scholarly journals Platelet Factor 4 Inhibits and Enhances HIV-1 Infection in a Concentration-Dependent Manner by Modulating Viral Attachment

2016 ◽  
Vol 32 (7) ◽  
pp. 705-717 ◽  
Author(s):  
Zahra F. Parker ◽  
Ann H. Rux ◽  
Amber M. Riblett ◽  
Fang-Hua Lee ◽  
Lubica Rauova ◽  
...  
Keyword(s):  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Platelet Factor ◽  
Viral Attachment ◽  
Hiv 1

Inhibition Of Contact Activation By Platelet Factor 4

1981 ◽  
Author(s):  
K Weerasinghe ◽  
M F Scully ◽  
V V Kakkar
Keyword(s):  
Platelet Factor 4 ◽  
Molar Ratio ◽  
Factor Xii ◽  
Dependent Manner ◽  
Dextran Sulphate ◽  
Contact Activation ◽  
Concentration Dependent Manner ◽  
Platelet Factor ◽  
Dependent Inhibition ◽  
Repeated Freezing

The contact activation of FXII by dextran sulphate (DS, MW 500,000) has been shown to be inhibited by platelet factor 4 (PF4). Plasma was prepared from blood taken with EDTA, theophylline and PGE1. Contact activation was initiated by the addition of an equal volume of DS (10μg/ml) and incubating at 0° (8 min) or 37° (2 min). The kallikrein generated was measured using S2302. Addition of purified PF4 to plasma caused a concentration dependent inhibition with complete inhibition at greater than 2.0μg/PF4/ml plasma (molar ratio of 20:1). PF4 was also observed in a concentration dependent manner to inhibit clotting of plasma as initiated by dextran sulphate.Repeated freezing of human PRP (Av. platelet count=3.55 × 108/ml) released PF4 (as measured by radioimmunoassay) and caused inhibition of contact activation. By dilution with PPP, the degree of inhibition was found to be related to the concentration of PF4. The amount needed for 50% inhibition varied from 0.2-1.5μg/ml in different donors. Platelet aggregation with epinephrine (2μg/ml) released 4.8μg/ml (±1.7) of PF4 and showed 55% (±22) inhibition of contact activation (n=ll). Collagen (5.7μg/ml) caused 4.9μg/ml (±1.3) of PF4 to be released, but the degree of inhibition varied (n=13). The discrepancy between this and the freezing experiment may be due to the “contact-product activator” properties of collagen and the intact platelet membrane. Control experiments performed using indomethacin or EDTA-PRP showed no release of PF4 or inhibition of contact activation.The effect observed appears to be related directly to the ability of PF4 to bind to negatively charged polysaccharides since PF4 was not found to inhibit activated factor XII or kallikrein. These results indicate another role for PF4 as an inhibitor of contact activation. Together with its known antiheparin properties it indicates that PF4 may be closely involved in the control of haemostasis.

scholarly journals Platelet Factor 4 Induces Leukocyte Responses through Integrin Mac-1 (CD11b/CD18)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2529-2529
Author(s):  
Nataly P. Podolnikova ◽  
Valentin P. Yakubenko ◽  
Tatiana P. Ugarova
Keyword(s):  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Structural Similarity ◽  
Peptide Library ◽  
Platelet Factor 4 ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner ◽  
Platelet Factor

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.

The Platelet Specific Chemokine Platelet Factor 4 Induces Expression of E-Selectin in an NF-κ B Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3908-3908
Author(s):  
Bruce S. Sachais ◽  
Peihong Ma ◽  
Ann H. Rux ◽  
Guangyao Yu
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Atherosclerotic Lesion ◽  
Surface Expression ◽  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Platelet Factor ◽  
Umbilical Vein Endothelial Cells ◽  
Electrophoretic Mobility Shift Assays

Abstract The involvement of platelets in the pathogenesis of atherosclerosis has recently gained much attention. Platelet factor 4 (PF4) is a platelet specific chemokine released upon platelet activation. PF4 has been localized to atherosclerotic lesions, including macrophages and endothelium. In this report, we demonstrate that E-selectin, an adhesion molecule involved in atherogenesis, is up-regulated in human umbilical vein endothelial cells exposed to PF4. Induction of E-selectin mRNA is time and dose dependent, and requires the presence of cell surface glycosaminoglycans. Surface expression of E-selectin, as measured by flow cytometry, is also increased by PF4. Activation of NF-κB is critical for PF4 induced E-selectin expression, as demonstrated by promoter activation studies and electrophoretic mobility shift assays. In summary, our data demonstrate that PF4 can increase expression of E-selectin by endothelial cells by activation of NF-κB. PF4 induction of endothelial E-selectin expression represents another mechanism by which platelets may participate in atherosclerotic lesion progression. These data also suggest that PF4 may participate in the proinflammatory functions of activated platelets.

