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PubMed1990

Two burgeoning families of platelet factor 4-related proteins: mediators of the inflammatory response.

The inflammatory response involves the recruitment and activation of various types of cells from the systemic circulation and from local tissues. One important component of the inflammatory response is the activation of platelets at sites of tissue injury and inflammation. In particular, activated platelets release large amounts of two proteins, platelet factor 4 (PF4) and beta-thromboglobulin (beta TG), which mediate several inflammatory processes. Recently, many novel proteins that are structurally related to PF4 and beta TG have been identified. The PF4-related proteins are secreted by white blood cells, endothelial cells, and fibroblasts in response to various inflammatory and mitogenic stimuli. Like PF4, these proteins appear to be inflammatory response mediators; several of them are potent chemoattractants, activating agents, or mitogens for specific cell types that are involved in the inflammatory response. The study of PF4-related proteins provides new insight into the mechanisms of the immune response, and may result in the development of new therapeutic agents.

Amino Acid Sequence↗
PubMed1990

Adhesion of Kirsten-ras+ tumor-progressing and Kirsten-ras- revertant 3T3 cells on fibronectin proteolytic fragments.

Adhesion has been evaluated for tumor cell populations derived from Kirsten murine sarcoma virus (KiMSV)-transformed BALB/c 3T3 cells responding to substrata coated with intact plasma fibronectin (pFN), a family of related proteolytic fragments from pFN or cellular fibronectins (FNs), and the heparan sulfate-binding platelet factor-4 (PF4). Both early-passage KiMSV cells, harboring the viral Kirsten ras oncogene (v-Ki-ras+), and late-passage KiMSV cells, in which most cells have lost the oncogene (v-Ki-ras-), are compared with primary tumor and lung metastatic tumor cells after three routes of injection into nude mice; nontumorigenic v-Ki-ras- revertant cells have been cloned from the late-passage KiMSV population. Attachment of early-passage KiMSV, primary tumor, and lung metastatic tumor cells was optimal and resistant to soluble RGDS peptide in the medium on intact pFN, on fragment F-155 from pFN containing the RGDS cell-binding domain and the heparinII domain, and on PF4 but decreased for metastatic cells on F110 containing only the RGDS domain (and sensitive to RGDS peptide). Cytoplasmic spreading of early-passage KiMSV and all tumor cells was good to excellent in polygonal patterns on pFN and on F155, while most cells remained round on F110. Responses for KiMSV and tumor cells varied on different heparin-binding proteins; cells remained rounded or detached on F38 derived from pFN or on PF4 but spread effectively with long linear process extension on cellular FN-derived fragments F44 + 47 harboring the extra domaina sequence. That F44 + 47 may contain a new cell-binding site for v-Ki-ras+ cells was also indicated by resistance to bacterial heparitanase in cell responses on F44 + 47 but not on PF4 and extensive catabolism of proteoglycans in the substratum-attached material of these cells. v-Ki-ras- revertant cells, nontumorigenic in nude mice, have reacquired 3T3-like responses to proteolytic fragments, including much more effective spreading on PF4 or on F38 substrata, and have reverted in generating microfilament stress fibers on pFN, a competence lacking in all v-Ki-ras+ cells. These results indicate that (a) v-Ki-ras+ primary and metastatic tumor cells respond similarly to most proteolytic fragments of FNs harboring known binding domains, with a few exceptions; (b) v-Ki-ras gene expression correlates with a new cell surface receptor activity recognized by extra domaina-containing fragments from cellular FNs; and (c) loss of the viral oncogene to generate v-Ki-ras- revertant cells reverts their FN-mediated adhesion responses.

Actins↗
PubMed1989

The three-dimensional structure of bovine platelet factor 4 at 3.0-A resolution.

