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PubMed2004

[The effects of culture supernatant of human GRC-1 cells transfected with PF4 cDNA on the VEGF expression and the growth of ECV304 cells].

AIM: To construct the eukaryotic expression vector pcDNA3-PF4-SS, and to detect the effects of the culture supernatant of transfected GRC-1 cells on the VEGF expression in transfected GRC-1 cells and the growth of ECV304 cells. METHODS: The eukaryotic expression vector pcDNA3-PF4-SS was constructed and identified with Bgl II/BamH I digestion. The pcDNA3-PF4-SS was transfected stably into GRC-1 cells with lipofectamine mediation. The VEGF expression in transfected GRC-1 cells was detected by immunohistochemical staining, and the effect of the culture supernatant of transfected GRC-1 cells on ECV304 cells was detected by MTT colorimetry. RESULTS: Restrictive enzyme (Bgl II/BamH I)digestion analysis showed that the recombinant expression vector pcDNA3-PF4-SS had been constructed successfully. RT-PCR detection proved that hPF4 cDNA had been transfected into GRC-1 cells. The result of immunohistochemical staining showed that the VEGF expression could be seen in the cytoplasm and on cytomembrane of GRC-1 cells transfected with pcDNA3-PF4-SS, but the expression obviously weakened as comparison with that before transfection. Cell counting and MTT colorimetry manifested that the culture supernatant of transfected GRC-1 cells could inhibit markedly growth of ECV304 cells. CONCLUSION: The eukaryotic expression vector pcDNA3-PF4-SS has been constructed successfully, and stably transfected into the GRC-1 cells. The culture supernatant of transfected GRC-1 cells has obviously inhibitory effect on the growth of ECV304 cells and the VEGF expression in the GRC-1 cells, which lays some foundation for exploring the mechanism for anti-tumor growth and developing tumor vaccine for kidney neoplasms.

Carcinoma, Renal Cell↗
PubMed2002

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.

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.

Animals↗
PubMed1996

In vivo effect of platelet factor 4 (PF4) and tetrapeptide AcSDKP on haemopoiesis of mice treated with 5-fluorouracil.

In vivo effects of platelet factor 4 (PF4) and tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) on haemopoietic progenitors were studied in mice treated with 5-fluorouracil (5-FU). The mice were injected with PF4 (40 micrograms/kg) or AcSDKP (4 micrograms/kg) twice at 6 h intervals, and 20 h after the second injection they were given one injection of 5-FU (150 mg/kg). 6, 8 and 13 d later the high proliferative potential-colony forming cell (HPP-CFC), burst-forming unit erythroid (BFU-E), colony forming unit granulocyte-macrophage (CFU-GM) colony forming unit megakaryocyte (CFU-MK), and megakaryocytes (MK) were examined. The results showed that the administration of PF4 or AcSKDP resulted in a significant increase in the number of HPP-CFC on days 6-8 and BFU-E and CFU-GM on day 8 when compared to 5-FU alone. Furthermore, PF4 was found to increase significantly the number of CFU-MK and MK on day 8, which was not observed with AcSDKP. However, both molecules had no obvious effect on peripheral blood cells. These data indicate that PF4 or AcSDKP accelerate the recovery in vivo of HPP-CFC, CFU-GM and BFU-E after 5-FU treatment but their effect may be different on megakaryocytic progenitors and suggests that both molecules may have a haemoprotective effect against chemotherapeutic agents.

Animals↗
PubMed1996

A fatal low-molecular-weight heparin-associated thrombocytopenia after hip surgery: possible usefulness of PF4-heparin ELISA test.

In 37 patients undergoing total hip replacement, a prophylactic treatment by a low-molecular-weight heparin (LMWH) was conducted for 2 weeks. They belonged to a group of 499 patients included in a multicenter clinically controlled trial comparing two LMWHs. Blood was collected 1 day before surgery (D-1) and at D+1 or D+2 and D+5 or D+6 as well as D+10 through D+14 after surgery for determinations of platelets counts and anti-Xa. Bilateral venography was performed between D+10 and D+14. A fatal heparin-associated-thrombocytopenia (HAT) occurred on D+9 in one patient and was associated with a positive platelet aggregation test. This finding was confirmed with a recent ELISA test which evidenced a high concentration of PF4-heparin dependent antibodies 72 h before the detection of thrombocytopenia. This led us to study retrospectively PF4-heparin ELISA results by testing the plasma samples of 36 other surgical patients treated under the same conditions and during the same period (four measurements per patient). Among these patients, seven had a venous thrombotic event as a treatment failure. Although some authors claimed that some post-operative thromboses may be facilitated by the presence of heparin-dependent antibodies associated with or without thrombocytopenia, no thrombocytopenia and no positive PF4-heparin ELISA test was observed in this group. Out of the 144 tests performed in these 36 patients for the detection of PF4-heparin complexes dependent antibodies, 15 results were borderline in ten patients and three results in two patients were positive. No relation was evidenced between a positive ELISA test and the occurrence of venous thrombosis. This study points out the possible usefulness of the PF4-heparin ELISA test for HAT-antibodies detection. A daily platelet count in a postoperative patient under heparin therapy, showing thrombocytopenia associated with the detection of heparin-dependent antibodies could allow an earlier and more reliable diagnosis of HAT.

