©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CXC Chemokines Connective Tissue Activating Peptide-III and Neutrophil Activating Peptide-2 are Heparin/Heparan Sulfate-degrading Enzymes (*)

(Received for publication, August 31, 1994; and in revised form, December 6, 1994)

Arlene J. Hoogewerf (1)(§) Joseph W. Leone (2) Ilene M. Reardon (2) W. Jeffrey Howe (3) Darwin Asa (4) Robert L. Heinrikson (2) (4) Steven R. Ledbetter (1)(¶)

From the  (1)Units of Cancer & Infectious Disease, (2)Biochemistry, (3)Computational Chemistry, and (4)Adhesion Biology, The Upjohn Company, Kalamazoo, Michigan 49001

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Heparan sulfate proteoglycans at cell surfaces or in extracellular matrices bind diverse molecules, including growth factors and cytokines, and it is believed that the activities of these molecules may be regulated by the metabolism of heparan sulfate. In this study, purification of a heparan sulfate-degrading enzyme from human platelets led to the discovery that the enzymatic activity resides in at least two members of the platelet basic protein (PBP) family known as connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2. PBP and its N-truncated derivatives, CTAP-III and neutrophil activating peptide-2, are CXC chemokines, a group of molecules involved in inflammation and wound healing. SDS-polyacrylamide gel electrophoresis analysis of the purified heparanase resulted in a single broad band at 8-10 kDa, the known molecular weight of PBP and its truncated derivatives. Gel filtration chromatography of heparanase resulted in peaks of activity corresponding to monomers, dimers, and tetramers; these higher order aggregates are known to form among the chemokines. N-terminal sequence analysis of the same preparation indicated that only PBP and truncated derivatives were present, and commercial CTAP-III from three suppliers had heparanase activity. Antisera produced in animals immunized with a C-terminal synthetic peptide of PBP inhibited heparanase activity by 95%, compared with activity of the purified enzyme in the presence of the preimmune sera. The synthetic peptide also inhibited heparanase by 95% at 250 µM, compared to the 33% inhibition of heparanase activity by two other peptides. The enzyme was determined to be an endoglucosaminidase, and it degraded both heparin and heparan sulfate with optimal activity at pH 5.8. Chromatofocusing of the purified heparanase resulted in two protein peaks: an inactive peak at pI 7.3, and an active peak at pI 4.8-5.1. Sequence analysis showed that the two peaks contained identical protein, suggesting that a post-translational modification activates the enzyme.


INTRODUCTION

The dynamic role of heparan sulfate proteoglycans (HSPGs) (^1)in biology has become increasingly apparent(1) . HSPGs are proteins with linear chains of repeating disaccharides, called glycosaminoglycans, and are found in abundance in the extracellular matrix and on cell surfaces. HSPGs participate in diverse processes, including blood coagulation, the anchoring of enzymes in the vascular lumen, and in cell adhesion and growth. Endothelial cell-associated HSPGs bind and potentiate antithrombin III, contributing to a nonthrombogenic vascular surface(2) . Vascular HSPGs also bind many enzymes, including lipoprotein lipase, elastase, and superoxide dismutase, anchoring and modulating the function of these enzymes in the vascular lumen(3, 4, 5) . HSPGs modulate cell-matrix adhesion by interacting with matrix proteins, such as vitronectin, fibronectin, laminin, and collagen(6, 7) . HSPGs also modulate cell-cell adhesion. HSPGs serve as ligands for L-selectin and P-selectin, adhesion molecules expressed on leukocytes, and platelets that are responsible for the initial loose attachment to endothelial cells(8) . Additionally, HSPGs bind and sequester cytokines and can present the bound cytokines to the loosely adhered leukocytes(9) . These cytokines can trigger events required for the tight adhesion and extravasation of the leukocytes. The HSPGs in the extracellular matrix/basement membrane provide a readily available tissue storage depot for growth factors and cytokines(10) . Finally, heparanase activity releases growth factors from their heparan sulfate storage sites in the extracellular matrix(11) , providing a mechanism for the induction of growth and migration of diverse cell types in normal or pathologic situations(11) .

Since HSPGs are now recognized as active biologic modulators, their degradation would be expected to have significant regulatory consequences. Indeed, HSPG catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes that degrade heparan sulfate play important roles in pathologic processes. Heparanase activity has been described in activated immune system cells and cancer cells, but research has been handicapped by the lack of biologic tools to explore potential causative roles of heparanase in disease. Although bacterial heparanases have been purified, earlier attempts to isolate mammalian heparanases have yielded only partial purifications and/or a lack of specific information such as amino acid sequences. In this paper, we report the purification of a mammalian heparanase from activated human platelet supernatants and demonstrate that the enzymatic activity resides in neutrophil activating peptide-2 (NAP-2) and connective tissue activating peptide-III (CTAP-III), two processed forms of the CXC chemokine platelet basic protein (PBP).


EXPERIMENTAL PROCEDURES

Materials

[S]Sulfate, sodium [^3H]borohydride, [^3H]glucosamine, [^3H]mannose, and [^14C]glucose were obtained from Amersham Corp. Superose 6 HR10/30, Superose 12 HR10/30, Superdex 75 HR10/30, Mono Q HR5/5, and Hi-Trap Heparin chromatographic columns and PBE 94/Polybuffer 74, heparin-Sepharose, Sepharose 6B, and DEAE-Sephacel matrices were obtained from Pharmacia Biotech Inc. Poros HS/F columns were obtained from Perseptive Biosystems. Bio-Gel P2 chromatographic matrix and silver stain kits were purchased from Bio-Rad. Glucuronic acid, Trasylol, leupeptin, antipain, benzamidine, chymostatin, and pepstatin were obtained from Sigma. 50 times lactalbumin hydrolysate and heparin (porcine intestine) were obtained from Life Technologies, Inc. Heparan sulfate (40 kDa, bovine intestine) was obtained from Celsus Laboratories, Cincinnati, OH. Ultrafree-MC 30,000 and 5000 NMWL filter units were obtained from Millipore Corp. ReadiSafe liquid scintillation mixture was obtained from Beckman. Peptide conjugation kits were obtained from Pierce. Precast polyacrylamide gels were obtained from Novex. Polyvinylidene difluoride was obtained from Novex and DuPont. Nitrocellulose was obtained from Schleicher & Schuell. Peroxidase-labeled goat anti-chicken IgG and TMB membrane peroxidase substrate system were obtained from Kierkegaard & Perry. Stirred cells and membranes were obtained from Amicon, Inc. Phosphocellulose chromatography paper was obtained from VWR Scientific.

