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PubMed2006

[Effects of stromal cell-derived factor 1 and platelet factor 4 on the adhesion characteristics and chemotactic function of ex vivo expanded umbilical cord blood CD34+ cells].

To investigate the effects of stromal cell-derived factor 1 (SDF-1) and platelet factor 4 (PF4) on the homing-related function of expanded ex vivo umbilical cord blood CD34(+) cells, purified cord blood CD34(+) cells were cultured in serum-free medium containing a HGF combination of FL + SCF + TPO (FST) with either 100 ng/ml SDF-1 alone, 100 ng/ml PF4 alone, or both of these 2 cytokines. The expansion rate of CD34(+) cells, colony formation, homing-related functions including expression of homing-related adhesion molecules of expanded CD34(+) cell, adhesion activity and chemotactic function of the re-selected expanded CD34(+) cells were evaluated at different time points. The results showed that expansion rate of CD34(+) cells and expansion multiple of CFU in SDF-1 groups were higher than those in control. The expression of CD49e on the expanded CD34(+) cells was remarkable up-regulated, in contrast, expression of CXCR-4 on the expanded CD34(+) cells was remarkable down-regulated in SDF-1 groups. The expression of CD49e, CD54 and CXCR-4 on the expanded CD34(+) cells were remarkably up-regulated in the PF4 groups. In all the SDF-1 group, PF4 group and SDF-1 plus PF4 group, the ability of expanded CD34(+) cells adhering to fibronectin layer were higher than those in the control on day 10. Spontaneous migration rate of expanded CD34(+) cells in SDF-1 groups were higher than those in control, while SDF-1-induced migration rate were lower than those in control on day 10. SDF-1-induced migration rate in PF4 groups were higher than those in control on day 10. Spontaneous and SDF-1-induced migration rate of expanded CD34(+) cells in the SDF-1 plus PF4 groups were higher than those in control on day 10. It is concluded that, SDF-1 and PF4 can up-regulate expression of adhesion molecules on expanded CD34(+) cells, and retain the adherent and migration ability of expanded CD34(+) cells, which is helpful for the homing of expanded CD34(+) cells. In short, SDF-1 and PF4 are helpful for the homing-related function of the expanded UCB HSPC.

Antigens, CD34↗
PubMed1990

Platelet factor 4 and the platelet secreted proteoglycan: immunologic characterization by crossed immunoelectrophoresis.

Platelet factor 4 (PF4) is a hydrophobic, alpha-granule protein with potent antiheparin activity. It also binds to a chondroitin sulfate-containing proteoglycan (PG) isolated from platelets. In order to evaluate further the relationship between PF4 and the chondroitin sulfate-containing proteoglycan in resting platelets, the PF4-binding proteoglycan from human platelets has been purified using purified PF4 as an affinity ligand and used to prepare polyclonal antiserum. Two antisera have been characterized: one reacts primarily with chondroitin sulfate (CS), the other reacts with the protein core of the platelet proteoglycan after chondroitinase AC digestion. PF4 and PG core protein antigen are present in separate, dissimilar precipitin arcs when triton-solubilized platelets are analyzed by crossed immunoelectrophoresis using polyclonal antisera to purified PF4 and PG. PF4 was demonstrated in a complex with a separate chondroitin sulfate antigen by crossed immunoelectrophoresis (CIE) experiments in which either anti-PF4 or anti-CS antisera was incorporated in the intermediate gel. Both the PF4-chondroitin sulfate complex and the proteoglycan are secreted from platelets when fresh, washed human platelets are stimulated by human alpha-thrombin. This second antigen may represent the PG after posttranslational modification of a precursor form of the proteoglycan.

Blood Platelets↗
PubMed1985

Inhibition of human neutrophil receptor-mediated uptake of N-formyl-met-leu-phe by platelet factor 4 (59-70).