Anti-Platelet Factor 4/Heparin Antibodies in Patients with Gram Negative Bacteremia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3391-3391
Author(s):  
Georgios Pongas ◽  
Swapan Dasgupta ◽  
Perumal Thiagarajan
Keyword(s):  
Conflicts Of Interest ◽  
Hospital Setting ◽  
Fluorescence Emission ◽  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Fluorescence Emission Spectrum ◽  
Dependent Manner ◽  
Platelet Factor ◽  
Gram Negative ◽  
Normal Controls

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.

scholarly journals Endothelial expression of E-selectin is induced by the platelet-specific chemokine platelet factor 4 through LRP in an NF-κB–dependent manner

Blood ◽  
2005 ◽  
Vol 105 (9) ◽  
pp. 3545-3551 ◽  
Author(s):  
Guangyao Yu ◽  
Ann H. Rux ◽  
Peihong Ma ◽  
Khalil Bdeir ◽  
Bruce S. Sachais
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Atherosclerotic Lesion ◽  
Density Lipoprotein ◽  
Nuclear Factor Κb ◽  
Platelet Factor 4 ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Platelet Factor

AbstractThe involvement of platelets in the pathogenesis of atherosclerosis has recently gained much attention. Platelet factor 4 (PF4), a platelet-specific chemokine released on platelet activation, has been localized to atherosclerotic lesions, including macrophages and endothelium. In this report, we demonstrate that E-selectin, an adhesion molecule involved in atherogenesis, is up-regulated in human umbilical vein endothelial cells exposed to PF4. Induction of E-selectin RNA is time and dose dependent. Surface expression of E-selectin, as measured by flow cytometry, is also increased by PF4. PF4 induces E-selectin expression by activation of transcriptional activity. Activation of nuclear factor-κB is critical for PF4-induced E-selectin expression, as demonstrated by promoter activation studies and electrophoretic mobility shift assays. Further, we have identified the low-density lipoprotein receptor-related protein as the cell surface receptor mediating this effect. These results demonstrate that PF4 is able to increase expression of E-selectin by endothelial cells and represents another potential mechanism by which platelets may participate in atherosclerotic lesion progression.

scholarly journals Studies of thromboxane B2, platelet factor 4, and fibrinopeptide A in bleeding-time blood of patients deficient in von Willebrand factor, platelet glycoproteins Ib and IIb-IIIa, and storage granules

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 481-490 ◽  
Author(s):  
HJ Weiss ◽  
B Lages
Keyword(s):  
Bleeding Time ◽  
Platelet Factor 4 ◽  
Thromboxane B2 ◽  
Initial Slope ◽  
Dependent Manner ◽  
High Shear ◽  
Fibrinopeptide A ◽  
Wound Surface ◽  
Platelet Factor ◽  
Fibrin Formation

The blood volumes and concentrations of thromboxane B2 (TxB2), platelet factor 4 (PF4), and fibrinopeptide A (FPA) were measured every 30 seconds in bleeding-time blood in normal subjects and in patients with idiopathic thrombocytopenic purpura (ITP), delta and alpha delta storage pool deficiency (SPD), Bernard-Soulier Syndrome (BSS), thrombasthenia (TSA), and von Willebrand's disease (vWD). Data were fitted to second-order (TxB2, PF4, and FPA) or third-order (volumes) polynomials. Average values for various parameters over fixed-time intervals were determined by numerical methods. The bleeding time was greater than 15 minutes in all patient groups and the initial bleeding, as reflected by the initial slope of the fitted blood volume curves, was increased in ITP, BSS, and SPD (delta-SPD in particular), but not in vWD and TSA. The increased values for both the initial slope and the volume of blood collected after 2 minutes in SPD suggest that vascular tone may be modulated, in part, by dense granule substances such as adenosine triphosphate (ATP) or serotonin. In TSA, uniquely, both platelet (TxB2 and PF4) and coagulation (FPA) values were increased in early bleeding samples (initial slope). In vitro studies of TxB2 production, together with previous flow studies of fibrin formation, also suggest enhanced activation and coagulant properties of thrombasthenic platelets. In other patients, reduced values of all substances at later times may reflect impaired platelet-fibrin plug formation in the high-shear regions at the ends of transected blood vessels. However, the initial slopes of the fitted curves for both TxB2 and PF4 were normal in vWD, suggesting that the early appearance of these substances may typically be from platelets that are adherent to collagen within the lower shear environment of the wound surface. The finding that FPA values were not decreased initially in any patient group, including ITP, but were decreased at later times (except for TSA), suggests that early fibrin formation occurs independently of platelets in the low-shear environment of the wound surface, whereas at later times fibrin is formed in a platelet-dependent manner in the high- shear regions at the ends of transected vessels.