Platelets

Platelet-rich plasma was obtained from healthy, informed volunteers by plasmapheresis at The Upjohn Jasper Research Clinic. 100-150 ml of platelet-rich plasma was obtained daily and had an average concentration of 10^9 platelets/ml. Platelets were centrifuged to remove the plasma(12) , suspended in 0.1 original volume of PBS (0.15 M NaCl, 10 mM sodium phosphate, pH 7.2), and stimulated with 1 unit/ml thrombin for 5 min at 37 °C. This concentration of thrombin was reported to release 100% of the heparanase activity from platelets(13) . Following stimulation, the thrombin was inactivated by the addition of 100 mM phenylmethylsulfonyl fluoride, and the platelets were centrifuged at 2000 times g for 30 min at 4 °C. The supernatant was stored at -80 °C.

Preparation of S-Labeled Proteoglycans and Glycosaminoglycans

[S]HSPG substrate for assaying heparanase activity was prepared from mice bearing the Engelbreth-Holm-Swarm tumor after injection with [S]sulfate, according to the method of Ledbetter(14) . The [S]HSPG obtained by this method was size-fractionated on a calibrated 1.0 times 50-cm column of Superose 6, run in 4 M guanidine hydrochloride, 20 mM Tris-HCl, pH 7.2, at 0.5 ml/min. Fractions containing S radioactivity that eluted with a molecular mass geq70 kDa were pooled. The proteoglycan was precipitated with 100% EtOH at -20 °C overnight. After centrifugation, the pellet was dissolved in PBS, and dialyzed against 3 times 100 volumes of PBS. Each preparation of [S]HSPG was confirmed to be geq98% heparan sulfate by susceptibility to low pH nitrous acid degradation(15) .

Preparation of heparan [S]sulfate glycosaminoglycan from [S]HSPG was achieved by beta-elimination. [S]HSPG (250,000 cpm) was incubated overnight with 1 M NaBH(4), 50 mM NaOH at 45 °C. The glycosaminoglycan was isolated by chromatography on Superose 6 HR 10/30 in PBS.

Chondroitin [S]sulfate glycosaminoglycan was prepared from human embryonic lung fibroblasts. Cells were cultured overnight with 0.1 mCi/ml sodium [S]sulfate, and the conditioned medium was collected and lyophilized. The lyophilizate was suspended in 6 M urea, 0.15 M NaCl, 20 mM Tris-HCl, pH 7.0, and chromatographed on a Mono Q HR5/5 column using a 40-ml linear gradient between 0.15 and 1.15 M NaCl in 6 M urea, 20 mM Tris-HCl, pH 7.0. Aliquots of each fraction were assayed for radioactivity, and fractions containing the radiolabel that eluted from the column between 0.85 and 1.15 M NaCl were pooled; 1 times lactalbumin hydrolysate carrier was added, and the chondroitin [S]sulfate was precipitated with 100% EtOH as described above. The precipitate was dissolved in 4 M guanidine hydrochloride, 20 mM Tris-HCl, pH 7.2, and chromatographed on a Superose 12 HR 10/30 column equilibrated in the same buffer. Aliquots of each fraction were used to determine radioactivity, and the fractions containing radioactivity that eluted with a calculated molecular mass >70 kDa were pooled and dialyzed against PBS.

Assay for Heparanase Activity

Heparanase activity was assessed by detection of lower molecular mass products following incubation of the geq70 kDa [S]HSPG with cell extracts or column fractions. Each digest contained 10 µl of sample, [S]HSPG (2000 cpm; 1 µg of HSPG), 0.15 M NaCl, 0.03% human serum albumin, 10 µM MgCl(2), 10 µM CaCl(2), and protease inhibitors (1 µg/ml leupeptin, 2 µg/ml antipain, 10 µg/ml benzamidine, 10 units/ml Trasylol, 1 µg/ml chymostatin, and 1 µg/ml pepstatin), in 0.05 M sodiumacetate buffer, pH 5.6. Digests were carried out for 3-21 h. Lower molecular mass radiolabeled fragments produced by the degradation of the geq70 kDa [S]HSPG were detected by centrifugation through Ultrafree-MC 30,000 NMWL filter units (Eppendorf centrifuge; 6200 rpm; 10 min). [S]HSPG degradation was evident by the presence of radioactivity in the filtrate that passed through the M(r) 30,000 membrane, as quantified by liquid scintillation counting using a Packard Tri-Carb TR1900 counter. Heparanase activity was operationally defined as the percent of total cpm < M(r) 30,000 for a given digest (1 unit = 1% cpm < M(r) 30,000/h). This definition of activity is not easily compared with bacterial heparinases, which differ mechanistically; the catalytic activity of bacterial heparinases is based on the measurement of ultraviolet absorption at 232 nm of alpha,beta unsaturated uronides formed during elimination reactions.

For determination of the optimal pH for heparanase activity, the 0.1 M sodium acetate buffer was replaced by 50 mM citrate, citrate-phosphate, or phosphate buffer at varying pH values. For samples from chromatographic steps performed under reducing conditions (1 mM GSH, 1 mM DTT), the concentration of a thiol oxidant (diamide) needed for optimum activity was determined. This concentration (100 µM diamide) was added to all assay tubes when reduced samples were assayed.

Commercial Platelet Basic Protein Derivatives

beta-Thromboglobulin was purchased from Celsus Laboratories, Cincinnati, OH; Haematologic Technologies, Essex Junction, VT; and Calbiochem; subsequent analysis showed that all three commercial products were, in fact, 95-100% CTAP-III. Recombinant NAP-2, produced in Escherichia coli, was purchased from BACHEM Bioscience Inc., Philadelphia, PA.

Antibody Preparation and Immunologic Methods

Synthetic peptides were synthesized by conventional solid phase methods and were conjugated to keyhole limpet hemocyanin utilizing a maleimide-activated carrier protein. Conjugated peptides (300 µg) were injected into chickens using Freund's complete adjuvant, with boosters administered at 2-week intervals (150 µg of conjugated peptide in incomplete adjuvant). The antisera were collected 5 weeks after initial immunization and every 4 weeks thereafter.

Verification that the antisera specifically recognized commercial CTAP-III (2.5 µg, Celsus Laboratories), isolated heparanase (1.5 µg), and 7-10-kDa proteins in 10 µl of the platelet supernatant used for the heparanase isolation was achieved by Western blotting. The proteins or platelet supernatant were separated on a reducing 18% polyacrylamide gel and transferred to nitrocellulose or polyvinylidene difluoride using a semi-dry transfer unit (Hoeffer Semi-Phor TE70). Western blotting was performed at 25° C according to the following protocol: 1 h Blocking Buffer (5% milk, 0.05% Tween-20, 0.15 M NaCl, 20 mM Tris-HCl, pH 7.0), 2 h preimmune or antisera (1:1500 in Blocking Buffer), 3 times 15-min rinses in Blocking Buffer, 1 h peroxidase labeled goat anti-chicken IgG (1:500 in Blocking Buffer), 3 times 15-min rinses in PBS, and 30 s membrane development with the TMB membrane peroxidase substrate system.