Human platelet factor 4 (PF4) and a substituent dodecapeptide designated PF4(59-70) elicited human neutrophil and monocyte chemotaxis with a similar concentration-dependence and maximal responses equal to that attained by chemotactic fragments of C5 (C5fr). At maximally chemotactic concentrations, PF4(59-70) stimulated the secretion by neutrophils of approximately 40% and 60% of the respective quantities of beta-glucuronidase and beta-glucosaminidase released by 10(-6) M N-formyl-methionyl-leucyl-phenylalanine (fMLP). In contrast to the deactivation of chemotaxis achieved by preincubation of neutrophils with other chemotactic factors, prior exposure to 10(-6)M PF4(59-70) for 2 min, or 20 min at 37 degrees, enhanced by 1.5- to 2-fold the chemotactic responses of neutrophils evoked by optimal concentrations of fMLP, C5fr, leukotriene B4, and PF4(59-70). Concentrations of PF4(59-70) which enhanced neutrophil chemotaxis inhibited the rate of receptor-mediated internalization of [3H]fMLP at 37 degrees and 18 degrees, but at 0 degrees failed to alter the binding affinity or the number of receptors for [3H]fMLP. Preincubation of neutrophils at 37 degrees with concentrations of PF4(59-70) which enhanced neutrophil chemotaxis also did not affect the subsequent binding of [3H]fMLP at 0 degrees. The inhibition by PF4(59-70) of the receptor-mediated internalization of [3H]fMLP was not mimicked by other positively charged compounds. The specific inhibition of receptor-mediated internalization of fMLP may explain the enhanced chemotactic responsiveness of neutrophils preincubated with PF4(59-70).

Acetylglucosaminidase↗
PubMed1995

Antibodies to macromolecular platelet factor 4-heparin complexes in heparin-induced thrombocytopenia: a study of 44 cases.

As heparin-PF4 (H-PF4) complexes are the target for antibodies associated to heparin-induced thrombocytopenia (HIT), an ELISA has been developed and optimised for testing antibodies binding to H-PF4. This test was consistently negative in 50 healthy subjects (A492 < 0.3) and 35 patients with other causes of thrombocytopenia (A492 < 0.5). In contrast, 43 out of 44 HIT patients showed antibodies to H-PF4 (A492 = 1.70 +/- 0.81) including 5 patients with a negative platelet aggregation test. In one patient with HIT, antibodies to H-PF4 were already present at day 7, whereas platelet counts dropped < or = 100 x 10(9)/l only at days 11-12. Surprisingly, among 41 patients under heparin for > 7 days, 5 showed antibodies to H-PF4, without HIT. These findings underline the major interest of this ELISA for the early diagnosis of HIT. We also showed that LMWH as well as other sulphated polysaccharides can bind to HIT antibodies in the presence of PF4 and that their reactivity is dependent on the molecular weight and the sulphation grade. The mechanism for HIT involves platelet PF4 receptors which bind the macromolecular H-PF4 complexes formed in the presence of a well defined heparin/PF4 ratio.

Adolescent↗
PubMed1994

Structural requirements of platelet chemokines for neutrophil activation.

Using recombinantly expressed proteins and synthetic peptides, we examined the structural/functional features of the platelet chemokines, neutrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4); that were important in their activation of neutrophils. Previous studies with the chemokine interleukin-8 (IL-8) had shown that the N-terminal region preceding the first cysteine residue was critical in defining neutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-terminus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybrid formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP-2, stimulated Ca2+ mobilization and elastase secretion. Furthermore, the effect of amino acid substitutions in the ELR motif differed from those seen with NAP-2 in that conserved substitutions of E or R in NAP-2 abolished activity, but only reduced neutrophil activation in the hybrid. These studies show that just as with IL-8, the N-termini of NAP-2 and PF4 are critical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP-2, which binds almost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensitize neutrophils to activation by IL-8 because IL-8 could bind to and activate via both type 1 and 2 IL-8R. AELR/PF4 appears to bind to both types of receptors because it desensitized neutrophils to NAP-2 activation; but was not desensitized by NAP-2, and because it desensitized to and was desensitized by IL-8. Thus, although NAP-2 and AELR/PF4 share approximately 60% amino acid homology, they have different receptor affinities. Studies were performed to define the role of the C-termini of these platelet chemokines in receptor binding. Heparin and a monoclonal antibody specific for the heparin-binding domain of PF4 both inhibited Ca2+ mobilization and elastase release, further suggesting that the C-terminus of these chemokines is important in receptor binding. Synthetic NAP-2(51-70) failed to mobilize Ca2+, whereas PF4(47-70) and PF4(58-70) induced Ca2+ mobilization and secretion of elastase at high concentrations. Pertussis toxin inhibited neutrophil activation by 40% to 50%, establishing a role for G-protein-coupled receptors such as the IL-8Rs in activation by the PF4 C-terminal peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

[Platelet factor 4, reversible inhibitor of megakaryocytogenesis, protector of megakaryocytes during chemotherapy].