Aged↗
PubMed1987

Relevance of platelet factor four (PF4) plasma levels in multiple sclerosis.

In order to study the role of platelets in Multiple Sclerosis (MS) we assessed, in a group of patients during a quiescent phase of the disease, the plasma levels of beta-thromboglobulin (beta-TG) and platelet factor four (PF4) both in absence of treatment and during administration of aspirin (ASA) at the dose of 50 mg/daily. In the MS patients studied, the basal plasma levels of beta-TG and PF4 were significantly higher than in control subjects. The increase in the beta-TG plasma levels occurred independently of the age, sex and severity of the disease, whereas the modification in the PF4 plasma levels was significantly correlated with the severity of the disease. Administration to the patients of ASA, at the dose that does not affect prostacyclin production, determined a decrease of beta-TG in 77% of the patients. Mean PF4 plasma levels remained unchanged. These results suggest that PF4 in the plasma of MS patients may originate not only from the platelets but also from the mast cells following platelet aggregating factor (PAF) stimulation and immunocomplex formation.

Adult↗
PubMed1987

Raised plasma concentrations of platelet factor 4 (PF4) in Crohn's disease.

Plasma platelet factor 4 (PF4), secreted by the platelets, is an index of platelet aggregation and thromboembolic risk. The authors assessed PF4 in 20 patients with Crohn's disease (ileitis in 13 patients, ileocolitis in seven) and in 20 healthy volunteers. Disease activity was low (Crohn's Disease Activity Index less than 150) in 11 patients and high in nine. Radioimmunoassay of PF4 using Abbott's Kit was performed on one sample of plasma from each subject (nv less than or equal to 0.324 nmol/ml), (nv less than or equal to 10 ng/ml). A significantly higher concentration of PF4 was found in Crohn's disease patients: 4.625 +/- 1.1 nmol/ml (142.5 +/- 36 ng/ml) than in the control group: 0.189 +/- 0.07 nmol/ml (5.6 +/- 4.8 ng/ml) (Z = 5.396, p less than 0.0001). No correlation was present between PF4 levels and activity, the site of disease, or medical treatment with or without prednisone.

Adult↗
PubMed1999

[The hemoprotective effect of platelet factor 4 (PF4) and tetrapeptide AcSDKP].

OBJECTIVE: To study the effects of platelet factor 4(PF4) and tetrapeptide N-acetyl-Ser-Asp-Lys-Pro(AcSDKP) on hemopoietic progenitors in mice treated with 5-Fluorouracil (5-FU). METHODS: Mice were injected with PF4 (40 micrograms/kg) or AcSDKP (4 micrograms/kg) twice at 6 h intervals, and 20 h after the second injection they were given one injection of 5-FU (150 mg/kg), and the high proliferative potential-colony forming cell (HPP-CFC), burst-forming unit erythroid (BFU-E), colony forming unit megakaryocyte (CFU-MK), and megakaryocyte (MK) were examined 6, 8 and 13 days later. RESULTS: The administration of PF4 or AcSDKP resulted in a significant increase of the number of HPP-CFC on days 6-8 and BFU-E and CFU-GM on day 8 when compared to 5-FU alone. Furthermore, PF4 was found to increase significantly the number of CFU-MK and MK on day 8, which was not observed with AcSDKP. CONCLUSION: PF4 or AcSDKP accelerate the recovery in vivo of HPP-CFC, CFU-GM and BFU-E after 5-FU treatment but their effect may be different on megakaryocytic progenitors. Both molecules may have a hemoprotective effect against chemotherapeutic agents.

Animals↗
PubMed1988

Serial determinations of PF4 and beta TG: comparisons between multiple venipunctures vs a catheter infusion system.