For antisera neutralization experiments, the preimmune and antisera were exchanged into PBS using a 100-kDa cut-off membrane in order to remove low molecular weight chicken heparanase normally present in the serum. Aliquots of isolated heparanase (15 ng) were preincubated for 30 min with 2 µl of either preimmune or antisera before adding the [S]HSPG to determine heparanase activity.

Chromatographic Methods

Heparin-Sepharose

Activated platelet supernatants were pooled, 1 mM exogenous GSH and 1 mM DTT were added, and the pooled supernatants were loaded (2.5 ml/min) onto a column of heparin-Sepharose (2.6 times 7.5 cm, 40 ml) equilibrated with PBS containing 1 mM GSH, 1 mM DTT. After sample loading, the column was washed sequentially with 200 ml of Buffer A (1 mM GSH, 1 mM DTT, 10 mM sodium acetate, pH 5) containing 0.15 M NaCl, and 60 ml of Buffer A containing 0.35 M NaCl, and eluted with a 160-ml linear gradient of increasing NaCl concentration (0.35-1.5 M) in Buffer A. Aliquots of each fraction were assayed for heparanase activity.

DEAE-Sephacel

Fractions from the heparin-Sepharose column that contained heparanase activity were pooled and concentrated, and the buffer was exchanged to 8 ml of Buffer B (0.15 M NaCl, 1 mM DTT, 1 mM GSH, 10 mM beta-octylglucoside, 10 mM sodium phosphate, pH 6.8) using a stirred cell fitted with a PM-10 membrane (YM-2 membranes were used in subsequent purifications after it was discovered that the molecular mass of heparanase was below 10 kDa). This solution was loaded onto a DEAE-Sephacel column (2.6 times 1.0 cm) equilibrated in Buffer B, and the column was washed to baseline absorbance (280 nm) with Buffer B. The column was then eluted with 10 ml of Buffer C (0.15 M NaCl, 10 mM beta-octylglucoside, 1 mM GSH, 1 mM DTT, 10 mM sodium acetate, pH 5), followed by 10 ml of Buffer C containing 1.5 M NaCl. Aliquots of the flow-through and each wash were used for determination of heparanase activity.

Poros HS/F (Cation Exchange)

The flow-through and pH 6.8 wash from the DEAE-Sephacel column were adjusted to pH 5 with glacial acetic acid and loaded onto a Poros HS/F column (4.6 mm times 50 mm), equilibrated with Buffer D (1 mM DTT, 1 mM GSH, 10 mM beta-octylglucoside, 10 mM sodium acetate, pH 5) containing 0.15 M NaCl. The column was washed (flow rate = 3.0 ml/min) with 35 ml of Buffer D containing 0.15 M NaCl, and was eluted with a 55-ml linear gradient of increasing NaCl concentration (0.15-1.5 M) in Buffer D. 3-ml fractions were collected, and 10-µl aliquots were assayed for heparanase activity.

Size Fractionation to <30 kDa and Concentration via Hi-trap Heparin-Sepharose

Fractions containing heparanase activity from the Poros HS/F column were size fractionated by centrifugation through ultrafree-MC 30,000 NMWL filter units (Eppendorf centrifuge, 6200 rpm, 2 h). The <30-kDa pool was concentrated by heparin-Sepharose chromatography. This was achieved by diluting the <30 kDa pool with Buffer B to reduce the [NaCl] to 0.15 M, loading it onto a 1-ml Hi-trap heparin-Sepharose column equilibrated with Buffer B containing 0.15 M NaCl and eluting with 2 ml of Buffer B containing 1.2 M NaCl.

Superdex 75 Gel Filtration

An aliquot of purified heparanase was chromatographed on a Superdex 75 HR 10/30 column. The column was run in 0.15 M NaCl, 0.5 mM DTT, 10 mM sodium acetate, pH 5, at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected, and aliquots were assayed for heparanase activity.

Bio-Gel P2 Gel Filtration

Gel filtration of heparin or heparan sulfate that had been enzymatically degraded and end-labeled with sodium [^3H]borohydride was performed on a column of Bio-Gel P2 (200 times 0.9 cm) run at 2.5 ml/h in 0.5 M NH(4)HCO(3). Fractions of 2 ml were collected, and aliquots were used for radioactivity determination.

Sepharose 6B Gel Filtration

Separations of the radiolabeled products resulting from degradation of either [S]HSPG (28,000 cpm) or heparan [S]sulfate (28,000 cpm) by the purified enzyme were performed on a column of Sepharose 6B (1 times 100 cm), equilibrated in 4 M guanidine hydrochloride, 20 mM Tris-HCl, pH 7.2. The column was run at 5 ml/h, and 1-ml fractions were collected. Entire fractions were used for the determination of radioactivity. Nitrous acid-treated [S]HSPG was used as a standard for the elution position of di- and trisaccharides, and [S]sulfate was used for the included volume determination.

Chromatofocusing

Samples were dissolved in 0.025 M imidazole buffer, pH 7.3, and were loaded onto a column of Polybuffer exchanger (PBE 94; 0.5 times 20 cm), equilibrated in the same buffer. Immediately after sample loading, Polybuffer 74 (1:8, pH 4.0) was pumped onto the column at 0.5 ml/min. Fractions of 2 ml were collected, and the pH of each was determined by narrow range pH paper (pH 4-7). Heparanase activity was determined on aliquots of each fraction. Portions of each protein peak eluted from this column were separated from ampholytes by C(4) reverse phase chromatography and subjected to Edman degradation.

Substrate Cleavage Site Determination

Heparin and heparan sulfate were reduced with unlabeled sodium borohydride by incubation of 360 µg of glycosaminoglycan in 10 mM Na(2)CO(3), 15 mM NaBH(4), 5 mM NaOH for 30 min at 45 °C. The reduced glycosaminoglycan was acidified to pH 5 by the addition of glacial acetic acid. Heparin and heparan sulfate (4 µg) that had been prereduced with unlabeled sodium borohydride were incubated with 1 µg of the purified heparanase for 24 h in 300 µl of 0.15 M NaCl, 10 µM MgCl(2), 10 µM CaCl(2), 10 mM sodium acetate, pH 5.6. Each sample was lyophilized, reduced with 1 mCi of sodium [^3H]borohydride under the conditions outlined above, and chromatographed on Bio-Gel P2 to remove the unreacted borotritide. Fractions corresponding to oligosaccharides were pooled and lyophilized. Monosaccharides were prepared from the lyophilizate by total acid hydrolysis/nitrous acid deamination, and phosphocellulose paper chromatography (ethyl acetate/pyridine/5 mM boric acid; 3:2:1) was used to identify the monosaccharide containing the new ^3H-reducing end as described by Conrad(16) . Radiolabeled standards for iduronic acid were prepared from heparin(16) ; standards for glucuronic acid were prepared by sodium [^3H]borohydride reduction of pure glucuronic acid, and standards for glucosamine were prepared from commercially available [^3H]glucosamine. [^14C]Glucose was used as internal standard for the paper chromatography.