Development of megakaryocyte (MK) from CD34+ cord blood (CB) cells in both plasma clot culture and liquid culture was significantly inhibited by human platelet factor 4 (PF4) and human transforming growth factor beta 1 (TGF beta 1). Inhibition of cell growth by PF4 was reversible judging from the fact that the CD34+ cells preincubated with PF4 could regenerate colonies after washing and replating into the cultures. By contrast, TGF beta 1-pretreated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF4 in liquid culture caused an increase in the number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells, and exhibited greater capacity to form MK colonies than control after the treatment of 5-FU. In vivo in mice, twice injections of PF4 at 40 micrograms/kg resulted in a significant increase in the number of colony-forming cells with high proliferative potential (HPP-CFC) and colony-forming unit-megakaryocyte (CFU-MK) in bone marrow. In exponentially growing human erythroleukemia cells (HEL), the addition of PF4 prolonged cell cycle progression and therefore resulted in an increased cell population in S phase, as determined by flow cytometric analysis. Different from PF4, TGF beta 1 blocked more cells in G1 phase. These results demonstrate that PF4 and TGF beta 1 inhibit MK development from CD34+ CB cells by different mechanisms and suggest that PF4, unlike TGF beta 1, exerts its inhibitory effect on cell growth in a reversible and S phase-specific manner by which it protects stem cells and MK progenitor cells from 5-FU cytotoxicity.

Animals↗
PubMed1998

Simultaneous binding of heparin and platelet factor-4 to platelets: further insights into the mechanism of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is mediated by antibody against complexes of platelet factor-4 (PF4) and heparin. Although it has been assumed that these complexes bind to platelets and provide a target for the antibody, this has never been demonstrated. Furthermore, there is evidence suggesting that heparin-PF4 complexes do not bind to platelets. We have analyzed the effect of each ligand on the platelet binding of the other. We particularly focused on the result when heparin and PF4 are in equimolar concentration because we had previously shown that this was the condition under which HIT-IgG increased on the platelet surface. We found that when the molar concentration of PF4 approximates or exceeds that of heparin, the ligands bind simultaneously to the cells and HIT-IgG binds also. However, when heparin is in molar excess, both PF4 binding and HIT-IgG binding are diminished. Our data are consistent with the hypothesis that heparin-PF4 complexes bind via their heparin component to heparin binding sites on the platelet membrane rather than by their PF4 component to PF4 sites. The conditions promoting the binding of the complexes also lead to binding of HIT-IgG.

Animals↗
PubMed1995

Inhibitory effects of nematode FMRFamide-related peptides (FaRPs) on muscle strips from Ascaris suum.

A large number of FMRFamide-related peptides (FaRPs) are found in nematodes, and some of these are known to influence tension and contractility of neuromuscular strips isolated from Ascaris suum body wall. Relaxation of these strips has been noted with five nematode FaRPs. The inhibitory actions of SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2) appear to be mediated by nitric oxide, as previously demonstrated with inhibitors of nitric oxide synthase (NOS). This present study showed that the effects of PF1 were also depended on external Ca++ and were reduced by the Ca(++)-channel blocker verapamil, observations consistent with the finding that nematode NOS is Ca(++)-dependent. KSAYMRFamide (PF3), KNIRFamide (PF4) and KNAFIRFamide (an alanine substituted analog of KNEFIRFamide, AF1, termed A3AF1) also relaxed A. suum muscle strips, but these responses were not affected by NOS inhibitors. PF3 inhibited the activity of strips prepared from the dorsal side of the worm, but contracted ventral strips. Both effects were dependent on the presence of ventral/dorsal nerve cords (unlike PF1/PF2) and were attenuated in medium which contained high K+ or low Ca++. PF4-induced muscle relaxation and hyperpolarization were independent of nerve cords, but were reversed in Cl-free medium, unlike PF1 or PF3. The PF4 effect physiologically desensitized muscle strips to subsequent treatment with PF4 and/or GABA. However, PF4 and GABA were not synergistic in this preparation. The effects of GABA, but not PF4, were reduced in muscle strips treated with the GABA antagonist, NCS 281-93. Following PF4 (or GABA) relaxation, subsequent treatment with higher doses of PF4 caused muscle strip contraction. A3AF1 was found to relax muscle strips and hyperpolarize muscle cells independently of the ventral and dorsal nerve cords, K+, Ca++, and Cl-, and mimicked the inhibitory phase associated with the exposure of these strips to AF1. On the basis of anatomical and ionic dependence, these data have delineated at least four distinct inhibitory activities attributable to nematode FaRPs. Clearly, a remarkably complex set of inhibitory mechanisms operate in the nematode neuromuscular system.