Epinephrine Promotes IL-8 Production in Human Leukocytes via an Effect on Platelets

1999 ◽  
Vol 81 (01) ◽  
pp. 139-145 ◽  
Author(s):  
Bjarne Østerud ◽  
Charlotte Engstad ◽  
Trine Lund
Keyword(s):  
Whole Blood ◽  
Mononuclear Cells ◽  
Inflammatory Diseases ◽  
Blood Platelets ◽  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Platelet Factor ◽  
Important Mediator ◽  
First Time ◽  
Dose Dependent

SummaryInterleukin-8 (IL-8) is generally accepted to be an important mediator of a number of acute and chronic inflammatory diseases and is produced by monocytes upon stimulation by lipopolysaccharide (LPS). Epinephrine has been reported by several groups to suppress activation of monocytes in response to LPS, and the aim of the present study was to examine the effect of epinephrine on LPS induced IL-8 production using whole blood as a model system. Epinephrine increased LPS induced IL-8 production in a dose-dependent manner in the whole concentration range (0.001–100 μM) and 1 μM epinephrine increased IL-8 levels with 125%. Epinephrine per se had no effect on IL-8 levels. The potentiating effect of epinephrine was mediated by blood platelets, since IL-8 levels in samples containing platelets and stimulated with LPS and epinephrine (1–100 μM) were significantly higher (p <0.05) than in control samples containing no platelets. This effect of platelets seemed to be due to platelet release products, since addition of 25 μl platelet lysate supernatant to whole blood increased LPS induced IL-8 production with 100% and a similar effect was observed in freshly isolated mononuclear cells resuspended in plasma. Upon addition of 50 μg/ml of the carboxyterminal peptide of platelet factor 4 (PF4(58-70)) to whole blood, LPS stimulated IL-8 levels were increased with 115%, whereas in mononuclear cells, 20 μg/ml PF4(58-70) enhanced IL-8 production with 40%. We demonstrate for the first time that epinephrine promotes LPS induced production of IL-8 in whole blood via an effect on blood platelets. This potentiating effect of platelets is shown to be due to platelet granule contents, and platelet factor 4 (PF4) is suggested to be one of several platelet granule proteins promoting LPS induced IL-8 production in whole blood.

scholarly journals Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

2015 ◽  
Vol 210 (4) ◽  
pp. 629-646 ◽  
Author(s):  
Jelle Hendrix ◽  
Viola Baumgärtel ◽  
Waldemar Schrimpf ◽  
Sergey Ivanchenko ◽  
Michelle A. Digman ◽  
...  
Keyword(s):  
Live Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Gag Polyprotein ◽  
Viral Particles ◽  
Infected Cells ◽  
Retroviral Replication ◽  
Gag Assembly ◽  
Reduced Mobility ◽  
Hiv 1

Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.

