Recombinant platelet factor 4, an angiogenic marker for human breast carcinoma.

The present study was designed to test the hypothesis that rhPF4 binds with high specificity to the neovasculature of breast cancer carcinoma. To achieve this goal, we used intravital microscopy to study the binding characteristics of systemically injected fluorescently labeled rhPF4 (FITC-rhPF4) to the microvasculature of dorsal skinfold chambers in nude mice implanted with tumor spheroids prepared from the human breast cancer cell line MCF-7. Our results show that intravenously as well as intra-arterially injected FITC-rhPF4 exclusively labeled, with high intensity and specificity, the endothelium of the breast cancer induced neovasculature.

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.

High activity suppression of myeloid progenitor proliferation by chimeric mutants of interleukin 8 and platelet factor 4.

The proliferation of human myeloid progenitor cells is negatively regulated in the presence of certain members of the chemokine family of molecules. This includes interleukin 8 (IL-8) and platelet factor 4 (PF4), which in combination are able to synergize, resulting in cell suppression at very low concentrations of these molecules. A series of PF4 and IL-8 mutant proteins were analyzed in an in vitro colony formation assay for myeloid progenitor cells to assess domains of these proteins that are required for activity.

Selective binding of platelet factor 4 to regions of active angiogenesis in vivo.

In a previous study we suggested that recombinant human platelet factor 4 (rhPF4) preferentially binds in vivo to regions of active angiogenesis/endothelial cell migration. To test this hypothesis, binding of fluorescently labeled rhPF4 to newly formed vessels was compared with that of the normal skin vasculature, using syngeneic Langerhans islets as inducers of angiogenesis. Islets were implanted in the dorsal skinfold chamber of the hamster, and the binding of rhPF4 was studied using intravital fluorescence microscopy.

Differences in binding of platelet factor 4 to vascular endothelium in vivo and endothelial cells in vitro.

The binding of fluorescein-labelled recombinant human platelet factor 4 (rhPF4) to the vasculature of the hamster cheek pouch in vivo was compared with that to cultured endothelial cells (EC) from human umbilical veins (HUVEC) and arteries (HUAEC) and from human aorta (HAEC). In vivo data: systemically injected rhPF4 rapidly disappeared from plasma in a biphasic pattern (t1/2 = 2 and 41 min). High intensity non-uniform binding of rhPF4 occurred at short specific sites along both arterioles and venules.

Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms.

The 121-amino acid form of vascular endothelial growth factor (VEGF121) and the 165-amino acid form (VEGF165) are mitogenic for vascular endothelial cells and induce angiogenesis in vivo. VEGF165 possesses a heparin binding ability and in the absence of heparin-like molecules does not bind efficiently to the VEGF receptors of vascular endothelial cells. The binding of 125I-VEGF165 to the VEGF receptors of endothelial cells, and the heparin-dependent binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1, were inhibited by the angiogenesis inhibitor platelet factor-4 (PF4).

Reversal of heparin anticoagulation by recombinant platelet factor 4 and protamine sulfate in baboons during cardiopulmonary bypass.

The ability of recombinant platelet factor 4 and protamine to neutralize heparin after cardiopulmonary bypass was compared in anesthestized baboons. Clotting titration curves of heparinized baboon blood demonstrate an anticoagulant effect of protamine that is not seen with recombinant platelet factor 4. Neither drug caused meaningful changes in central pressures or cardiac output within 30 minutes after injection.

The complete primary structure of glycosylated porcine platelet factor 4.

We have purified platelet factor 4 from porcine platelets and shown that it is glycosylated. The purified protein migrated as a broad band at approximately 14,000 Da, characteristic of glycoproteins. Electrospray mass spectroscopy of the intact protein gave a predominant mass of 11,111 Da, with a minor component of 10,804 Da.

Inhibition of development of murine melanoma lung metastases by systemic administration of recombinant platelet factor 4.

Background: When administered locally, recombinant platelet factor 4 (rPF4), a known angiogenesis inhibitor, has been shown to effectively suppress murine melanoma and human colon carcinoma primary tumor growth in mice. It was tentatively concluded that this effect was due to the inhibition of tumor neovascularization.

Purpose: This study has evaluated the effects of systemically administered rPF4 on the growth and establishment of experimental B16F10 melanoma lung metastases in syngeneic mice.

Recombinant platelet factor 4 reversal of heparin in human cardiopulmonary bypass blood.

The ability of recombinant platelet factor 4, a protein of human origin with high heparin affinity, and the present clinical heparin reversal agent, protamine, to neutralize heparin in human whole blood was studied by means of three standard whole blood coagulation tests: whole blood clotting time, heparin assay, and activated clotting time. Ten subjects were chosen at random among patients undergoing cardiopulmonary bypass operations. Heparinized blood, free of protamine, was obtained from the bypass reservoir for testing.

Crystal structure of recombinant human platelet factor 4.

The crystal structure of human platelet factor 4 (PF4) has been solved to a resolution of 2.4 A by molecular replacement and refined to an R-factor of 24.1%.

Evaluation of recombinant platelet factor 4 and protamine sulfate for heparin neutralization: clotting and clearance studies in rat.

Recombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in rats without the adverse effect of protamine sulfate (PS) (Circulation 1992; 85: 1102). This study confirmed that rPF4 and PS neutralized heparin in rats. In vitro addition of excess PS but not rPF4 to plasma prolonged the activated partial thromboplastin time.

Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association.

Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state.

Platelet factor 4 efficiently reverses heparin anticoagulation in the rat without adverse effects of heparin-protamine complexes.