Animals↗
PubMed2002

Signal transduction of chemokine platelet factor 4 in human erythroleukemia cells.

Previous data have demonstrated that CXC-chemokine platelet factor 4 (PF4) inhibits the proliferation of the human erythroleukemia cell line (HEL). However, the mechanism of action is unclear at present. The signaling transduction induced by PF4 in the HEL was compared with that induced by transforming growth factor beta1 (TGF-beta1), which is also a potent inhibitor of HEL growth. It was found that PF4 had no inhibitory effect on intracellular calcium levels in resting HEL cells. When HEL cells were stimulated with interleukin-3 (IL-3), a rapid increase in the intracellular level of free calcium occurred within 15 to 20 seconds, and this increase was followed by a sustained increase that gradually declined until resting levels were reached 30 to 40 minutes later. PF4 dramatically decreased the transient rise of [Ca2+] and protein kinase C (PKC) activity of HEL cells induced by IL-3. However, PF4 had no inhibitory effect on PKC activation in resting HEL cells. Furthermore, PF4 was found to down-regulate significantly protein tyrosine kinase (PTK) activity. In contrast, TGF-beta1 induced an increase in intracellular free calcium concentration and PKC and PTK activity in HEL cells. Furthermore, PF4 significantly increased the messenger RNA (mRNA) level of p21waf1 in HEL cells. These data demonstrate that PF4 acts on HEL cells through a signaling transduction pathway, which is different from that of TGF-beta1 and is related to the up-regulatory mRNA level of p21waf1 in HEL cells.

Calcium↗
PubMed1984

Complementary adhesive responses of human skin fibroblasts to the cell-binding domain of fibronectin and the heparan sulfate-binding protein, platelet factor-4.

Plasma fibronectin (pFN) contains binding domains for an unidentified receptor on the surface of fibroblasts and for heparan sulfate chains of proteoglycans on these same cells. A series of experiments were designed to assess the relative importance of these activities in mediating substratum adhesion of human skin fibroblasts (strain 4449) grown in the absence of ascorbate (asc-) or in its presence (asc+) to minimize or maximize collagen production-maturation, respectively. The cell-binding fragment (CBF) of pFN was purified from chymotryptic digests free of any heparan sulfate-binding activity. The responses of cells to CBF were then compared with those mediated by the heparan sulfate-binding protein, platelet factor-4 (PF4). At early time points when cells had spread effectively on pFN, both asc- or asc+ cells extended spiky projections on PF4 and long projections on CBF with actively ruffling membranes at their tips. By 4 h, asc+ cells had spread much more effectively on CBF than asc+ cells on PF4 or asc- cells on either binding activity. Mixtures (w/w) of CBF:PF4 between 1:1 and 9:1 generated a more physiologically normal response than to either of the binding proteins alone, particularly for asc+ cells. Examination of cytoskeletal reorganization by fluorescence analysis with an antibody to 7S tubulin (for microtubules) and NBD-phallacidin (for F-actin) revealed condensations of microfilaments at the ruffling edges of asc- cells on CBF or on PF4 and for asc+ cells on PF4; in contrast, asc+ cells on CBF generated long bundles of microfilaments in their spreading lamellae within 4 h. Microtubule networks reorganized very well on CBF but only partially on PF4 with either cell type. Microfilament reorganization was comparable to that on intact pFN with CBF:PF4 mixtures of 1:1 and 9:1 for asc+ cells, whereas asc- cells generated condensations of microfilaments but little bundling. These studies reveal that the adhesive responses to mixtures of these two binding activities are significantly greater than to the individual activities and that the responses of asc+ cells approach the properties of cells on intact pFN, whereas asc- cells remain incapable of forming stress fiber-like bundles of microfilaments under all conditions.