Platelet factor 4 (PF4), which is released by platelets during coagulation, binds very tightly to negatively charged oligosaccharides such as heparin. To date, six other proteins are known that are homologous in sequence with PF4 but have quite different functions. The structure of a tetramer of bovine PF4 complexed with one Ni(CN)4(2-) molecule has been determined at 3.0 A resolution and refined to an R factor of 0.28. The current model contains residues 24-85, no solvent, and one overall temperature factor. Residues 1-13, which carried an oligosaccharide chain, were removed with elastase to induce crystallization; residues 14-23 and presumably 86-88 are disordered in the electron density map. Because no heavy atom derivative was isomorphous with the native crystals, the complex of PF4 with one Ni(CN)4(2-) molecule was solved using a single, highly isomorphous Pt(CN)4(2-) derivative and the iterative, single isomorphous replacement method. The secondary structure of the PF4 subunit, from amino- to carboxyl-terminal end, consists of an extended loop, three strands of antiparallel beta-sheet arranged in a Greek key, and one alpha-helix. The tetramer contains two extended, six-stranded beta-sheets, each formed by two subunits, which are arranged back-to-back to form a "beta-bilayer" structure with two buried salt bridges sandwiched in the middle. The carboxyl-terminal alpha-helices, which contain lysine residues that are thought to be intimately involved in binding heparin, are arranged as antiparallel pairs on the surface of each extended beta-sheet.

Amino Acid Sequence↗
PubMed1988

The expression in Escherichia coli of recombinant human platelet factor 4, a protein with immunoregulatory activity.

In order to establish more firmly the immunoregulatory effect of platelet factor 4 (PF4) and develop a means to provide material for possible clinical use, the nucleotide sequence for PF4 was synthesized utilizing a ligation strategy of six duplexes ranging from 27 to 43 base pairs in length. The individual oligodeoxynucleotides were synthesized on an automated system. The resultant gene segment (226 base pairs), which incorporated convenient HindIII and BamHI overhangs at the 5' and 3' ends, respectively, was cloned into the pIN-III-ompA-2-expression vector in Escherichia coli, affording a fusion protein of Mr = 8900 with 7 additional amino acids at the amino terminus and 4 at the carboxyl terminus and with aspartic acid rather than asparagine in position 47. The recombinant PF4 (rPF4) was purified by heparin-agarose affinity chromatography and reverse-phase high performance liquid chromatography. It reacted with a monoclonal mouse anti-human PF4 antibody on a Western blot and in an enzyme-linked immunosorbent assay. The rPF4 protein exhibited an immunoregulatory effect like that of human PF4 in its ability to reverse concanavalin A-induced immunosuppression in BALB/c mice.

Amino Acid Sequence↗
PubMed1984

Detection of enhanced in vivo platelet alpha-granule release in different patient groups--comparison of beta-thromboglobulin, platelet factor 4 and thrombospondin assays.

During the platelet release reaction beta-thromboglobulin (beta TG), platelet factor 4 (PF4) and thrombospondin (TSP) are released from the platelet into plasma and assays of these proteins can be used to monitor in vivo platelet activation. We have assessed their relative merits as markers of the in vivo platelet alpha-granule release reaction in a number of patient groups which have previously been shown to have elevated plasma beta TG and/or PF4 levels. It is concluded that in diseases or conditions not complicated by its reduced clearance, beta TG is the most sensitive marker of in vivo platelet alpha-granule release. However, the TSP assays may be the least ambiguous when monitoring the platelet alpha-granule release reaction in patients with renal failure who are undergoing haemodialysis with heparin anticoagulation. Under these circumstances plasma beta TG, but not PF4 or TSP, levels are elevated because of impaired renal catabolism, and the presence of a heparin-releasable reservoir of PF4 on the endothelium complicates the use of the PF4 assay. In liver failure none of these assays may accurately reflect platelet alpha-granule release because of impaired hepatic or renal elimination of the proteins.

Adolescent↗
PubMed1980

Plasma concentrations of platelet-specific proteins correlated with platelet survival.

Relationships between 51Cr platelet survival and plasma concentrations of beta-thromboglobulin (betaTG) and platelet factor 4 (PF4) were analyzed in 91 studies of patients with coronary artery disease. betaTG was significantly correlated with platelet life-span, turnover, and the number of hits in the multiple hit model. PF4 was significantly correlated with life-span and turnover. The most significant relationship involving platelet-specific protein concentrations and life-span estimates was between betaTG and life-span estimated using the multiple hit model (r = -0.39, p less than 0.001). There was a high correlation between betaTG and PF4 (r = 0.62, p less than 0.001), and no improvement could be obtained by combining the measurements of the two proteins in any regression with life-span or turnover. The results indicate that the patients with the shortest platelet survival time in this group tended to have the highest plasma concentration of betaTG and PF4 and thus probably increased in vivo release of betaTG and PF4. They strengthen the claim that these platelet-specific proteins may be indicators of platelet involvement in disease.