Platelet factor 4 (PF4) and beta thromboglobulin (beta TG) are platelet-specific proteins which are released upon platelet aggregation and which can be accurately measured by radioimmunoassay. We devised a catheter-infusion system that enables serial determinations of these proteins. In 20 subjects (10 healthy volunteers and 10 patients with stable coronary artery disease), we compared samples collected by individual venipunctures with those simultaneously obtained by means of a simple catheter-infusion system. At least 5 samples were obtained over a period of time which was as long as 60 min, and at least 30 min. Subjects with stable coronary artery disease were selected so that they would be expected to have stable and normal PF4 and beta TG levels. Thus, elevations of either PF4 or beta TG would represent artifacts secondary to sampling technique. Analysis of the results demonstrated that the catheter-infusion system was equivalent to individual venipunctures for determination of PF4 and beta TG. 16.8% of samples obtained via the catheter and 17.2% of those obtained by individual venipunctures were spuriously elevated. A second series of studies were performed to refine the technique further by examining the impact of infusion rate and the addition of citrate phosphate dextrose (CPD) to the infusate. Ten additional subjects had catheter systems utilized in both arms simultaneously. The addition of CPD resulted in significantly less abnormal values at slower infusion rates (1 and 2.5 cc/min). At 5 cc/min D5/w or saline alone are suitable. These investigations confirm that this simple catheter system is equivalent to individual venipunctures for determination of PF4 and beta TG while avoiding patient discomfort.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1982

Immunohistological comparison of platelet factor 4 (PF4), fibronectin (Fn) and factor VIII related antigen (VIIIR:Ag) in human platelet granules.

Immunofluorescence microscopy was used to study the localization of platelet factor 4 (PF4), fibronectin (Fn) and factor VIII related antigen in washed normal platelets and those from patients with severe von Willebrand's disease (vWd). Platelets were prepared by an improved multi-unit modification of the Sayk chamber. This facilitated the preparation of samples with minimum disruption of platelets and readily permitted the demonstration of intact platelet granules by the immunological techniques used. The antigens were demonstrated by treating the same preparations sequentially with appropriate heterologous antisera and species specific fluorescein or rhodamine conjugated antisera. Comparison of the different antigens in identical platelets indicated that Fn and VIIIR:Ag were localized to the same granules as PF4 and the results were thus consistent with their presence in alpha granules. Fn and PF4, but not VIIIR:Ag, were present in platelet granules of patients with severe vWd. The antigens were always detected in the same granules, and major sub-populations of differently stained granules were not observed. The methods were applied to investigate normal platelets aggregated with collagen. Fn and VIIIR:Ag were detected in platelet granules after aggregation although the granules themselves were possibly differently distributed in these samples compared with the non-aggregated platelets. The localization of PF4 could not be reliably assessed in aggregated platelets by these methods. The techniques may be useful in localizing platelet antigens and studying release during aggregation.

Antigens↗
PubMed1992

Inhibition of thrombin generation by heparin and low molecular weight (LMW) heparins in the absence and presence of platelet factor 4 (PF4).

The ability of several low molecular weight (LMW) heparins and unfractionated heparin (UFH) to inhibit thrombin generation, and their anti-Xa and anti-IIa activities, were measured in the absence and presence of platelet factor 4 (PF4). The LMW heparins studied were 2-5 times less potent, on a weight basis, than UFH as inhibitors of thrombin generation in platelet-poor plasma; the inhibition of thrombin generation by LMW heparins correlated better with their anti-IIa activity (r = 0.98) than with their anti-Xa activity (r = 0.69). At low concentrations of PF4, the activity of LMW heparins in the thrombin generation test was neutralized less than that of UFH, but at higher PF4 concentrations all their activities could be neutralized except in anti-Xa assays. These observations support the hypothesis that anti-IIa activity is important for inhibition of thrombin generation by LMW heparins in vitro. However, when all the anti-IIa activity of LMW heparins was neutralized by PF4, considerable inhibitory activity remained in thrombin generation and anti-Xa assays, indicating that a portion of the anti-Xa activity of LMW heparins also contributes towards inhibition of thrombin generation.

Factor Xa Inhibitors↗
PubMed1995

Megakaryocyte-specific positive regulatory sequence 5' to the human PF4 gene.