Cell Adhesion↗
PubMed2006

Prevalence of heparin-induced thrombocytopenia in patients undergoing cardiac catheterization.

BACKGROUND: Heparin is ubiquitously used in cardiac catheterization but predisposes to the development of heparin-induced thrombocytopenia. The objective was to examine prospectively the prevalence of anti-platelet factor 4 (PF4)/heparin antibodies and heparin-induced thrombocytopenia in the population undergoing cardiac catheterization. METHODS: This is a prospective study of 500 consecutive patients presenting for cardiac catheterization at our institution who were enrolled over the course of 1 year. Anti-PF4/heparin antibodies and concurrent platelet counts were measured at catheterization and 5 days thereafter. Thrombotic complications were assessed 30 days after the procedure via telephone interview. All patients presenting for cardiac catheterization at our institution were screened. Inclusion criteria were (a) males and nonpregnant females with age >18 years and (b) patients scheduled to undergo cardiac catheterization. Patients with a known history of heparin-induced thrombocytopenia, with documented bleeding or hypercoagulability, and those at high risk for bleeding were excluded. RESULTS: Of 500 patients, 15 (3%) had anti-PF4/heparin antibodies before catheterization. After catheterization, the prevalence of anti-PF4/heparin antibodies increased to 10.1% (36 of 357) of the patients. Overall rates of thrombotic complications were low (4 of 445, 0.9%) and did not correlate with anti-PF4/heparin antibody status. Patients with an initial positive test for anti-PF4/heparin antibodies were more likely to have prior coronary disease (73.3% vs 45.2%; P < .05). Patients who developed anti-PF4/heparin antibodies after catheterization were more likely to have increased length of stay (3.7 vs 2.4 days; P = .02). The platelet count at the time of catheterization was lower in the cohort of patients who developed the second positive anti-PF4/heparin antibody test versus patients without a second positive antibody test (mean values of 191,800/microL vs 222,300/microL; P = .008). CONCLUSIONS: The prevalence of antibodies to PF4/heparin is low in the population presenting for cardiac catheterization. However, a significant proportion of patients develop antibodies to PF4/heparin after a small exposure to heparin during catheterization. Clinically significant thrombotic complications were rare and did not correlate with antibody status.

Aged↗
PubMed1995

Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding.

Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin. PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal alpha-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process.

Amino Acid Sequence↗
PubMed1996

Direct and reversible inhibition of platelet factor 4 on megakaryocyte development from CD34+ cord blood cells: comparative studies with transforming growth factor beta1.

Mechanisms of the action of platelet factor 4 (PF4) on the growth of megakaryocyte (MK) progenitor cells in CD34+ cord blood (CB) cells were studied in comparison with transforming growth factor beta1 (TGFbeta1). Development of MK from CD34+ CB cells in both plasma clot culture and liquid culture was significantly inhibited by either purified human PF4 and by recombinant human TGFbeta1. Inhibition of MK colony formation by PF4 was reversible because CD34+ cells preincubated with PF4 could regenerate colonies after washing and replating into secondary cultures. In contrast, TGFbeta1-preincubated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF4 in liquid culture caused the increased number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells. In addition, PF4-preincubated CD34+ cells exhibited a higher potential in MK colony formation in the presence of 5-fluorouracil (5FU). These results demonstrate that both PF4 and TGFbeta1 inhibit MK development from CD34+ CB cells by different mechanisms, and suggest that PF4, unlike TGFbeta1, exerts its inhibitory effect on the growth of the target cells in a reversible manner which results in a preservation of a more immature and 5FU-resistant cell population.

