basis of in vitro studies. The goal of this study was to create a mouse model that recapitulates the disease process in humans in order to understand the Factors that predispose some patients to develop thrombocytopenia and thrombosis and to investigate new therapeutic approaches. Mice that express both human Platelet FcγRIIA and hPF4 were generated. The FcγRIIA/hPF4 mice and controls, transgenic for either FcγRIIA or hPF4, were injected with KKO, a mouse monoclonal antibody specific for hPF4/heparin complexes, and then received heparin (20 U/d). Nadir Platelet counts for KKO/heparin–treated FcγRIIA/hPF4 mice were 80% below baseline values, significantly different ( P