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. Only on rare occasions (0.7 +/- 1.5 site per cm2 skinfold), short (37 +/- 48 microns) intense labeled sites were found in the normal vasculature of the skinfold. Heparin could displace most of the label if injected within 10 min after the rhPF4-injection, but not 30 min after. In conclusion, our results show that rhPF4 preferentially binds to regions of active angiogenesis in vivo, supporting the concept of using rhPF4 conjugates to target tumors in cancer patients. Certain rhPF4 conjugates could have applications as imaging agents, with potential utility in identification, screening, detection, prognosis or staging of breast cancer and other cancers. Other similar conjugates, bearing therapeutic isotopes or toxins might be useful in selective treatment strategies.

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. Mutation of either of the two DLQ motifs within PF4 resulted in an inactive protein. Perturbations within the IL-8 dimer interface region also resulted in mutants that were incapable of suppressing colony formation. A class of chimeric mutants consisting of domains of either PF4 and IL-8, Gro-alpha and PF4, or Gro-beta and PF4 were observed to inhibit myeloid cell proliferation at concentrations which were between 500- and 5000-fold lower than either the IL-8 or PF4 wild-type proteins alone. These chimeric mutants possessed activities that were comparable to or better than the activity observed when IL-8 and PF4 were added together in vitro. One of these highly active chimeric proteins was observed to be 1000-fold more active than either IL-8 or PF4 alone in suppressing not only the proliferation but also the cell cycling of myeloid progenitor cells following intravenous injection of the mutant into mice. Examination of additional IL-8-based mutants in the colony formation assay, which centered on the perturbation of the amino-terminal "ELR" motif, resulted in the observation that the highly active IL-8 mutant required both aspartic acid at amino acid residue 4 and either glutamine or asparagine at residue 6. Single mutations at either of these positions resulted in mutants with myelosuppressive activity equivalent to wild-type IL-8. Mutants such as IL-8M1 and IL-8M10 were observed to be significantly reduced in their ability to activate isolated human neutrophils, suggesting that separate mechanisms may exist by which myeloid progenitor cells and neutrophils are affected by chemokines.

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. Intra-arterially injected rhPF4 labeled, with high intensity, the endothelium along newly formed vessels of the islets (1,632 +/- 617 microns labeled vessel length per islet), and only on rare occasions (1 +/- 2 sites per cm2 skinfold) were short (62 +/- 48 microns) intense-labeled sites found in the normal vasculature of the skinfold. Heparin could displace most of the label if injected within 10 min after the rhPF4 injection, but not 30 min after. In conclusion, rhPF4-preferentially binds to regions of active angiogenesis in vivo. On binding, rhPF4 is internalized as judged from a decreasing heparin sensitivity with time after rhPF4 injection. The infrequent rhPF4-labeling sites in the normal skin vasculature most likely represent regions of newly formed cells/migration, i.e., normal endothelial turnover, supporting our previous findings demonstrating that the occurrence of such regions is rare in the normal microvasculature. Furthermore, despite the previously demonstrated short half-life in plasma, systemically injected rhPF4 will target regions of angiogenesis with high affinity, thereby facilitating the antiangiogenic effect of PF4.

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. The length of the intense sites was 76 +/- 46 microns and their frequency was 10 +/- 4 per cm2 cheek pouch. Heparin was injected at 4 and 9 min, but not 30 min, post-rhPF4 displaced most of the high intensity labelling indicating internalization with time. Neither pretreatment with more than 50-fold excess of unlabelled rhPF4 nor histamine- or LTB4-induced vascular macromolecular leakage changed the frequency of short intense sites. In vitro data: uniform time-dependent intense binding of rhPF4 occurred in a similar fashion in subconfluent HUVEC, HUAEC and HAEC. All cell types showed nuclear staining, demonstrating internalization. When heparin was given to EC prior to rhPF4, binding was delayed in time but not blocked. In conclusion, rhPF4 does not bind uniformly with high intensity along pre- and post-capillary vessels of the hamster cheek pouch in vivo as predicted by the rhPF4-labelling of subconfluent (migrating/proliferating) human EC in vitro. The short infrequent sites of intense rhPF4-labeling in vivo may represent regions of endothelial cell migration/proliferation similar to subconfluent EC in culture.