Heparin-Induced Thrombocytopenia: Prevalence in a Large Cohort of Patients and Confirmed Role of PF4/Heparin Complex as the Main Antigen for Antibodies

1997 ◽  
Vol 3 (3) ◽  
pp. 203-209 ◽  
Author(s):  
Fabrizio Fabris ◽  
Immacolata Cordiano ◽  
Federica Salvan ◽  
Leopoldo Saggin ◽  
Giuseppe Cella ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Count ◽  
Platelet Factor 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Molecular Weight ◽  
Dependent Manner ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Heparin Treatment ◽  
Daily Dose

We performed a retrospective study on the prevalence of heparin-induced thrombocytopenia (HIT) in 233 patients receiving hog mucosa heparin therapy. Of these, 82 patients received s.c. calcium heparin, 130 patient received unfractionated (UF) i.v. heparin, and 21 patients received low molecular weight heparins (LMWH). An additional four patients, referred to our consultation and diagnosed by us as having clinically active type II HIT (HIT-II) were also studied. The mean platelet count of the 233 patients receiving heparin showed a significant decrease after 2 days of heparin treatment and a following significant increase 6 days later (basal: 257 ± 147 x 109 platelets/L; day 2: 239 ± 122, p < 0.0002; day 6: 286 ± 119, p < 0.004). Of the 212 patients receiving UF heparin, 13 (6%) fulfilled the criteria for HIT-II: seven of these had received i.v. heparin (mean daily dose 26,600 ± 4,082 IU ± SD) and six had received s.c. heparin (mean daily dose 21,428:t 6,900 IU). Their mean basal platelet count was 226 ± 100 SD × 109 platelets/L and the nadir during heparin treatment was 78 ± 39 x 10 9 platelets/L. Thrombotic complications occurred in four (30.7%) of the 13 patients with HIT-II. Since the immunological mechanism has been demonstrated for HIT-II and since platelet factor 4 (PF4) was identified as the co-factor for the binding of heparin-related antibodies, we set up our own enzyme-linked immunosorbent assay (ELISA) for testing antibodies against PF4/heparin complex bound through electrostatic bridges to the solid phase. The highest binding capacity of HIT-related IgG to the multimolecular complex was obtained at 20 μg/ml for PF4 and 3 μg/ml for heparin, corresponding to 250 ng of PF4 and 42 ng of heparin in each microtiter well. Such binding was inhibited in a dose-dependent manner by increasing amounts of heparin, protamine hydrochloride, and a monoclonal antibody anti-human PF4 clone 1OB2. We observed that HIT-related antibodies bound also to PF4/LMWH complexes but the optimal PF4/glycosaminoglycan ratio appeared more critical for LMWH (enoxaparin, fraxiparin, and pamaparin) than for UF heparin. Sera from eight patients with HIT-II were tested by PF4/heparin ELISA; six of these had IgG against the complex PF4/heparin and three also had IgM. The persistence of HIT-related antibodies was investigated in three patients: in one such antibodies were still detectable 3 years after the acute episode, while in the other two, they disappeared after 6 months and 1 year, respectively. Key Words: Heparin-related anti body—Platelet factor 4 (PF4)—Heparin—Low molecular weight heparin—Thrombocytopenia—Thrombosis.

scholarly journals Identification of the natural product berberine as an antiviral drug

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jiping Shao ◽  
Debin Zeng ◽  
Shuhong Tian ◽  
Gezhi Liu ◽  
Jian Fu
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Antiviral Drug ◽  
Host Cells ◽  
Heptad Repeat ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Antiviral Activities ◽  
Hiv 1

Abstract Drugs targeting the fusion process of viral entry into host cells have been approved for clinical use in the treatment of AIDS. There remains a great need to improve the use of existing drugs for HIV therapy. Berberine is traditionally used to treat diarrhea, bacillary dysentery, and gastroenteritis in clinics, here our research shows that berberine is effective in inhibiting HIV-1 entry. Native polyacrylamide gel electrophoresis studies reveal that berberine can directly bind to both N36 and C34 to form a novel N36-berberine-C34 complex and effectively block the six-helix bundle formation between the N-terminal heptad repeat peptide N36 and the C-terminal heptad repeat peptide C34. Circular dichroism experiments show that binding of berberine produces conformational changes that damages the secondary structures of 6-HB. Computer-aided molecular docking studies suggest a hydrogen bond with T-639 and two polar bonds with Q-563 and T-639 are established, involving the oxygen atom and the C=O group of the indole ring. Berberine completely inhibits six HIV-1 clade B isolates and exhibits antiviral activities in a concentration-dependent manner with IC50 values varying from 5.5 to 10.25 µg/ml. This compound-peptide interaction may represent a mechanism of action of antiviral activities of berberine. As a summary, these studies successfully identify compound berberine as a potential candidate drug for HIV-1 treatment. As a summary, antiviral activity of berberine in combination with its use in clinical practice, this medicine can be used as a potential clinically anti-HIV drug.

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