Three-dimensional Modeling

Three dimensional modeling of a NAP-2 dimer was based on the structure of IL-8(17, 18) . Coordinates for the IL-8 NMR solution structure were obtained from entry 1IL-8 in the Brookhaven Protein Data Bank(19) . The NAP-2 dimer model was constructed by a simple replacement of IL-8 amino acid sidechains for those of NAP-2, where appropriate. Backbone angles were left unchanged. There is only one insertion in IL-8/NAP-1 relative to NAP-2, a Pro between amino acids 71 and 72 (see Fig. 3), and this surface loop residue was retained in our NAP-2 model to simplify the replacement of residues downstream. Coloring of charged residues (see Fig. 9) is based on the NAP-2 sequence.


Figure 3: N-terminal sequence of purified heparanase and CXC chemokines. An aliquot of heparanase from each of two Step 5 preparations was desalted using a M(r) 5000 membrane and was subjected to N-terminal sequencing as described under ``Experimental Procedures.'' The published sequence for platelet basic protein is reviewed by Miller and Krangel(31) , and N-terminal cleavages producing PBP, CTAP-III, beta-thromboglobulin, and NAP-2 are indicated by arrows. Calculated masses of the derivatives are based on 1 amino acid = 110.5 Da. The asterisk denotes a blank sequencing cycle.




Figure 9: A view of a smoothed backbone model of a NAP-2 dimer. The backbone of the model is identical to that of the parent IL-8 structure from which it was constructed, as described under ``Experimental Procedures.'' Positive and negative amino acid residues are shown in blue and red, respectively. The side chains of possible catalytic residues (Glu-93, Asp-107, and Asp-109) are highlighted in yellow.



Other Methods

Protein concentration was determined by the method of Lowry (20) . Uronic acid assays were performed by incubating 20 µl of sample and 150 µl of sulfuric acid/borate (2.39 g of sodium tetraborate decahydrate in 250 ml of H(2)SO(4)) for 15 min at 90 °C, adding 20 µl of carbazole/ethanol (0.125% carbazole in 100% EtOH), heating at 90 °C for 15 min, and reading the absorbance at 520 nm. Unless otherwise indicated, the molecular mass of heparin or heparan sulfate chains was assumed to be 10 kDa. SDS-polyacrylamide gels were run according to the method of Laemmli(21) , and molecular weight standards were from Amersham Corp. and Bio-Rad. Gels were fixed and stained using a Silver stain kit and protocol from Bio-Rad. N-terminal sequencing was performed by automated Edman degradation in a gas/liquid phase protein sequencer (Applied Biosystems). Phenylthiodantoin amino acids were resolved and quantitated by an on-line high pressure liquid chromatography system (Applied Biosystems) with data analysis on a Nelson analytical system. The isolated heparanase was exchanged into 5% acetic acid using 5-kDa membranes prior to composition and sequence analysis.


RESULTS

Purification of Platelet Heparanase

Platelet heparanase was isolated from the supernatant of thrombin-activated fresh platelets by chromatography on columns of heparin-Sepharose, DEAE-Sephacel, and Poros HS/F, followed by size fractionation through M(r) 30,000 membranes, and concentration by Hi-trap Heparin chromatography.

Step 1: Preparation of Washed, Activated Platelets

Fresh platelets were obtained daily and activated as described under ``Experimental Procedures.'' The supernatant from 1850 ml of washed, activated platelets was used for the isolation of heparanase. As shown in Table 1, the washed activated platelet supernatant contained only 0.5% of the original protein while retaining 97% of the heparanase activity.



Step 2: Heparin-Sepharose Chromatography

Reducing agents (1 mM GSH, 1 mM DTT) were added to the activated platelet supernatant. This was done because earlier attempts at heparanase purification resulted in significant contamination with human albumin that could be separated under the reducing conditions used in SDS-PAGE. The supernatant was loaded onto a heparin-Sepharose column and eluted using a linear gradient of increasing NaCl concentration under reducing conditions. As shown in Fig. 1A, heparanase activity eluted in two discrete peaks: 15% of the eluted activity was between 0.45 and 0.7 M NaCl, while 85% of the eluted activity was between 0.9 and 1.15 M NaCl. The activity eluting between 0.9 and 1.15 M NaCl was used for further purification.


Figure 1: Heparin-Sepharose and cation exchange chromatography of heparanase from activated platelet supernatants. A, heparin-Sepharose chromatography. Heparanase was eluted under reducing conditions (1 mM GSH, 1 mM DTT) with a linear NaCl gradient () at pH 5 as described under ``Experimental Procedures.'' 8-ml fractions were collected, and a 10-µl aliquot was assayed for heparanase activity (bullet). Activity that eluted between 0.9 M and 1.15 M NaCl (indicated by solidbar on lowerpanel) was used for further purification. B, cation-exchange chromatography. The flow-through from the DEAE-Sephacel column was adjusted to pH 5, loaded onto a column of Poros HS/F, and eluted with a linear NaCl gradient (), as described under ``Experimental Procedures.'' 3-ml fractions were collected, and 10-µl aliquots were assayed for heparanase activity (bullet).



Step 3: DEAE-Sephacel Chromatography

This step was performed to remove highly charged glycosaminoglycans from the sample. Under the conditions employed, glycosaminoglycans are expected to bind to DEAE-Sephacel, whereas 88% of the loaded heparanase activity and 90% of the total protein did not bind and could be recovered in the flow-through. An additional 10% of the activity was eluted from the column by lowering the pH of the eluent to 5, and another 2% was eluted when the pH was lowered to 5 and the NaCl concentration adjusted to 1.5 M (data not shown). The flow-through pool was used for further purification.

Step 4: Poros HS/F (Cation Exchange Chromatography)

The flow-through from the DEAE-Sephacel column was loaded directly onto the cation exchange column, and the column was developed with a linear NaCl gradient using detergent and reducing conditions at pH 5. The heparanase activity eluted broadly between 0.45 M and 0.85 M NaCl, and these fractions were pooled and used for further purification (Fig. 1B).