Antigens, CD34↗
PubMed1999

Heparin dodecasaccharide binding to platelet factor-4 and growth-related protein-alpha. Induction of a partially folded state and implications for heparin-induced thrombocytopenia.

alpha-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-alpha (Gro-alpha) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied. In the absence of H12, both PF4-M2 and Gro-alpha give diffusion coefficients consistent with the presence of mostly dimers. As the PF4-M2:H12 ratio is increased from 1:6 to 2:1, the diffusion coefficient increases, indicating dissociation to the monomer state. On addition of H12 to either protein, (15)N/(1)H heteronuclear single quantum coherence NMR data demonstrate loss of (1)H resonance dispersion and intensity, particularly at protein:H12 ratios of 2:1 to 4:1, indicating significant perturbation to native structures. For Gro-alpha in particular, (1)H resonance dispersion appears random coil-like. At these same ratios, circular dichroism (CD) data show general retention of secondary structure elements with a slight shift to additional helix formation. Random coil NMR resonance dispersion suggests a shift to a less compact, partially folded, and/or more flexible state. Further addition of H12 causes resonance intensity and dispersion to return making NMR spectra appear native-like. At low PF4-M2:H12 ratios, loss of resonance intensity for residues proximal to Arg-20 and Arg-22 in three-dimensional NMR HCCH-TOCSY spectra suggests that the Arg-20-Arg-22 loop either interacts most strongly with H12 and/or that binding at this site is heterogeneous. This domain was previously shown to be crucial to heparin binding. Of particular interest to the biology of PF4-heparin complex formation, heparin-induced thrombocytopenia antibody binding occurs at about the same PF4-M2:H12 ratio as does this transition to a partially folded PF4-M2 state, strongly suggesting that heparin-induced thrombocytopenia antibody recognizes a less folded, lower aggregate state of the protein.

Chemokine CXCL1↗
PubMed2005

Evidence for a granule targeting sequence within platelet factor 4.

Platelets achieve bleeding arrest at sites of vascular injury via secretion of secretory proteins from their storage granules, termed alpha-granules. We have recently analyzed granule targeting of platelet factor 4 (PF4), a secretory alpha-granule chemokine, and demonstrated that PF4 alpha-granule storage relied upon determinants within PF4 mature sequence. To define these determinants, PF4 mutants fused to the fluorescent reporter protein green fluorescent protein were generated by progressive deletions and site-directed mutagenesis. They were then transfected in AtT20 cells and assessed for granule targeting by colocalization with ACTH-containing granules, using laser scanning confocal microscopy. This strategy identified the amino acid 41-50 (LIATLKNGRK) sequence as most critical for PF4 granule targeting and/or storage; its deletion from PF4 induced a marked decrease in granule storage (from 81 +/- 2% to 17 +/- 3%, p < or = 0.0001). Ala-scanning mutagenesis of LIATLKNGRK narrowed down the targeting motif to LKNG. A direct role for LKNG in alpha-granule targeting was confirmed in the thrombopoietin-induced human megakaryocytic Dami cells, in which the LKNG-green fluorescent protein chimera exhibited an 82.5 +/- 1.8% colocalization with the alpha-granule proteins von Willebrand factor and P-selectin. LKNG is poorly conserved within the chemokine family. However three-dimensional alignments of the human alpha-granule chemokines Nap-2 (neutrophil-activating peptide) and RANTES (Regulated upon Activation Normal T Cell Expressed and Secreted) with PF4 revealed that LKNG, a surface-exposed hydrophilic turn/loop, matched Nap-2 (LKDG) and RANTES (TRKN) peptides with similar features. Moreover Nap-2 and RANTES peptides exhibited the same alpha-granule targeting efficiency than LKNG. We therefore postulate that the three-dimensional and physicochemical characteristics of PF4 LKNG are of general relevance to alpha-granule targeting of chemokines and possibly of other alpha-granule proteins.

Alanine↗
PubMedDate not supplied

Patients with paroxysmal atrial fibrillation but not paroxysmal supraventricular tachycardia display evidence of platelet activation during arrhythmia.