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). In contrast, PF4 was not able to inhibit the binding of VEGF121, a VEGF isoform which lacks a heparin binding capacity, to the VEGF receptors of the cells or to KDR/flk-1. These results indicate that PF4 may inhibit VEGF165 binding to VEGF receptors by disrupting the interaction of VEGF165 with cell surface heparan sulfates. Since PF4 mutants lacking a heparin binding ability retain their anti-angiogenic activity, alternative inhibitory mechanisms were also examined. 125I-PF4 bound with high affinity (Kd 5 x 10(-9) M) to VEGF165-coated wells. The binding of 125I-PF4 to the VEGF165-coated wells was inhibited by several types of heparin binding proteins, including unlabeled PF4 and unlabeled VEGF165. The binding was not inhibited by proteins which lack a heparin binding capacity, nor was it inhibited by VEGF121. Heparinase did not inhibit the binding of 125I-PF4 to VEGF165, indicating that heparin-like molecules are not required. These experiments suggest that PF4 can bind to heparin binding proteins such as VEGF165 leading to an inhibition of their receptor binding ability. In agreement with these results, we have observed that PF4 inhibits efficiently the VEGF165 induced proliferation of vascular endothelial cells. Unexpectedly, PF4 also inhibited efficiently the VEGF121-induced proliferation of the cells, indicating that PF4 can disrupt VEGF receptor mediated signal transduction using an unknown mechanism which does not interfere with VEGF121 binding.

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. After 30 minutes of cardiopulmonary bypass, recombinant platelet factor 4 normalized thrombin times and activated partial thromboplastin times within minutes of injection, but protamine did not. Neither drug altered bleeding times. Recombinant platelet factor 4 caused a species-specific leukopenia in baboons and significantly increased activated complement protein 3 (C3a) more than protamine. However, the increase in plasma C3a was small and neither drug caused a significant increase in plasma neutrophil elastase-alpha 1 proteinase inhibitor complex. We conclude that recombinant platelet factor 4 is effective and safe in baboons, does not have an anticoagulant effect with excess concentration, and reverses in vivo heparin more rapidly than protamine. The data support progression to a clinical trial.

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. Sialidase digestion reduces both forms to a single mass of 10,497 Da. Upon Edman degradation, the amino terminus was found to be blocked by the presence of a pyroglutamate residue. We have determined the complete primary structure of platelet factor 4 by peptide mapping and Edman degradation, thereby completing information on the amino-terminal and carboxy-terminal regions which is missing in the previously published partial sequence. Sequencing of the intact and deglycosylated protein show that the glycosylation site is at Thr8. The amino acid composition accounts for a mass of 9623 Da, and the carbohydrate moeity was found to contribute 1490 Da. The biological activity of the porcine protein has been compared to recombinant human platelet factor 4 in an endothelial cell proliferation assay; both inhibit at a concentration giving half the maximal inhibition of 0.1 microM. Removal of the 19 amino-terminal residues carrying the carbohydrate moiety results in no change in the biological activity.

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. METHODS: B16F10 cells (0.5-1.0 x 10(5) were administered intravenously to mice; 21 days later, the lungs were removed and the tumor foci were counted. Treatments with rPF4 were given to C57BL/6J mice immediately following tumor inoculation either (a) intravenously as a single dose (0.375, 0.75, 1.5, or 2.0 mg) or as multiple doses (6 mg total) over a 48-hour period, (b) subcutaneously or intramuscularly as multiple doses (6 mg total) over a 72-hour period, or (c) subcutaneously as multiple doses (6 mg total) over a 92- or 96-hour period following a delay of 4, 24, or 48 hours. (BALB/cByJ x C57BL/6)F1 (CByB6F1/J) athymic nude mice received rPF4 subcutaneously over a 72-hour period. The ability of rPF4 to block binding of 51Cr-labeled B16F10 tumor cells to matrix-coated microtiter plates was evaluated in vitro. The in vivo effect of intravenously injected rPF4 on the retention of 51Cr-labeled B16F10 cells was examined by determining the remaining lung-associated radioactivity after 30 minutes or 1, 2, or 4 hours. RESULTS: Intravenous administration of rPF4 significantly inhibited the development of metastatic lung nodules in a dose-dependent fashion as assessed by both lesion number (P < .03) and lung weight (P < .05). When initiation of subcutaneous treatment with rPF4 was delayed 24-48 hours, the number of metastatic foci was significantly reduced (P < .05). The antitumor effect of rPF4 was not dependent on a T-lymphocyte-mediated process, since subcutaneous rPF4 treatment also suppressed the number of lung metastases in T-cell-deficient athymic CByB6F1/J nude mice. In vitro experiments demonstrated modest inhibitory effects (28%) of rPF4 on B16F10 tumor cell binding to purified murine vitronectin. However, lung clearance experiments at early time points (0.5 hour and 1 hour) showed that tumor lodging was not affected by rPF4 treatment. CONCLUSIONS: The administration of rPF4 by systemic routes inhibited the development of experimental lung metastases. These findings are consistent with the known angiostatic properties of rPF4, and we conclude that inhibition of metastatic tumor formation may be due to inhibition of tumor-induced neovascularization. IMPLICATIONS: These results support the testing of rPF4 as an angiostatic agent in the treatment of metastatic tumors.