Platelet factor 4 (PF4) is only expressed in platelets and is an appropriate marker for studying megakaryocytic differentiation. We previously characterized cDNA and genomic clones for human PF4 (hPF4) and now present transient expression studies defining the promoter of the gene. 12-O-tetradecanoyl-phorbol-13- acetate (TPA) induces megakaryocytic differentiation of human erythroleukemia (HEL) cells, providing an excellent model system for the study of megakaryocyte-specific promoter activity. Luciferase reporter-gene constructs containing sequences from -2074 to +49 were used to map regions that may regulate PF4 gene expression. The sequence in the region -239 to -107 increased basal promoter activity by four- to five-fold in TPA-induced HEL cells. The sequence between -239 and -107 contains 53 consecutive thymidine residues. Functional studies using constructs in this region show that poly(T) and the region -187 to -107 are necessary for the total increase in activity in TPA-induced HEL cells. Mobility-shift assays show that the poly(T) tract binds TPA-inducible proteins. The results suggest a complex promoter for the PF4 gene involving a basal nonspecific promoter element between -107 and +49, a positive promoter element between -239 and -107 binding specific nuclear proteins from megakaryocyte-lineage cells, and a silencer-like region between -2074 and -1653.

Base Sequence↗
PubMed1990

Negative regulation of human megakaryocytopoiesis by human platelet factor 4 (PF4) and connective tissue-activating peptide (CTAP-III).

We have previously reported that human platelet factor 4 (PF4) and beta-thromboglobulin inhibit human megakaryocyte (meg) colony formation in vitro. Here, we report further findings concerning the effect of PF4 as well as another platelet-derived factor: connective tissue-activating peptide (CTAP-III). Addition of these factors (2.5-10 micrograms/ml) into normal marrow cultures resulted in a significant decrease of meg colonies, especially the mixed-meg colonies, BFU-meg and large CFU-meg, suggesting that they inhibit both proliferation and maturation of meg progenitor cells, with a predominant effect on earlier progenitor cells. Comparison of the effects of the two factors showed that their major effects were similar, with some difference in inhibitory degree. These results indicate that both PF4 and CTAP-III are potent inhibitors of meg colony formation and involved in negative autocrine regulation of megakaryocytopoiesis.

Bone Marrow Cells↗
PubMed2000

Nematode neuropeptide modulation of the vagina vera of Ascaris suum: in vitro effects of PF1, PF2, PF4, AF3 and AF4.

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 > or = 10 nM were quantitatively the greatest. Both AF3 and AF4 (1 microM) 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.

Animals↗
PubMed2001

Anti-PF4-heparin immunoglobulin G is the major class of heparin-induced thrombocytopenia antibody: findings of an enzyme-linked immunofiltration assay using membrane-bound hPF4-heparin.

An enzyme-linked immunofiltration assay (ELIFA) was developed for detecting anti-human platelet factor 4 (hPF4)-heparin antibody in sera of patients with heparin-induced thrombocytopenia (HIT). The immunofiltration assay was developed to capture HIT antibody by hPF4-heparin complex adsorbed onto a positively charged nylon membrane, as an alternative to plastic bound hPF4. Of 75 sera with a positive serotonin-release assay (SRA), anti-PF4-heparin of the immunoglobulin (Ig)G class was detected in 72 (96%) sera. With SRA-negative sera from thrombocytopenic patients treated with heparin, anti-hPF4-heparin IgG and IgA were detected in 16% (n = 126) and 14% (n = 74) of sera respectively; 6% (n = 71) of SRA-negative sera contained both IgG and IgA anti-hPF4-heparin antibodies. The detection of anti-hPF4-heparin IgG in all HIT sera supports the assay of anti-PF4-heparin IgG as being a sensitive screening test for HIT. Alternatively, the absence of anti-hPF4-heparin IgA cannot be used as a test for excluding HIT, as it was detected in only 48% of SRA-positive HIT sera. However, it may be used to support the diagnosis of HIT, when HIT IgG is weak. This study emphasized the need to use different immobilizing media for the capture of anti-PF4-heparin antibody.

Antibodies↗
PubMed1999

Hemostasis and Thrombosis: Clinical Usefulness of Combined use of Platelet Aggregation Test and Anti PF4-H. Antibodies Elisa test for the Diagnosis of Heparin Induced Thrombocytopenia.