Step 5: Size Fractionation to <30 kDa and Concentration by Hi-trap Heparin Chromatography

The active fractions from the cation exchange step were size-fractionated to <30 kDa by the use of ultrafree-MC 30,000 NMWL filter units. 90% of the heparanase activity was <30 kDa. The <30 kDa pool was diluted to reduce the concentrations of NaCl, and detergent and was loaded onto a Hi-trap heparin column. All of the loaded protein bound to the column, based on absorbance (280 nm). A single protein peak was eluted with 1.2 M NaCl under reducing conditions. The heparanase activity was not associated with the flow-through from this column, but it did coincide with the peak that eluted at 1.2 M NaCl (data not shown).

The final yield of heparanase protein from 1850 ml of platelet-rich plasma was 2.7 mg. The overall recovery of activity was 8%, with a 4150-fold purification (Table 1). The preparation was judged to be homogeneous by silver-staining of SDS-PAGE (Fig. 2, inset).


Figure 2: Molecular size and oligomerization of human platelet heparanase. An aliquot of human platelet heparanase was chromatographed on Superdex 75 under reducing conditions and assayed for heparanase activity as described under ``Experimental Procedures.'' Elution positions of standards are indicated at the top of the figure: bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20.1 kDa), alpha-lactalbumin (14.2 kDa), and aprotinin (6.5 kDa). Inset, an aliquot of Step 5 heparanase was desalted using M(r) 5000 membranes and electrophoresed under reducing conditions on an 18% SDS-polyacrylamide gel. Migration positions of molecular mass markers are shown on the left of the inset: bovine serum albumin (69 kDa), ovalbumin (45 kDa), soybean trypsin inhibitor (21 kDa), myoglobin I + II (14.4 kDa), myoglobin I (8.1 kDa), and myoglobin II (6.2 kDa).



Molecular Mass of Human Platelet Heparanase

Of the total heparanase activity, 90% passed through a 30-kDa membrane, suggesting that the molecular mass of the enzyme is below 30 kDa. This observation was confirmed by SDS-PAGE. An aliquot of the purified heparanase from Step 5 was desalted using a 5-kDa membrane and separated on an 18% SDS-polyacrylamide gel under reducing conditions. A major broad band was detected between 8500 and 10,100 Da (Fig. 2, inset). Upon storage at 4 °C, a faint band at 7700 Da was visible, suggesting that the purified protein breaks down upon storage (data not shown). Gel filtration chromatography of the purified heparanase yielded three peaks of heparanase activity, at approximate molecular masses indicative of the formation of monomers, dimers, and tetramers, even though the column was run in the presence of reducing agents (Fig. 2). Heparanase that eluted in positions corresponding to dimers and tetramers resulted in broad 8-10 kDa bands when analyzed by reducing SDS-PAGE (data not shown), suggesting that noncovalent dimers and tetramers were formed.

N-terminal Sequence and Amino Acid Composition of Heparanase

Edman degradation of the purified enzyme shown in Fig. 2(Step 5, preparation) revealed that it was a mixture of the known chemokine PBP (15%) and an N-truncated form of PBP known as CTAP-III (85%) (Fig. 3). N-terminal sequencing of a second heparanase preparation, which utilized platelets that had been stored frozen, revealed that it was also an N-terminal truncation of PBP known as NAP-2. PBP is a CXC chemokine family member, which is proteolytically processed to give rise to CTAP-III, beta-thromboglobulin, and NAP-2, proteins with calculated molecular masses between 7.7 and 10.4 kDa. Amino acid analysis of the preparations gave compositions consistent with that expected for the PBP derivatives identified by sequencing (linear regression of expected versus found resulted in r values of >0.97), thus confirming that the N-terminal sequence obtained was not due to a minor contaminant in the preparation.

Confirmation of Heparanase Activity in PBP Derivatives

To provide confirmation of the identity of heparanase as the previously described PBP derivatives, three different commercial preparations of CTAP-III (labeled by the suppliers as beta-thromboglobulin) were tested for heparanase activity. Aliquots of each preparation were taken for heparanase activity assays, reverse phase chromatography, and amino acid sequence analysis to confirm enzymatic activity, homogeneity, and identity. The CTAP-III from all three suppliers had enzymatic activity, although the specific activity was 100-200-fold less than that isolated by the method described in this paper. A graph displaying increasing heparanase activity with increasing protein concentrations is shown in Fig. 4. The three commercial preparations were 95-100% homogeneous by reverse phase analysis. As judged by Edman degradation, CTAP-III accounted for greater than 95% of the total protein, the remaining 5% being beta-thromboglobulin. One commercial preparation (Haematologic Technologies) was 100% CTAP-III, and, since the second of our preparations was 100% NAP-2, heparanase activity was established in at least these two N-truncated derivatives of PBP. The specific activity of each of our two preparations was not appreciably different. It was not possible to determine the specific activity of each of the N-truncated derivatives since the conditions that could separate the individual derivatives were reverse phase chromatography with trifluoroactic acid/acetonitrile, and these conditions destroyed the enzymatic activity.


Figure 4: Degradation of S-HSPG by commercial CTAP-III. Increasing amounts of commercial CTAP-III were assayed for heparanase activity. Shown are the results of a 16-h assay with protein obtained from Calbiochem. Similar results were obtained using protein obtained from either Celsus Laboratories or Haematologic Technologies.



To rule out the possibility that the heparanase activity was attributable to a minor contaminant and not to CTAP-III, we prepared antisera from synthetic peptides of CTAP-III, and tested the ability of these antisera to neutralize heparanase activity. Keyhole limpet hemocyanin conjugates of C-terminal (CNQVEVIATLKDGRKICLDPDAPRIKKIVQKK) and N-terminal (NLAKGKEE-SLDSDLC) synthetic peptides of CTAP-III/NAP-2 (residues 89-120 and 44-57 in Fig. 3, respectively) were used for immunizing chickens. These antisera, but not the preimmune sera, bound specifically to 8-10-kDa proteins on immunoblots of SDS-PAGE separations of purified platelet heparanase and commercial CTAP-III (data not shown). An experiment was designed to measure the heparanase activity of the purified enzyme in the presence of preimmune or antisera. In the presence of the preimmune sera, the enzyme had 14.3 ± 0.1 units of heparanase activity, while in the presence of the C-terminal peptide antisera, only 0.8 ± 0.2 units of heparanase were detected (p < 0.001; results confirmed in a second experiment). The N-terminal peptide antiserum was not able to neutralize the heparanase activity. Similar results were obtained when the synthetic peptides themselves were tested for their ability to neutralize heparanase activity. Heparanase assays conducted with 3 nM enzyme, 47 nM [S]HSPG substrate, and varying concentrations of peptides showed that heparanase activity was inhibited by 95% in the presence of 250 µM C-terminal peptide. By contrast, heparanase activity in the presence of 250 µM of either the N-terminal peptide or an unrelated peptide (PLALWAR) was inhibited by only 33%. The ability of both the C-terminal peptide or antisera from a chicken immunized with the C-terminal synthetic peptide to neutralize heparanase activity demonstrates conclusively that CTAP-III and NAP-2 possess heparanase activity, and suggests that the C-terminal region may be essential for catalysis.

