heparin inhibits bmp-2 osteogenic bioactivity by binding to both bmp-2 and bmp receptor

ORIGINAL ARTICLE 844J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology
Heparin Inhibits BMP-2
Osteogenic Bioactivity by Bindingto Both BMP-2 and BMP Receptor
SHIN KANZAKI,1,2 TETSU TAKAHASHI,1 TAKAHIRO KANNO,1,3 WATARU ARIYOSHI,1
KOUHEI SHINMYOUZU,1 TOSHIYUKI TUJISAWA,2 AND TATSUJI NISHIHARA2*1Division of Oral and Maxillofacial Reconstructive Surgery, Department of Oral and Maxillofacial Surgery, Kyushu Dental College,

Kitakyushu, Japan2Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu, Japan3Division of Oral and Maxillofacial Surgery, Kagawa Prefectural Central Hospital, Takamatsu, Japan

Heparin demonstrates several kinds of biological activities by binding to various extracellular molecules and plays pivotal roles in bonemetabolism. However, the role of heparin in the biological activity of bone morphogenetic protein (BMP) remains unclear. In the presentstudy, we examined whether heparin has the effects on osteoblast differentiation induced by BMP-2 in vitro and also elucidated the precisemechanism by which heparin regulates bone metabolism induced by this molecule. Our results showed that heparin inhibited alkalinephosphatase (ALP) activity and mineralization in osteoblastic cells cultured with BMP-2. Heparin was found to suppress the mRNAexpressions of osterix, Runx2, ALP and osteocalcin, as well as phosphorylation of Smad1/5/8 and p38 MAPK. Further, heparin bound toboth BMP-2 and BMP receptor (BMPR). These results suggest that heparin suppresses BMP-2-BMPR binding, and inhibits BMP-2osteogenic activity in vitro.

J. Cell. Physiol. 216: 844–850, 2008. � 2008 Wiley-Liss, Inc.

*Correspondence to: Tatsuji Nishihara, 2-6-1 Manazuru,Kokurakita-ku, Kitakyushu 803-8580, Japan.E-mail: [email protected]

Received 14 October 2007; Accepted 6 March 2008

DOI: 10.1002/jcp.21468

Proteoglycans are involved in a variety of physiologicalconditions by binding to a litany of extracellular molecules(Takada et al., 2003), including growth factors, adhesionmolecules, and cytokines. Recently, the potential roles ofproteoglycans in some biological processes (Sasisekharan,2000; Shriver et al., 2002), including angiogenesis (Saisekharanet al., 1997), viral invasion (Shukla et al., 1999), tumor growth(Vlodavsky et al., 1999), and bone metabolism (Bi et al., 2005;Ariyoshi et al., 2005; Shinmyouzu et al., 2007), have beenreported.

Proteoglycans are abundant cell-surface molecules thatconsist of a core protein and highly sulfated glycosaminoglycan(GAG) chains. GAGs are long-chain compounds composed ofrepeating disaccharide units with a carboxyl group and one ormore sulfates, in which one sugar is N-acetylgalactosamine orN-acetylglucosamine (Bellows et al., 1986). Heparin, heparansulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, and hyaluronic acid are well known asendogenous GAGs.

Recent genetic study has indicated that heparan sulfate/heparin chains are involved in bone morphogenetic protein(BMP)-mediated developmental processes. For example,heparan sulfate/heparin chains bound to BMP-4 and restrictedthe expression pattern of BMP-4 in Xenopus embryos(Ohkawara et al., 2002). Heparan sulfate also binds to noggin, asecreted polypeptide that inhibits the function of BMP, resultingin modification of BMP-4 activity (Paine-Saunders et al., 2002),while heparan sulfate chains bind to BMP-7 and heparan sulfate/BMP-7 interaction is required for BMP-7 signaling (Irie et al.,2003). In addition, heparan sulfate and heparin modulate BMP-2osteogenic activity by sequestering BMP-2 on the cell surfaceand mediate its internalization (Takada et al., 2003; Zhao et al.,2006; Jiao et al., 2007).

BMPs were originally identified as unique proteins indemineralized bone matrix that induce ectopic bone formationwhen implanted into muscular tissues (Urist, 1965), and werelater shown to regulate the differentiation and function of cellsthat are involved in bone and cartilage formation and

� 2 0 0 8 W I L E Y - L I S S , I N C .

deformation (Canalis et al., 2003). Signaling of BMPs is initiatedby binding to the specific transmembrane receptors, type I andtype II serine/threonine kinase receptors (Wrana, 2000). Type Ireceptors are activated by ligand bound-type II receptors, andthen phosphorylate downstream molecules in the cytoplasm.Further, Smad1/5/8 transcription factors are phosphorylated bythe BMP receptor (BMPR) in the cytoplasm as substrates andaccumulate in the nucleus within 1 h after BMP stimulation(Waite and Eng, 2003). The phosphorylated Smads directlyregulate expression of primary target genes through binding totheir promoter or enhancer elements together with Smad4 andother transcription factors (Ito, 2003).

Although proteoglycans are characterized by variousGAG chains, it remains unclear how heparin regulates bonemetabolism induced by BMP-2. In the present study, weexamined the effects of heparin on osteoblast differentiationinduced by BMP-2 in vitro and also elucidated the precisemechanism by which heparin regulates the signals transductionsof osteoblastic cells cultured with BMP-2 for a short time.