Background: It has been observed that the reversal of heparin anticoagulation in humans by protamine sulfate (PS) results in various adverse reactions including leukopenia, thrombocytopenia, activation of complement, increased vascular permeability, systemic hypotension, pulmonary vasoconstriction, and pulmonary edema. The purpose of this study was to compare the efficacy and effects of native platelet factor 4 (PF4) and recombinant platelet factor 4 (rPF4) with those of PS in heparin neutralization in vivo, using a rat model.

Methods And Results: Sprague-Dawley rats were anesthetized with sodium pentobarbital, and the right femoral vein and carotid artery were cannulated.

Induction of local inflammation by recombinant human platelet factor 4 in the mouse.

Platelet factor 4 (PF-4) has been shown to be chemotactic for neutrophils and monocytes in vitro. To assess whether these observations have in vivo relevance, we tested the ability of recombinant human PF-4 (rPF-4) to induce acute and chronic dermal inflammation in the mouse. When injected as a single dose intradermally, rPF-4 induced an acute inflammatory response that peaked at 6 to 12 hr and which resolved by 36 hr.

Inhibition of tumor growth in mice by an analogue of platelet factor 4 that lacks affinity for heparin and retains potent angiostatic activity.

An analogue of human platelet factor 4 (PF4) lacking affinity for heparin was specifically designed to evaluate the importance of this property in the antitumor effects of recombinant PF4. The purified protein, recombinant PF4-241 (rPF4-241), failed to bind heparin but retained the ability to suppress the growth of tumors in mice. Daily intralesional injections of rPF4-241 significantly inhibited the growth of the B-16 melanoma in syngeneic mice without direct inhibitory effects on B-16 cell growth in vitro.

The lentivirus regulatory proteins REV and REX are site specific RNA binding proteins.

These studies demonstrate that HIV-1 REV and HTLV-1 REX are site specific RNA binding proteins. In addition, the REV protein studies indicate a protein domain essential for biological function that is not involved in RRE binding. Lentiviruses are unique among retroviruses in providing gene products to switch expression from early to late genes.

Development of angiogenesis inhibitors for clinical applications.

Angiogenesis, the development of new blood vessels, is associated with many life-threatening pathologies. The neovascularization of tumors for example, allows a blood supply to deliver the required nutrients for tumor development. Inappropriate blood vessel growth also contributes to the pathology of other diseases such as atherosclerosis and arthritis.

Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site.

The circular dichroism (CD) spectrum of the Rev protein from HIV-1 indicates that Rev contains about 50% alpha helix and 25% beta sheet at 5 degrees C in potassium phosphate buffer, pH 3, and 300 mM KF. The spectrum is independent of protein concentration over a 20-fold range. At neutral pH, Rev is relatively insoluble but can be brought into solution by binding to its specific RNA binding site, the Rev-responsive element (RRE), at a Rev:RNA ratio of about 3:1.

Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4.

Although it is well established that angiogenesis is essential to tumor development, no human protein with high specificity and efficacy for prevention of angiogenesis has been characterized. In a previous study, we demonstrated that recombinant platelet factor 4 (rPF 4) inhibited angiogenesis in the chicken chorioallantoic membrane. In the present study, we have extended that finding to the use of recombinant human platelet factor 4 (rHuPF 4) to inhibit solid tumor growth in the mouse.

Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides.

Recombinant human platelet factor-4 (rhPF4), purified from Escherichia coli, inhibited blood vessel proliferation in the chicken chorioallantoic membrane in a dose-dependent manner. Treatment of several cell types with rhPF4 in vitro suggested that the angiostatic effect was due to specific inhibition of growth factor-stimulated endothelial cell proliferation. The inhibitory activities were associated with the carboxyl-terminal, heparin-binding region of the molecule and could be abrogated by including heparin in the test samples, an indication that sulfated polysaccharides might modulate the angiostatic activity of platelet factor-4 in vivo.

Specific binding of HIV-1 recombinant Rev protein to the Rev-responsive element in vitro.

The human immunodeficiency virus type 1 (HIV-1) genome encodes the regulatory protein Rev, of relative molecular mass 13,000, which is synthesized from fully processed viral transcripts before synthesis of HIV-1 structural proteins. Rev has been postulated to exert control within the nucleus at the level of messenger RNA processing. The availability of Rev in the nucleus serves to increase the proportion of unspliced and singly spliced mRNA species relative to fully spliced mRNA molecules, resulting in an increased synthesis of viral structural proteins.

Cyclosporine inhibits basic fibroblast growth factor-driven proliferation of human endothelial cells and keratinocytes.

Keratinocytes and endothelial cells produce basic fibroblast growth factor (b-FGF), and this cytokine is mitogenic for both cell types. Additionally, b-FGF is stored in the vicinity of keratinocytes and endothelial cells in basement membrane and extracellular matrix, and can be displaced from these "buffers" by various stimuli. Displacement of b-FGF by physical stimuli, such as scratching or rubbing, could explain koebnerization in diseases such as psoriasis.

Association of the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardii with Photoreduction and the Oxyhydrogen Reaction.

The hydrogenase-dependent processes, photoreduction and the dark oxyhydrogen reaction, both of which can support CO(2) assimilation, were compared with aerobic photosynthesis and respiration for their sensitivity to electron transport inhibitors in cells and intact chloroplasts of Chlamydomonas reinhardii 11-32/6. Photoreduction but not photosynthesis was inhibited in chloroplasts and the oxyhydrogen reaction detected only in cells was inhibited up to 75 and 90%, respectively, by 150 micromolar rotenone, indicating the involvement of a NAD(P)H-plastoquinone oxidoreductase in the hydrogen utilizing pathways. The oxyhydrogen reaction coupled to CO(2) fixation was inhibited more than 95% by 10 micromolar 2,5 - dibromo - 3 - methyl - 6 - isopropyl - p - benzoquinone (DBMIB), a concentration which did not affect respiratory activity.