Isoelectric Point of Active Human Platelet Heparanase

When the Step 5 preparation of heparanase (Table 1) was subjected to chromatofocusing (Fig. 5), 90% of the protein eluted in a peak displaying a pI of greater than 7.3, as predicted by the amino acid sequence. This peak was inactive. Ten percent of the protein recovered in this procedure eluted in a second peak, displaying a pI of 4.8-5.1 and containing all of the heparanase activity. N-terminal sequence analysis showed that the protein in both peaks was an essentially identical mixture of PBP (15%) and N-truncated derivatives of PBP (85%). The same pattern was observed with CTAP-III obtained from commercial sources. Thus, the heparanase activity resides in PBP or an N-truncated derivative of PBP that is modified such that the pI is lowered to 4.8-5.1. The specific enzyme activity of the low pI heparanase was raised to approximately 6000 units/mg.


Figure 5: Chromatofocusing of platelet heparanase. An aliquot of Step 5 heparanase was chromatofocused on PBE 94 between pH 7.3 and 4.0, as described under ``Experimental Procedures.'' Fractions of 2 ml were collected, and the pH (box) and heparanase activity () for each fraction was plotted. Data shown are respresentative of three experiments.



Enzymatic Properties of Human Platelet Heparanase

The enzyme cleaves heparan sulfate and heparin glycosaminoglycan chains. Incubation of Step 5 heparanase with the in vivo radiolabeled [S]HSPG (size fractionated to >70 kDa) for 3-21 h resulted in the production of lower molecular weight radiolabeled products (Fig. 6). This heparanase activity was assessed by two methods. Fig. 6A shows the Sepharose 6B profile of the radiolabeled products from a digest of [S]HSPG with suboptimal concentrations of heparanase. The peak of undigested [S]HSPG elutes from the column at 17 ml, while digestion of [S]HSPG with low pH nitrous acid, which specifically degrades heparin and heparan sulfate to di- and trisaccharides(15) , results in a shift of all radioactivity toward the included volume of the column (peak at 47 ml). The profile of the digestion with heparanase results in S fragments of intermediate size. These data provide evidence that the enzyme has an endoglycosidic nature since an exoglycosidic activity would result in fragments that elute as small saccharides, similar to the nitrous acid digestion. Addition of 20 mMD-saccharic acid 1,4-lactone, a reported inhibitor of exoglycosidases (22) had no effect of the degradation of [S]HSPG (data not shown), providing further evidence of the endoglycosidic nature of the enzyme. The gel filtration profile of the lower molecular weight radiolabeled fragments produce by the enzyme also precludes the possibility that the enzyme acts as a sulfatase, since the enzymatic release of free SO(4) would result in the elution of the radiolabel at the included volume of the column, and intermediate size fragments were observed in this digest.


Figure 6: Enzymatic degradation of intact [S]HSPG and heparan [S]sulfate chains by platelet heparanase. Sepharose 6B chromatography was used to monitor the lower molecular weight products formed during an incubation of 28,000 cpm of intact >70 kDa [S]HSPG (A) or heparan [S]sulfate chains (B) for 16 h with (down triangle) or without (circle) the Step 5 heparanase, or for 45 min with HNO(2) (bullet), as described under ``Experimental Procedures.'' One-ml fractions were collected, and the radioactivity was quantitated by liquid scintillation counting. The arrow denotes the included volume of the column, determined with [S]sulfate. C, lower molecular weight products formed during an incubation of >70 kDa [S]HSPG with the Step 5 heparanase for 3 h (down triangle), 6 h (bullet), or 21 h (circle) were detected by centrifugation through 30-kDa membranes, described under ``Experimental Procedures.'' Each point represents the mean ± S.D. for triplicates. Graphs shown are representative of at least four determinations.



The buffer used for determination of heparanase activity contains six different protease inhibitors, so it was unlikely that the S-labeled fragments produced by heparanase digestion resulted from cleavage of the protein backbone of the heparan sulfate proteoglycan. To provide further evidence that this hypothesis was correct, S-heparan sulfate glycosaminoglycan (no protein backbone) was prepared from the [S]HSPG by beta-elimination and was used as the substrate for a heparanase digestion. Fig. 6B shows that the Step 5 enzyme was able to cleave the protein-free S-labeled heparan sulfate glycosaminoglycan to intermediate-sized fragments, confirming that the enzyme is a glycosidase.

A second method of detecting the presence of S-labeled low molecular weight fragments produced by digestion of heparanase with the >70 kDa [S]HSPG was to centrifuge the digest through 30-kDa membranes. The percent of radioactivity that passed through the 30-kDa membrane was used as a measure of heparanase activity. Fig. 6C shows an example of this type of assay using increasing amounts of the Step 5 enzyme for 3, 6, or 21 h. Increased heparanase activity corresponded with increased protein concentration and time. Since analysis of heparanase activity using this method was rapid, reproducible, and allowed simultaneous analysis of several digests, this method was routinely used to assay for heparanase activity throughout the isolation of heparanase.

The platelet heparanase was determined to be an endoglucosaminidase. To show this, heparin and heparan sulfate, prereduced with sodium borohydride at alkaline pH, were incubated for 24 h with Step 5 enzyme under conditions designed to achieve complete enzymatic cleavage. After 24 h, the sample was lyophilized, the new reducing ends produced by enzymatic cleavage were labeled with sodium [^3H]borohydride, and the unincorporated sodium [^3H]borohydride was separated from the saccharides by gel filtration on Bio-Gel P2 (Fig. 7A). Cleavage of both heparin and heparan sulfate by the platelet heparanase resulted principally in disaccharides. The disaccharides were lyophilized and subjected to total acid hydrolysis/nitrous acid deamination to produce monosaccharides, which were separated on phosphocellulose paper chromatography under conditions that distinguish the derivatives of sugars found in heparin and heparan sulfate (uronic acid, glucuronic acid, and glucosamine). As shown in Fig. 7B, the [^3H]monosaccharide derived from heparan sulfate that had been cleaved with heparanase and labeled with sodium [^3H]borohydride eluted in a position similar to the glucosamine standard, thus identifying this platelet heparanase as an endoglucosaminidase. Similar results were obtained when the heparin was the glycosaminoglycan under study (data not shown). The Step 5 enzyme was unable to cleave [S]chondroitin sulfate (data not shown).