Background. Heparin is the most commonly used drug in patients requiring therapeutic anticoagulation. But the use of heparin has serious side effects such as heparin-induced thrombocytopenia (HIT). The diagnosis of HIT is often difficult. This study was designed to test the diagnosis value of 2 laboratory tests: the platelet aggregation test and the ELISA test (Enzym Linked Immuno-Sorbent Assay). Methods and Results. In order to evaluate the diagnostic value of these 2 tests, we prospectively performed both tests in all patients referred to our laboratory with suspected type II HIT, and compared their results with clinical outcome. Plasmas from 60 patients suspected of HIT, were tested for heparin-dependent platelet-reactive antibodies with a platelet agregation test (PAT) and with an ELISA which detects anti heparin platelet factor 4 antibodies (Anti PF4-H Ab). Among the 60 explored patients, the clinical diagnosis of HIT was confirmed in 27 patients by clinical criteria. In 16 of these 27 patients, PAT and anti PF4-H. Ab were both positive, and in the 11 other patients, one of the 2 tests was negative. In 29 of the 33 patients with no clinical HIT, PAT and anti PF4-H. Ab were both negative, in the 4 remaining patients, one of the 2 tests was positive. Conclusion. For 75% of patients, biological results were concordant with the final clinical diagnosis. The combination of both tests is more reliable than the use of a single test; in the present series, all patients with positive results on both tests had clinically confirmed HIT, and all patients with negative results on both tests had not clinically confirmed HIT.

Journal Article↗
PubMed1991

[The changes in plasma beta-thromboglobulin (beta-TG), platelet factor 4 (PF4) and thromboxane B2 (TXB2) after a bronchial provocation test (BPT) with house dust (HD) allergen].

In order to investigate the role of platelets in allergic asthma, the time related changes in plasma levels of beta-TG, PF4 and TXB2 were evaluated following BPT with HD in 19 patients with bronchial asthma who were positive in skin test and RAST to HD. The results obtained were as follows. 1) Plasma beta-TG and PF4 levels tended to increase following BPT with HD at the time of immediate asthmatic response (IAR) in patients showing IAR alone. 2) Plasma beta-TG and PF4 levels increased significantly (p less than 0.05) at IAR and tended to increase at the time of late asthmatic response (LAR) in patients showing a dual asthmatic response (DAR). 3) The levels of plasma TXB2 in patients showing IAR alone significantly increased at IAR (p less than 0.05) and gradually decreased and the levels of plasma TXB2 in patients showing a DAR increased in each period of IAR, 3 hr after BPT and LAR, and the peak of TXB2 was observed in 3 hours after BPT. 4) These results suggest that platelets are activated at IAR and there was also a possible activation in platelets at LAR.

Adult↗
PubMed1995

rmIL-6 stimulates the transcriptional activity of the rat PF4 gene.

We have examined the effect of recombinant murine interleukin-6 (rmIL-6) on megakaryocytopoiesis in a liquid rat bone marrow culture system. At concentrations of IL-6 up to 100 ng/mL, no stimulation of megakaryocyte ploidy was observed. However, transient expression studies revealed that IL-6 did have a significant effect on the transcriptional activity of the platelet factor four (PF4) gene, a platelet alpha-granule protein gene uniquely expressed in megakaryocytes. Furthermore, when the amount of PF4 message was directly measured in megakaryocytes, it was increased three-fold in response to IL-6. We also note that the PF4 promoter contains a hexamer, CTGGGA, described as the IL-6-responsive element in other genes. Our results suggest that the platelet progeny of IL-6-stimulated megakaryocytes may have altered alpha-granule constituents.

Animals↗
PubMed1999

Significantly elevated expression of PF4 (platelet factor 4) and eotaxin in the NOA mouse, a model for atopic dermatitis.

The NOA (Naruto Research Institute Otsuka Atrichia) mouse, an animal model of allergic or atopic dermatitis, exhibits ulcerative skin lesions associated with accumulation of mast cells and eosinophils, a significantly increased level of serum IgE, and scratching behavior. To investigate genetic contributors to the pathological process of dermatitis in this murine model, we looked for genes that were expressed differently in spleens of NOA mice compared with controls, by means of a differential display method. We cloned and characterized one gene that revealed a significantly higher expression in the NOA mouse than in control strains. Its cDNA consisted of 570 nucleotides, including 315 nucleotides of open reading frame encoding 105 amino acids. The deduced amino acid sequence identified this gene as the murine homologue of rat and human platelet factor (PF) 4s (89% identity and 64% identity in 105 amino acids, respectively). PF4 is a heparin-binding protein that is released from alpha-granules of activated platelets and belongs to the family of chemokine molecules that contain a CXC motif. Our results suggested that increased expression of PF4 may play an important role in the etiology of allergic dermatitis.

Amino Acid Sequence↗