Blood Platelets↗
PubMed1982

Platelet factor four and protamine sulfate neutralization of heparin fractionated according to anionic charge density.

The role of heparin structure in neutralization by the neutralizing substances (NS) platelet factor 4 (PF4) and protamine sulfate (PS) was investigated using a thrombin clotting assay and a series of more homogeneous heparin fractions varying systematically in charge density (Z). For a given heparin, plotting inverse clotting times measured without NS, and in the presence of PF4 or PS, vs heparin concentration yielded approximately parallel straight lines displaced horizontally according to the amount of NS. Potencies of heparin fractions in the absence of NS, or in the presence of PF4 of PS, depended almost identically upon Z2. Small but significant quantitative differences in potency among equivalent fractions from different heparins showed both PF4 and PS had a slight preference for the least active subfraction of decolorized heparins, but for the most active subfraction of undecolorized heparins. Neutralization of heparin by PF4 and PS probably proceeds by similar mechanisms, but the details of structure outside the antithrombin III-binding oligosaccharide of heparin may enter in differently.

Animals↗
PubMed1978

In vitro and in vivo effects of adrenaline and phentolamine on platelet function.

In vitro and in vivo effects of adrenaline (ADR) on platelet aggregation, on platelet factor 3 (PF3) availability and on platelet factor 4 (PF4) release were studied in man. Inhibitory action of an alpha-blocker, phentolamine (PHEN) was investigated in the same conditions. The threshold concentration (TC) of ADR inducing the typical two-phase response in aggregation tests when added to platelet-rich plasma (PRP) varied in different pools of plasma, but always induced an evident PF4 release and increased PF3 availability. A further increase in both parameters was obtained with higher concentrations but without any significant dose/response correlation. Adding PHEN alone to PRP did not induce platelet aggregation or modify PF4 release induced by stirring, but it reduced PF3 availability. On the other hand, PHEN prevented the effects of ADR in different platelet tests, at appropriate concentrations. Intravenous infusion of ADR lowered the TC, and increased PF3 availability and PF4 release. In vivo administration of PHEN, in contrast, increased TC and reduced PF3 availability, while PF4 remained unchanged.

Blood Platelets↗
PubMed1995

Induction of ICAM-1 expression on the eosinophilic leukemia cell line-3 (EoL-3) by platelet-activating factor and platelet factor 4.

BACKGROUND: Eosinophils are assumed to transmigrate to local tissue under the direction of the eosinophil chemotactic factor present in allergic inflammatory lesions. OBJECTIVE: We examined whether PAF and PF4, which are related to eosinophil localization, affect the expression of ICAM-1 on the human eosinophilic leukemia cell line (EoL-3). METHODS: ICAM-1 expression on EoL-3 cells in the presence or absence of PAF or PF4 was assessed by immunofluorescent analysis. The presence of sICAM-1 in culture supernatants of EoL-3 cells was detected with an ELISA kit. RESULTS: Both PAF and PF4 enhanced the expression of ICAM-1 on EoL-3 cells. We found no difference in sICAM-1 concentration with or without PAF or PF4. CONCLUSIONS: The eosinophil chemotactic factors PAF and PF4 not only may attract eosinophils, but they may enhance the expression of ICAM-1 on eosinophils at the site of the lesion. Further, it is suggested that the shedding of ICAM-1 has little relation to the induction of ICAM-1 expression in vitro.

Culture Media↗
PubMed1993

[Characterization of expression of adhesion molecules on eosinophils in bronchial asthma].