Figure 7: Gel filtration and paper chromatography of heparin and heparan sulfate after cleavage by heparanase and labeling with sodium [^3H]borohydride. A, Bio-Gel P2 chromatography of heparan sulfate (upper) and heparin (lower) after cleavage by heparanase and labeling with sodium [^3H]borohydride, as described under ``Experimental Procedures.'' Two-ml fractions were collected, and 100 µl was used to determine radioactivity by liquid scintillation counting. Positions of native heparin and disaccharides produced by HNO(2) digestion of heparin were determined by a carbazole assay, and [^3H]mannose was determined by liquid scintillation counting. B, cellulose phosphate paper chromatography of labeled monosaccharides obtained from the labeled disaccharide produced by enzymatic digestion and borotritide labeling. Chromatography and preparation of standards is described under ``Experimental Procedures.'' The upperpanel shows the results obtained with monosaccharides obtained from heparan sulfate. The lowerpanel shows the elution positions of gulonic acid derived from glucuronic acid (down triangle), idonic acid derived from iduronic acid (box), and anhydromannitol derived from glucosamine (circle). Data shown are representative of two independent experiments.



The pH optimum of the heparanase was determined by conducting activity assays between pH 3.5 and 8.0, using a citrate buffer (pH 3.5-6.0), citrate-phosphate buffer (pH 6.5-7.0), and phosphate buffer (pH 7.5-8.). Heparanase was active between pH 5.0 and 7.5, with the optimum pH at 5.8 (Fig. 8). The enzyme rapidly and irreversibly lost activity above pH 7.5.


Figure 8: The HSPG-degrading activity of isolated platelet heparanase as a function of pH. Heparanase activity was assayed by incubating the [S]HSPG with 10 µl of Step 5 heparanase for 16 h at varying pH values. Assays were performed as described under ``Experimental Procedures,'' except that the 0.1 M sodium acetate, pH 5.6, buffer was replaced with 0.05 M citrate buffer between pH 3.5 and 6.0; 0.05 M citrate-phosphate buffer between pH 6.5 and 7.0; or 0.05 M phosphate buffer between pH 7.5 and 8.0. Data shown are representative of three separate experiments.




DISCUSSION

This report is important in that it describes the purification and identification of a mammalian heparanase, an enzyme that has been difficult to isolate. Furthermore, this is the first demonstration of enzymatic activity in a member of the CXC chemokine family.

One of the more striking observations from these studies is the small molecular mass of this heparanase. That the mass of heparanase is below 10 kDa may account for our low recovery after heparin-Sepharose chromatography, which included concentration and buffer exchange through 10-kDa membranes. Subsequent isolations utilized membranes with a 2-kDa cut-off. Furthermore, in our earlier unsuccessful attempts to isolate a heparanase, we routinely used 10% SDS-polyacrylamide gels, which would not resolve proteins with molecular masses below 20 kDa. Other estimations of the molecular mass of heparanase in nonhomogeneous preparations, measured by gel filtration chromatography, are from 50 to 134 kDa(23, 24, 25) . CTAP-III/NAP-2 also exist as higher order aggregates, and aggregation may account for the various higher estimates of molecular mass made by other researchers.

Another observation is the amount of heterogeneity displayed by this protein. At least four different N-truncated forms of this protein have been isolated, and the protein exists as monomers, dimers, and tetramers. Moreover, there is heterogeneity in its isoelectric point, which others have also observed(26) . SDS-PAGE results in a broad band from 8.5 to 10.1 kDa, consistent with the molecular mass of processed forms of PBP. Other researchers have found that storage increases the amounts of processed forms(27) . We found 100% NAP-2, a highly processed form, in a second Step 5 preparation that used activated platelets that had been stored frozen for several months. Gel filtration chromatography of the Step 5 heparanase resulted in peaks of enzymatic activity that corresponded to higher order aggregates. Analysis of the Step 5 enzyme by chromatofocusing showed variations in the isoelectric point of the protein, suggesting that a post-translational modification that lowers the pI is responsible for the enzymatic activity. The sequence of PBP contains potential sites for protein kinase C phosphorylation (Ser-35, Ser-36, Thr-41, Ser-70, Thr-87, and Thr-97), N-myristoylation (Gly-86), amidation (Asp-100), and O-linked glycosylation (Ser-70), but no sites for N-linked glycosylation. Of these potential modifications, phosphorylation is attractive, since it would lower the pI and is known to contribute to the regulation of many enzymes. In our experience, gradient elution of this protein from various chromatographic matrices results in either more than one protein peak (heparin-Sepharose) or in broad peaks (Poros S). This may be the result of variations in the isoelectric point or aggregation state. This large reservoir of potential microheterogeneity may have complicated the isolation of active heparanase in the past and may make it difficult to express this protein recombinantly. Native, commercial CTAP-III displayed heparanase activity, but with a specific activity reduced at least 100-fold. Commercial recombinant NAP-2 produced in E. coli did not have activity (data not shown). This lower specific activity or lack of activity may be accounted for by either the methods used to isolate the protein, including chromatography above pH 7.5, reverse phase chromatography with trifluoroactic acid/acetonitrile, and lyophilization, or by expression in systems that would not be expected to correctly fold or modify the protein. We are now in the process of producing the protein in mammalian expression systems.

Because the purification to homogeneity and characterization of mammalian heparanases has been difficult, other purified heparanases are not available for comparison. Most reports contain only partial descriptions. In one of the more well characterized reports, Oosta et al.(23) described the purification of a human platelet heparanase that was homogeneous with respect to charge by acidic disc gel electrophoresis, although determination of homogeneity by SDS-PAGE or N-terminal sequencing was not reported. The protein was estimated to have a molecular mass of 134 kDa by gel filtration chromatography and was found to be an endoglucuronidase, not an endoglucosaminidase as shown for our platelet heparanase. Since amino acid sequence data are not available for the earlier protein, sequence comparison is not possible. A melanoma heparanase, whose molecular size of 94 kDa and 25 N-terminal amino acid residues are identical to glucose-regulated protein/endoplasmin, has also been described to be an endoglucuronidase (24) , although the presence of heparanase activity in this protein has recently been challenged(28) . It is entirely possible that platelets, as well as other cells, contain several heparanases that are regulated differently and/or have different specificities. Three bacterial heparinases from Flavobacterium heparinum have been isolated and characterized(29) . They are similar to the present enzyme in that they also are endoglucosaminidases, but the bacterial enzymes have molecular masses of 42.5, 84, and 70 kDa, and the present heparanase shares no amino acid homology with the one bacterial heparanase of known amino acid sequence(30) .