It is well known that chronic atrial fibrillation (CAF) and paroxysmal atrial fibrillation (PAF) are associated with hypercoagulable state. However, pathological hemostatic changes during the paroxysmal supraventricular tachycardia (PSVT) have not yet been elucidated. To determine platelet activity in patients with PSVT, PAF and CAF, we examined the levels of beta-thromboglobulin (BTG) and platelet factor 4 (PF4) during tachyarrhythmia attacks. We measured the levels of BTG and PF4, as an index of platelet activation in 15 patients with PAF (9 men, mean age 45+/-11), and 14 patients with PSVT (8 men, mean age 40+/-10). Levels were compared to 22 age and sex matched healthy controls in sinus rhythm and with 25 patients with CAF (16 men, mean age 51+/-12). Blood samples were taken during arrhythmia and 24 hours after conversion to sinus rhythm. Patients taking medications or have clinical conditions that may affect the BTG and PF4 levels were excluded. In patients with PAF, BTG and PF4 levels were significantly higher than in controls (p<0.009, and p=0.002, respectively), and in patients with PSVT (p<0.004, and p=0.009, respectively), however, BTG and PF4 levels were significantly lower than CAF patients (p=0.002, and p=0.02, respectively). Moreover, BTG and PF4 levels were significantly decreased 24 hours after conversion to sinus rhythm (p<0.0001), and p=0.004, respectively). Although BTG and PF4 levels in patients with PSVT were significantly lower than in patients with PAF (p=0.04, and p=0.009, respectively) and CAF (p=0.0001, and p=0.0001, respectively), BTG and PF4 levels were similar to controls and did not change significantly after recovery to sinus rhythm (p=NS for all). These results indicate that there was no platelet activation in patients with PSVT during tachyarrhythmia but significantly increased platelet activity in PAF and CAF patients. There was a significant decrement of the platelet activity to a level of control subjects twenty-four hours after cardioversion of PAF.

Adult↗
PubMed2003

Suppression of tumor growth by viral vector-mediated gene transfer of N-terminal truncated platelet factor 4.

Platelet factor four (PF4), an inhibitor of endothelial cell proliferation in vitro, inhibits angiogenesis and tumor growth in vivo in experimental animals. The present study was designed to determine whether gene therapy-mediated expression of a form of PF4 lacking 16 amino acids of N-terminus from tumor cells could inhibit angiogenesis and tumor growth in vivo. Two replication-defective recombinant retroviral vectors were constructed. One encodes human PF4 (rRV-PF4) and the other encodes the N-truncated peptide (rRVp17-70). These vectors were then used to transduce KB cells, a human head and neck squamous carcinoma cell line. Expression of PF4 and p17-70 transgenes was confirmed by Western blot analysis. In vitro, both rRV-PF4 and rRVp17-70 were able to inhibit selectively the proliferation of human umbilical vascular endothelial cells (HUVEC) but not KB cells. In vivo activity was assessed by injecting 10(7) KB cells subcutaneously into nude mice and by monitoring subsequent tumor growth, xenograft vascular histochemistry, and animal survival. Viral vector-mediated cDNA transfer of PF4 and p17-70 resulted in inhibiting solid tumors through an anti-angiogenic action in vivo. Our data indicate that targeting tumor angiogenesis using viral-mediated gene transfer of full-length and N-terminal truncated PF4 represents a promising strategy for cancer gene therapy.

Animals↗
PubMed1995

Heparin neutralization by recombinant platelet factor 4 and protamine.

Protamine is the only available drug to reverse heparin-induced anticoagulation. Platelet factor 4 (PF4) is a basic polypeptide stored in platelets that reverses heparin. To investigate its potential as a reversal drug, we studied recombinant PF4 on anticoagulated blood obtained during cardiac surgery. Blood was obtained from 33 different venous reservoirs, and activated clotting time (ACT), heparin concentrations, and heparinase-ACT were determined. Anticoagulation was reversed by adding incremental PF4:heparin and protamine:heparin ratios to the heparinized blood, and the ACTs were determined (n = 21). Viscoelastic analysis of anticoagulation reversal was performed by adding protamine or PF4 at reversal ratios of 1.3:1 protamine:heparin, and 3.2:1 PF4:heparin using thromboelastography (n = 12). PF4 reversal ratios of 3:1 and 3.5:1 and protamine reversal ratios of 1:1, 1.5:1, 2:1 were not statistically different from heparinase-ACT values. There were no significant differences in viscoelastic measurements of clot formation between protamine and PF4. Recombinant PF4 at a 3.0:1 ratio reverses heparin-induced anticoagulation after cardiopulmonary bypass, and represents a potential alternative, especially for the protamine allergic patient.

Blood Coagulation↗