It is well known that inflammatory processes play an important role in allergic reactions such as bronchial asthma (BA). Eosinophils are considered to the main players in such allergic inflammation. Moreover, adhesion molecules have recently been found to play an important role in the inflammatory processes in allergic and immune reactions. Thus, in this study, the characteristics of the adhesion molecules expression on human eosinophils obtained from BA patients were examined. High intensity expressions of adhesion molecules (CR3, LFA-1 alpha, LFA-1 beta) on eosinophils obtained from the patients were observed. Furthermore, ICAM-1, which is the ligand for LFA-1 molecules, was also expressed on eosinophils from BA patients. To extend our understanding of this high intensity expression of adhesion molecules (CR3, LFA-1 alpha, LFA-1 beta, ICAM-1) on such eosinophils, we examined whether chemotactic agents might be involved in adhesion molecule expression using an eosinophilic cell line named EoL-3. These characteristics of adhesion molecules on eosinophils were induced by the chemotactic agent, platelet factor 4 (PF4). Taken together, we could conclude that chemotactic agents might be involved in the high intensity expressions of adhesion molecules on eosinophils from BA patients. Moreover, to investigate the mechanisms of the induction of adhesion molecules on EoL-3 cells by PF4, sICAM-1 levels in the supernatants of EoL-3 with PF4 and without PF4 were examined. In our results, the presence or absence of PF4 made no difference in sICAM-1 levels, suggesting that the shedding of ICAM-1 has little relation to the induction of adhesion molecules.

Adult↗
PubMed1994

Functional and ligand binding specificity of the rabbit neutrophil IL-8 receptor.

IL-8 mediates migration and activation of neutrophils. This study describes the functional and ligand binding specificity of the human intercrine peptides IL-8, neutrophil-activating peptide 2 (NAP-2), melanoma growth stimulatory activity (GRO), and platelet factor 4 (PF4) to rabbit neutrophils and mammalian cell lines transfected with rabbit IL-8 receptor cDNA (F3R). Rabbit neutrophil membranes bound 125I-labeled IL-8 and 125I-labeled NAP-2 but did not bind 125I-labeled GRO or 125I-labeled PF4. Rabbit neutrophils mobilized intracellular Ca2+ in response to IL-8 and NAP-2 but not to GRO or PF4. Monkey kidney cells (COS-7) and hamster lung fibroblasts (CCL-39) were transiently and stably transfected with the rabbit neutrophil IL-8 receptor F3R cDNA. COS-7 cells transfected with F3R cDNA bound 125I-labeled IL-8 but did not bind other IL-8-related peptides such as 125I-labeled NAP-2, 125I-labeled GRO, or 125I-labeled PF4. Furthermore, bound 125I-labeled IL-8 was only displaced by unlabeled IL-8 but not by unlabeled NAP-2, GRO alpha, or PF4. Consistent with this observation, stably transfected CCL 39 cells expressing F3R cDNA mobilized Ca2+ only in response to IL-8. We conclude that F3R cDNA encodes a functional IL-8 receptor isotype with strict ligand binding specificity for IL-8, that rabbit neutrophils do not bind human GRO alpha, and it is suggested that rabbit neutrophils contain in addition to the F3R protein another IL-8 receptor isotype with broad ligand specificity or a distinct NAP-2 receptor.

Animals↗
PubMed1994

The interaction of GATA-binding proteins and basal transcription factors with GATA box-containing core promoters. A model of tissue-specific gene expression.

The core promoters of the rat platelet factor 4 (PF4), mouse erythropoietin and chicken beta globin genes contain a GATA motif in place of the consensus TATAAA site. In the case of the PF4 gene, this site has been shown to play a critical role in restricting transcription to the megakaryocyte lineage. In order to understand the mechanism of tissue specificity, we investigated the function of the GATA box-containing promoters in vitro. Our studies show that the TATA-binding protein of TFIID is required for initiation of transcription from the GATA box-containing promoters. GATA-1 interacts with the core promoter GATA motif and inhibits generation of preinitiation complexes. The functional significance of the inhibition of preinitiation complexes is supported by in vitro transcription assays in which transcription from the PF4 and erythropoietin core promoters is suppressed by GATA-1. We also demonstrate that GATA-2 inhibits initiation of transcription from the PF4 core promoter. Based on these results, we propose a model in which repression of PF4 expression in nonmegakaryocytes is mediated, in part, by competition between GATA-binding proteins and basal factors for the core promoter.

Animals↗
PubMed1993

Measurement of beta-thromboglobulin and platelet factor 4 to follow up patients with artificial heart valves.