The most provocative information from this study is that members of the PBP family are heparan sulfate/heparin-degrading enzymes. PBP and naturally occurring N-truncated derivatives of PBP are members of the large chemokine (chemoattractant and cytokine) superfamily (for review, see (31) ) which functions in inflammation, wound healing, and growth regulation. The superfamily includes platelet factor 4, PBP and N-truncated derivatives, IL-8, GRO-alpha, beta, , 9E3, ENA-78, IP-10, macrophage inflammatory proteins 1alpha (MIP-1alpha), MIP-1beta, MIP-2, monocyte chemoattractant proteins-1 (MCP-1) MCP-2, MCP-3, and RANTES. All family members are basic heparin-binding proteins with molecular masses below 12 kDa. The members share similar tertiary structures and are known to form dimers and tetramers, consistent with our gel filtration data showing that the isolated protein exists as higher order aggregates.

Since a high degree of sequence identity is shared among members of the chemokine family, it is possible to model the three-dimensional structure of NAP-2 based on the NMR solution structure of IL-8/NAP-1 (17, 18) . We derived an approximate NAP-2 dimer model by a pairwise replacement of residues, which differed between NAP-2 and IL-8/NAP-1 (Fig. 9). Two antiparallel alpha-helices can be seen corresponding to the C-terminal regions of each monomer. Truncation of these alpha-helices in IL-8 led to the loss of heparin-binding activity(32) , and it is probable that the same heparin-binding function exists for the C-terminal alpha-helices in NAP-2. It is tempting to speculate as to what amino acids might play a role in catalysis. As a group of enzymes, most glucosidases function via an acid-base catalytic mechanism analogous to that for lysozyme where a Glu and an Asp participate in the hydrolysis (for review, see (33) ). Interestingly, 2 Asp and 1 Glu appear at each end of the groove defined by the pair of alpha-helices in NAP-2. Accordingly, some combination of these Asp and Glu residues (residues 93, 107, and 109 in Fig. 4; highlighted in Fig. 9) could serve as catalytic residues in the breakdown of heparan sulfate. These residues are contained within the C-terminal peptide that was effective in neutralizing heparanase activity. This model may be useful in predicting whether other chemokines may be expected to have heparanase activity. Among PBP, IL-8, GRO, 9E3, MCPs, MIPs, PF4, RANTES, IP-10, and LD78, only PBP and its derivatives contain both Glu-93 and Asp-109 and would be expected to be active if these residues are involved in catalysis. If Glu-93 and Asp-107 conferred activity, then PBP, IL-8, 9E3, MIP-2, and MCP-1 may be expected to have heparanase activity.

Most of the research described for the chemokines has focussed on the mechanisms responsible for their chemoattractant and activating abilities. CTAP-III has also been described as a mitogen for a variety of cell types(34) , but the mechanism by which it promotes growth is not well understood. That the chemokine CTAP-III has the ability to degrade heparan sulfate suggests a novel molecular mechanism that may account for the growth promoting activities of CTAP-III. Heparanase cleavage of immobilized HSPGs in the extracellular matrix could promote growth by allowing the heparan sulfate and any bound growth factors to become soluble and interact with growth factor receptors on adjacent cells.

The dual function of CTAP-III/NAP-2 as both a heparanase and a neutrophil chemoattractant suggests that a complex relationship may exist where each activity could alter the activity or bioavailability of the other function in various pathologies. For example, chemokines are known to be immobilized by proteoglycans, which can present the chemokines to inflammatory cells(9) . The heparanase activity of the chemokine could down-regulate inflammation by degrading focal sites of chemokine anchoring on the surface of the inflamed endothelium. In vascular pathologies, postinjury adherence of platelets to the subendothelium causes platelet activation and release of granule contents, including 20 µg of PBP (and derivatives) per 10^9 cells(34) . Degradation of vascular heparan sulfate by CTAP-III would remove antithrombin III binding sites and promote thrombogenesis. NAP-2 may attract neutrophils, consistent with the concept that pathophysiological events associated with arterial injury are controlled by multiple cell types and pathways, including neutrophils(35) . In some situations, such as the inflamed synovial joint, where CTAP-III and NAP-2 are found in abundance(36, 37) , it is difficult to predict the consequences of having both activities. For example, others have reported that chemokine activity is enhanced following binding to heparin(32) . It could be envisioned that built-in heparanase activity would degrade the heparin ``enhancer'' and down-regulate the activity of the chemokine. It could also be envisioned that chemokines are stored in the extracellular matrix in a fashion analogous to growth factors (10) and that heparanase activity would mobilize and therefore make the chemokine more available. In tumor growth and metastasis, it is also difficult to predict the effects of the dual functions. Heparanase activity may degrade the heparan sulfate needed for the binding of growth factors to high affinity receptors (38) and inhibit growth. Alternatively, the dual functions may work synergistically to both attract leukocytes and to mobilize growth factors from extracellular matrix storage sites. In regard to these apparent paradoxes, an essential question is whether the heparanase activity is a critical part of the mitogenic and/or chemoattractant activity ascribed to CTAP-III/NAP-2 or if the enzymatic, mitogenic, and chemoattractant functions on this single protein function independently.

HSPGs associate with diverse biologically active molecules. The enzymatic release of bioactive complexes may be a critical regulatory mechanism in controlling the availability of these molecules. Our observations that the previously known proinflammatory chemokines CTAP-III and NAP-2 are heparanases suggest that this class of proteins may have multiple mechanistic capabilities and biologic functions, and this may profoundly influence our understanding of vascular biology.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Glaxo IMB S.A., 14 chemin des Aulx, 1228 Plan-les-Oates, Geneva, Switzerland.

To whom correspondence should be addressed: The Upjohn Co., Cancer & Infections Diseases, 7252-267-410, 301 Henrietta St., Kalamazoo, MI 49001. Tel.: 616-385-7212; Fax: 616-385-6492; srledbet{at}upj.com.

(^1)
The abbreviations used are: HSPG, heparan sulfate proteoglycan; CTAP-III, connective tissue activating peptide-III; PBP, platelet basic protein; NAP-2, neutrophil activating peptide-2; PBS, phosphate buffered saline; DTT, dithiothreitol; IL-8, interleukin-8; PAGE, polyacrylamide gel electrophoresis; 9E3, chicken 9E3 chemokine; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; RANTES, regulated on activation, normal T-cell expressed, and secreted.


ACKNOWLEDGEMENTS

We thank Drs. Mike Bienkowski and Gabriel Vogeli for their expert advice, Dr. Clark Smith and associates for providing synthetic peptides, and C. Ann Kiewiet for assistance in preparing figures.


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