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. Whole blood aliquots, without reversal agents (controls) or with either protamine (10, 20, 30, or 40 micrograms/ml) or recombinant platelet factor 4 (10, 20, 40, or 80 micrograms/ml), were analyzed. The quantity of each agent required to reverse the ten samples, using 95% upper confidence bounds (t distribution) was determined for each method. Recombinant platelet factor 4 reversed heparin at 40 micrograms/ml and protamine at 20 micrograms/ml, suggesting a reversal ratio for recombinant platelet factor 4/protamine of 2:1 on a milligram basis. Further, currently available methods for testing coagulation should be reliable, without modification, to monitor the restoration of normal coagulation parameters with recombinant platelet factor 4 after cardiopulmonary bypass.

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%. The structure consists of four polypeptide chains which form a tetrameric unit. N-terminal residues, previously defined as a random coil or extended loop region, form antiparallel beta-sheet-like structures that form noncovalent associations between dimers. These antiparallel beta-sheet-like structures are positioned lateral to the beta-bilayer motif and stabilize the tetrameric unit. A positively charged ring of lysine and arginine side chains encircles the PF4 tetramer sphere, presenting multiple potential sites and orientations for heparin binding. The electrostatic interactions of multiply charged amino acid side chains and hydrogen bonding interactions at the AB/CD dimer interface serve to stabilize the tetrameric structure further.

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. Injection of rPF4 or PS 2 min following injection of 3H-heparin augmented loss of radioactivity from the circulation over the first 2 min but did not affect the half life of 3H-heparin for the next 58 min. PS was coupled to 4-(p-Azidosalicylamido)butylamine (ASBA), radioiodinated and purified by means of heparin-agarose chromatography. Heparin prevented the rapid loss of 125I-rPF4 from the circulation within the first 2 min but modestly increased loss of radioiodinated derivatized PS. Heparin extended the half-life of derivatized radioiodinated PS (measured between 2 and 60 min after injection) while modestly shortening that of 125I-rPF4. Both radioiodinated heparin binding proteins accumulated predominantly in liver and kidney. A greater percentage of radioactivity was found in these organs with rPF4 than with PS but more PS was found in urine. A larger percentage of radioiodinated derivatized PS than 125I-rPF4 was undetected. These results indicate that rPF4 and PS affect the kinetics of heparin clearance similarly but that organ deposition of the two agents may differ and offer an explanation of different physiological effects seen previously.

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. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.

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. For determination of activated partial thromboplastin time, platelet count, white blood cell count, and complement titer, arterial blood samples were taken before and immediately after heparin (10 units/100 g) infusion and at several time points after the infusion of the neutralizing agent (PS, 0.1 mg/100 g; PF4, 0.5 mg/100 g). In separate groups of animals, mean arterial blood pressure was monitored throughout identical protocols and the lungs were prepared for histological examination. The anticoagulant activity of heparin was effectively reversed by all of the neutralizing agents (PS, PF4, and rPF4). Platelet count (48% of initial), white blood cell count (52% of initial), complement titer (60% of initial), and mean arterial pressure (20% decrease) decreased significantly in heparinized animals receiving PS but not in those receiving PF4 or rPF4. Lung interstitium appeared normal when heparin was followed by PF4; however, interstitial edema and hemorrhage were observed with heparin-PS. CONCLUSIONS: These results suggest that PF4 efficiently reverses heparin anticoagulation in the rat without the adverse effects of heparin-protamine complexes. Therefore, rPF4 may be an appropriate substitute for PS in patients undergoing cardiovascular surgery and other procedures that require heparin anticoagulation.