Efficient antithrombotic therapies are necessary for the prevention of thrombotic complications in patients with artificial heart valves. Antiplatelet drugs are commonly used since platelet activation is a feature frequently found in these pathologic conditions. The measurement of beta TG and PF4 is useful for the diagnosis of platelet activation, which may increase the thrombotic risk. Two commercially available ELISA kits for beta TG and PF4 were tested and compared with the corresponding RIA methods used previously. Coefficients of correlation were 0.976 for beta TG and 0.985 for PF4. Both methods gave identical results, although RIA was expressed in nanograms per milliliter, whereas ELISA results were in international units per milliliter by comparison with International Standards. For both parameters 1 IU/ml was found to be almost equal to 1 ng/ml. beta TG was elevated in the group of patients with heart valves (57.28 IU/ml); so was PF4 but to a lesser extent (11.28 IU/ml). The mean concentrations on a normal population were, respectively, 22.40 (+/- 5.15) IU/ml and 4.10 (+/- 2.02) IU/ml. The monitoring of patients with heart valves allowed the identification of two distinct groups: those who had persistent elevated levels of beta TG and PF4, and those whose levels of the same parameters were in the subnormal ranges. All the thrombotic complications were observed in the first group and were preceded by an elevation of platelet markers. beta TG was the most predictive and correlated closely with the evolution of the clinical state. Cerebrovascular accidents were the commonly observed complications. These patients were treated by hypocoagulant therapy and with antiplatelet drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1997

Modulation of blood cell activation by four commonly used anticoagulants.

In the past years, our group has made several observations suggesting that blood cells behave differently and when stimulated, release different levels of cytokines, depending on which anticoagulant the blood has been drawn into. The aim of this study was therefore to compare the effect of the four anticoagulants EDTA, citrate, heparin and hirudin on monocyte, neutrophil (PMN), and platelet function in human whole blood. Human whole blood was employed as an ex vivo model of cytokine production and protein secretion, and lipopolysaccharide (LPS) induced tissue factor (TF) activity in monocytes and LPS induced tumor necrosis factor alpha (TNF alpha) release were chosen as parameters of monocyte activation. Platelet factor 4 (PF4) secretion and LPS induced lactoferrin release were chosen as parameters of platelet and PMN activation, respectively. When human whole blood was stimulated with 5 ng/ml LPS for 2 h, TF activity in monocytes isolated from EDTA blood was found to be 2.9 mU/10(6) cells, whereas TF activity in monocytes isolated from citrated, heparinized and hirudinized blood was 14.7, 24.7 and 28.5 mU/10(6) cells, respectively. TNF alpha concentrations in platelet poor plasma (PPP) isolated from whole blood stimulated with 5 ng/ml LPS for 2 h was increased with 200, 400 and 350% in citrated, heparinized and hirudinized blood respectively, as compared to EDTA blood. Next, the effect of the anticoagulant on PMN secretion was measured. PPP isolated from whole blood incubated with 5 ng/ml LPS for 90 min contained 1170 ng/ml (EDTA blood), 2880 ng/ml (citrated blood), 4220 ng/ml (heparinized blood), and 5520 ng/ml lactoferrin (hirudinized blood). When studying the platelet parameter PF4, whole blood was incubated without any stimuli for 60 min, and we found that heparin PPP contained 1180 ng/ml PF4, while hirudin PPP contained 469 ng/ml, citrate PPP 440 ng/ml, and EDTA PPP 217 ng/ml PF4, respectively. Finally, the low molecular weight heparin compound Fragmin had no enhancing effect on PF4 levels in whole blood. It is concluded that the anticoagulant used in in vitro experiments has a large influence on the parameters measured.

Anticoagulants↗
PubMed1997

Heparin-induced platelet aggregation vs platelet factor 4 enzyme-linked immunosorbent assay in the diagnosis of heparin-induced thrombocytopenia-thrombosis.

