Binding of heparin to human platelet factor 4.
Ontology highlight
ABSTRACT:
SUBMITTER: Cowan SW
PROVIDER: S-EPMC1146592 | biostudies-other | 1986 Mar
REPOSITORIES: biostudies-other
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Project description:Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.
| S-EPMC1154112 | biostudies-other
Project description: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 G2b? 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.
| S-EPMC5744055 | biostudies-literature
Project description:Heparin (HP) is a polysaccharide that is widely used in the clinic as an anticoagulant. A major side effect associated with HP is the heparin-induced thrombocytopenia (HIT), which is initiated by the immune response to complex formed by HP and platelet factor 4 (PF4). Low molecular weight heparins (LMWHs) are the depolymerized version of HP, which have reduced risks of inducing HIT. However, it is still necessary to evaluate the immunogenicity of LMWHs to ensure their drug safety. Since HIT involves very complicated processes, the evaluation of HP and LMWH immunogenicity requires experiments from multiple aspects, of which the binding affinity between HP and PF4 is a key property to be monitored. Herein, we developed a novel competitive biolayer interferometry (BLI) method to investigate the binding affinity between HP and PF4. The influence of different domains in HP on its immunogenicity was compared for better understanding of the molecular mechanism of HP immunogenicity. Furthermore, the half maximal inhibitory concentration (IC50) of HP and LMWH can be measured by competitive combination, which is important for the quality control during the developing and manufacturing of HP and LMWH drugs.
| S-EPMC9458964 | biostudies-literature
Project description:BackgroundHeparin-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.ObjectiveTo 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 resultsUsing 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.ConclusionsTaken 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.
| S-EPMC4516590 | biostudies-literature
Project description:Heparin, a widely used anticoagulant, carries the risk of an antibody-mediated adverse drug reaction, heparin-induced thrombocytopenia (HIT). A subset of heparin-treated patients produces detectable levels of antibodies against complexes of heparin bound to circulating platelet factor 4 (PF4). Using a genome-wide association study (GWAS) approach, we aimed to identify genetic variants associated with anti-PF4/heparin antibodies that account for the variable antibody response seen in HIT. We performed a GWAS on anti-PF4/heparin antibody levels determined via polyclonal enzyme-linked immunosorbent assays. Our discovery cohort (n = 4237) and replication cohort (n = 807) constituted patients with European ancestry and clinical suspicion of HIT, with cases confirmed via functional assay. Genome-wide significance was considered at α = 5 × 10-8. No variants were significantly associated with anti-PF4/heparin antibody levels in the discovery cohort at a genome-wide significant level. Secondary GWAS analyses included the identification of variants with suggestive associations in the discovery cohort (α = 1 × 10-4). The top variant in both cohorts was rs1555175145 (discovery β = -0.112 [0.018], P = 2.50 × 10-5; replication β = -0.104 [0.051], P = .041). In gene set enrichment analysis, 3 gene sets reached false discovery rate-adjusted significance (q < 0.05) in both discovery and replication cohorts: "Leukocyte Transendothelial Migration," "Innate Immune Response," and "Lyase Activity." Our results indicate that genomic variation is not significantly associated with anti-PF4/heparin antibody levels. Given our power to identify variants with moderate frequencies and effect sizes, this evidence suggests genetic variation is not a primary driver of variable antibody response in heparin-treated patients with European ancestry.
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Project description:A major limitation in biopharmaceutical development is selectively targeting drugs to diseased tissues. Growth factors and viruses have solved this problem by targeting tissue-specific cell-surface heparan sulfates. Neuregulin (NRG), a growth factor important in both nervous system development and cancer, has a unique heparin-binding domain (HBD) that targets to cell surfaces expressing its HER2/3/4 receptors (Esper, R. M., Pankonin, M. S., and Loeb, J. A. (2006) Brain Res. Rev. 51, 161-175). We have harnessed this natural targeting ability of NRG by fusing the HBD of NRG to soluble HER4. This fusion protein retains high affinity heparin binding to heparin and to cells that express heparan sulfates resulting in a more potent NRG antagonist. In vivo, it is targeted to peripheral nerve segments where it blocks the activity of NRG as a Schwann cell survival factor. The fusion protein also efficiently blocks autocrine and paracrine signaling and reduces the proliferation of MCF10CA1 breast cancer cells. These findings demonstrate the utility of the HBD of NRG in biopharmaceutical targeting and provide a new way to block HER signaling in cancer cells.
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Project description:We have shown that autocrine proliferation of human keratinocytes (KCs) is strongly dependent upon amphiregulin (AREG), whereas blockade of heparin-binding EGF-like growth factor (HB-EGF) inhibits KC migration in scratch wound assays. Here we demonstrate that expression of soluble HB-EGF (sHB-EGF) or full-length transmembrane HB-EGF (proHB-EGF), but not proAREG, results in profound increases in KC migration and invasiveness in monolayer culture. Coincident with these changes, HB-EGF significantly decreases mRNA expression of several epithelial markers including keratins 1, 5, 10, and 14 while increasing expression of markers of cellular motility including SNAI1, ZEB1, COX-2, and MMP1. Immunostaining revealed HB-EGF-induced expression of the mesenchymal protein vimentin and decreased expression of E-cadherin, as well as nuclear translocation of ?-catenin. Suggestive of a trade-off between KC motility and proliferation, overexpression of HB-EGF also reduced KC growth by >90%. We also show that HB-EGF is strongly induced in regenerating epidermis after partial-thickness wounding of human skin. Taken together, our data suggest that expression of HB-EGF in human KCs triggers a migratory and invasive phenotype with many features of epithelial-mesenchymal transition (EMT), which may be beneficial in the context of cutaneous wound healing.
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Project description:Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin leading to a reduction in circulating platelets with an increased risk of thrombosis. It is precipitated by polymerized immune complexes consisting of pathogenic antibodies that recognize a small chemokine platelet factor 4 (PF4) bound to heparin. Characterization of these immune complexes is extremely challenging due to the enormous structural heterogeneity of such macromolecular assemblies and their constituents. Native mass spectrometry demonstrates that up to three PF4 tetramers can be assembled on a heparin chain, consistent with the molecular modeling studies showing facile polyanion wrapping along the polycationic belt on the PF4 surface. Although these assemblies can accommodate a maximum of only two antibodies, the resulting immune complexes are capable of platelet activation despite their modest size. Taken together, these studies provide further insight into molecular mechanisms of HIT and other immune disorders where anti-PF4 antibodies play a central role.
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