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. Injection of an equivalent amount of cytochrome c, buffer alone, or an amino-terminal PF-4 peptide failed to elicit a significant inflammatory response; however, the carboxy-terminal PF-4 peptide retained proinflammatory properties. The inflammatory infiltrate induced by a single injection of either rPF-4 or the 41 amino acid carboxy-terminal peptide was composed of neutrophils and smaller numbers of mononuclear cells. Repeated injection of rPF-4 resulted in nearly equal numbers of neutrophils and mononuclear cells. Moreover, marked dermal fibrosis developed after only 5 days of daily injection of rPF-4. Although relatively high concentrations of rPF-4 were required to elicit an inflammatory response, these concentrations may be locally attainable during platelet aggregation. Our findings thus support the hypothesis that PF-4 may contribute to the development of inflammatory responses at sites of platelet aggregation.

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. Similar antitumor effects were observed with the human colon carcinoma, HCT-116, grown in nude mice, indicating that the inhibitory activity was neither tumor-type specific nor T-cell dependent. rPF4-241 inhibited endothelial cell proliferation in vitro with dose dependence similar to the native sequence rPF4. Both rPF4 and rPF4-241 inhibited angiogenesis in the chicken chorioallantoic membrane. The analogue, however, was inhibitory at lower concentrations than rPF4 in the chorioallantoic membrane system and its inhibitory effects were not abrogated by the presence of heparin. The present findings support the conclusion that both rPF4 and rPF4-241 inhibit tumor growth by suppression of tumor-induced neovascularization. The finding that this activity is independent of heparin binding may allow the development of PF4-based angiostatic agents with reduced toxicity and improved bioavailability. These results also suggest that PF4 may play a more specific role in modulation of blood vessel development than previously recognized.

Expression and purification of active recombinant platelet factor 4 from a cleavable fusion protein.

A synthetic gene for human platelet factor 4 (hPF4) has been expressed at high levels as a fusion protein in Escherichia coli. The hPF4 sequence has been cleaved from the fusion protein by cyanogen bromide treatment and purified by column chromatography. Like hPF4, our recombinant hPF4 (rhPF4) is tetrameric under physiological conditions, binds heparin, and inhibits angiogenesis. Extensive purification to remove trace amounts of uncleaved fusion protein completely from the desired product rhPF4 was difficult. We have exploited recombinant DNA technology by modifying the fusion moiety to accomplish separation. This type of modification, which did not affect expression level, could be applied to other recombinant fusion proteins.

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. The process of angiogenesis is beginning to be better understood, and as Ted Maione and Richard Sharpe explain, this understanding has led to the identification of several lead compounds that inhibit this process. At present all of these candidate drugs exhibit severe host toxicity, but more selective angiogenesis inhibitors might be expected to be extremely useful therapeutic agents.

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. Nonspecific binding to tRNA does not solubilize Rev. As judged by difference CD spectra, the conformation of Rev when bound to the RRE at neutral pH is similar to the conformation of unbound Rev at pH 3, although changes in the RNA may also contribute to the difference spectrum. Indeed, some difference is observed near 260 nm, consistent with a conformational change of the RRE upon Rev binding. Rev alone at pH 3 shows irreversible aggregation as the temperature is raised, while Rev bound to the RRE at neutral pH shows a reversible transition with a Tm of 68 degrees C.

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. rHuPF 4 effectively suppressed the growth of the B16-F10 murine melanoma in syngeneic C57BL/6J hosts and prevented the growth of primary tumors of both B16-F10 murine melanoma and HCT 116 human colon carcinoma in semisyngeneic CByB6F1/J female athymic nude mice. These two transformed cell lines were completely insensitive to rHuPF 4 in vitro at levels (50 micrograms/mL) that extensively inhibit normal endothelial cell proliferation. The migration of human endothelial cells was also inhibited at these concentrations of rHuPF 4, suggesting a second mechanism by which rHuPF 4 may modulate capillary development. The observed antitumor effects of rHuPF 4 might be due to the inhibition of angiogenesis. This finding could have implications for the development of novel therapeutic approaches to angiogenic diseases. Alternative, and possibly concurrent, mechanisms of the rHuPF 4 antitumor effect include lymphokine-activated killer cell activation and the induction of other cytokines.

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. Understanding of the mechanisms of control of angiogenesis by endogenous proteins should facilitate the development of effective treatments for diseases of pathogenic neovascularization such as Kaposi's sarcoma, diabetic retinopathy, and malignant tumor growth.