Thrombosis occurs in an unpredictable subset of patients with heparin-induced thrombocytopenia (HIT). The diagnosis of HIT requires clinical suspicion and laboratory confirmation. Although the "gold-standard" diagnostic test is considered to be the serotonin release assay (SRA), most laboratories use heparin-induced platelet aggregation (HIPA), which is highly specific but reported to be less sensitive than the SRA. Recently, the heparin-platelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA) has been reported to have comparable sensitivity to the SRA. We compared the HIPA and PF4 ELISA in serum samples from 146 patients examined for HIT and assessed whether either test predicted thrombotic risk. Results for 81 patients were positive for HIPA, PF4 ELISA, or both. Of these, 91% were HIPA-positive, while only 60% were PF4 ELISA-positive. Clinical information was available on 63 patients, 17 of whom had thrombotic events (10 venous, 6 arterial, and 1 both). Neither the HIPA nor the PF4 ELISA predicted thrombotic risk, but the HIPA proved to be a more sensitive test for laboratory confirmation.

Enzyme-Linked Immunosorbent Assay↗
PubMed1999

Angiogenesis inhibitors overcome tumor induced endothelial cell anergy.

We report here that tumor angiogenesis-mediated endothelial cell (EC) anergy can be overcome by inhibitors of angiogenesis. We found previously that tumor growth, known to be dependent on angiogenesis, results in down-regulation of endothelial adhesion molecules and tumor EC anergy to inflammatory signals. We hypothesized that counteracting angiogenesis induces re-expression of adhesion molecules and normalizes responses to inflammatory cytokines. Here, we present data to show that the angiogenesis inhibitor platelet factor-4 (PF4) is able to prevent basic fibroblast growth factor (bFGF)-induced down-regulation of intercellular adhesion molecule-1 (ICAM-1). Furthermore, PF4 restores ICAM-1 expression following bFGF-induced down-regulation of ICAM-1. This PF4 effect occurs at the protein level and the RNA level and it has functional impact on leukocyte adhesion. In addition, PF4 overcomes the tumor-induced EC anergy to inflammatory signals such as tumor necrosis factor alpha (TNF alpha). Our findings may be the basis of new cancer therapies by combining anti-angiogenic therapy and immunotherapy to decrease blood vessel formation and to increase the effectiveness of inflammatory reactions against tumors.

Animals↗
PubMed1991

Platelet factor 4 mRNA expression in cells from a patient with megakaryoblastic crisis of chronic myelogenous leukemia.

A 61-year-old man with Philadelphia chromosome-positive chronic myelogenous leukemia developed megakaryoblastic leukemia. In the blast phase, his blast cells showed undifferentiated megakaryoblastic characteristics with no alpha-granules or demarcation membranes but with detectable platelet peroxidase (PPO) activity and surface glycoprotein (GP) IIb/IIIa. The patient has remained reasonably well for at least 12 months after blastic crisis, and 6-mercaptopurine alone has been effective in controlling leukocytosis and megakaryoblast proliferation. The expression of mRNA for platelet-specific proteins, such as GPIIb and platelet factor 4 (PF4), was studied in the patient's blast cells by the Northern blot analysis. Both GPIIb and PF4 mRNA were detected in the blast cells. Cytoplasmic maturation occurs later than the synthesis of the surface GP during megakaryocyte maturation. Therefore, PF4 mRNA expression should be a marker of mature megakaryoblasts. The PF4 mRNA expression in megakaryoblastic leukemia may indicate that a patient will have long survival and a good response to chemotherapy.

Antibodies, Monoclonal↗
PubMed2002

Frequency of anti-heparin-platelet factor 4 antibodies in hemodialysis patients and correlation with recurrent vascular access thrombosis.

Heparin-induced thrombocytopenia (HIT), characterized by the formation of antibodies to a complex of platelet factor 4 (PF4) and heparin, is a well-recognized risk factor for thromboembolic complications. The frequency of antibody development varies among patient populations. Hemodialysis patients have repeated heparin exposure and should be at risk of developing HIT. This might, contribute to the development of vascular access thrombosis. We prospectively evaluated 88 hemodialysis patients for the presence of anti-PF4/heparin antibodies. Eighteen patients (20%) had a prior history of 1 or more prior access thrombosis. One patient (1.14%), without a history of graft thrombosis, tested positive for anti-PF4/heparin antibodies. In our study, the presence of anti-PF4/heparin antibodies was rare and was not increased in patients with a history of vascular access thrombosis.

Antibodies↗