Materials and MethodsReagents

Porcine intestinal mucosal heparin and low molecular weightheparin were purchased from Sigma Chemical Co. (St Louis, MO).Recombinant human BMP-2 was kindly supplied by AstellasPharmaceutical Inc. (Tokyo, Japan). Activin A was kindly supplied byAjinomoto Co. (Tokyo, Japan). Anti-phosphorylated Smad1/5/8,

E F F E C T S O F H E P A R I N O N B M P - 2 A C T I V I T Y 845

anti-p38 mitogen-activated protein kinase (MAPK) and anti-phosphorylated p38 MAPK were obtained from Cell SignalingTechnology, Inc. (Beverly, CA). Anti-Smad5 was obtained fromSanta Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-6xHis wasobtained from Nacalai Tesque, Inc. (Kyoto, Japan). Recombinanthuman BMPR type I and BMPR type II, each containing a6xHis-tagged Fc region, were obtained from R&D systems, Inc.(Minneapolis, MN).

Cell culture

The murine osteoblastic cell line MC3T3-E1 cells were cultured ina-minimum essential medium (a-MEM; Gibco, Grand Island, NY)containing 10% fetal calf serum (FCS; Gibco), penicillin G(100 U/ml), and streptomycin (100 mg/ml). The cells weremaintained at 378C in 5% CO2.

Alkaline phosphatase (ALP) activity

The ALP activity was evaluated after an initial cell seeding (1� 105/well) in a 24-well plate. The cultured cells were washed twice withHank’s balanced salt solution (HBSS), and solubilized with HBSScontaining 0.2% Nonidet P-40. The ALP activity of the lysate wasdetermined using p-nitrophenylphosphate (pNPP; Wako, Osaka,Japan) with the Lowry method. After a 30-min incubation at 378C,the absorbance of pNPP at 405 nm was measured with amicro-plate reader. The specific activity of ALP was calculated asmM/mg protein.

Mineralization

For a bone nodule formation assay, a mineralized extracellularmatrix was stained using the Von Kossa technique, as previouslydescribed (Bellows et al., 1986). In brief, the cells were seeded at adensity of 4� 105 in a 12-well plate. After 4 weeks of culture with50 mg/ml of ascorbic acid, 10 nmol/L of dexamethasone, and10 mmol/L of b-glycerophosphate, the specimens were stained todetect minerals in the bone nodules. Next, the specimens werewashed with phosphate-buffered saline (PBS, pH 7.2) 3 times, fixedwith 3.7% formaldehyde in PBS for 10 min, washed with distilledwater 3 times, and then incubated in 0.5% silver nitrate for 60 min.They were washed with distilled water 3 times, incubated in 0.3%sodium thiosulfate pentahydrate for 3 min, washed with distilledwater, and dried.

RT-PCR analysis

Gene expression levels were determined using a reversetranscription-polymerase chain reaction (RT-PCR) method. TotalRNA was extracted using a Total RNA Extraction Miniprep System(Viogene Co., Sunnyvale, CA), according to the manufacturer’sinstructions, and the reverse transcript was subjected to PCR.Oligonucleotide primers were designed to amplify cDNAfragments encoding Runx2 (381 bp), Osterix (497 bp), ALP(447 bp) and osteocalcin (371 bp). The primers used were asfollows: Runx2, 50-CCAGATGGGACTGTGGTTACC-30 and50-ACTTGGTGCAGAGTTCAGGG-30; Osterix,50-CTGGGGAAAGGAGGCACAAAGAAG-30 and50-GGGTTAAGGGGAGCAAAGTCAGAT-30; ALP,50-ACTGCTGATCATTCCCACGTT-30 and50-GAACAGGGTGCGTAGGGAGA-30; osteocalcin,50-CAAGTCCCACACAGCAGCTT-30 and50-AAAGCCGAGCTGCCAGAGTT-30; and GAPDH,50-ACCACAGTCCATGCCATCAC-30 and50-TCCACCACCCTGTTGCTGTA-30.

Western blot analysis

Cells (8� 105 cells/well) were cultured in 6-well plates ina-MEM inthe presence or absence of BMP-2 (100 ng/ml) and heparin (100mg/ml), then washed with PBS and lysed in lysis buffer (75 mM Tris–HClcontaining 2% SDS and 10% glycerol, PH 6.8). Protein contentswere measured using a DC protein assay kit (Bio-Rad, Hercules,

JOURNAL OF CELLULAR PHYSIOLOGY

CA). The samples were subjected to 10% SDS-PAGE andtransferred to polyvinylidene difluoride membranes (MilliporeCorp., Bedford, MA). Non-specific binding sites were blocked byimmersing the membranes in 10% skimmilk in PBS for 60 minat room temperature, after which the membrane was washed 4times with PBS, followed by incubation with the diluted primaryantibody overnight at 48C. Anti-Smad5, anti-phospho-Smad1/5/8,anti-p38 MAPK, and anti-phospho-p38 MAPK primary antibodies,as well as horseradish peroxidase-conjugated anti-mouse andanti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology,Inc.) were used in this experiment. After washing the membranes,chemiluminescence was produced using ECL reagent (AmershamPharmacia Biotech, Uppsala, Sweden) and detected withHyperfilm-ECL (Amersham Pharmacia Biotech).

Kinetic analysis using quartz-crystal microbalance (QCM)

A 27-MHz QCM (AffinixQ; Initium Inc., Tokyo, Japan) wasemployed to analyze the affinity of heparin and BMP-2. Heparin(3 ml, 100 mg/ml) was placed on the gold electrode surface ofthe QCM ceramic sensor chip and kept overnight at roomtemperature. After washing with distilled water, the sensor chipwas soaked in a chamber containing 10 ml of PBS at 258C untilfrequency equilibrium was attained. BMP-2 (250 mg/ml; volume20 ml) was added into the equilibrated solution containing theheparin-immobilized sensor chip. The binding of BMP-2 to heparinwas determined by monitoring the alterations in frequencyresulting from changes in mass on the electrode surface.

Next, we examined the binding of heparin and BMPR. In thiscase, anti-6xHis was immobilized to strengthen the binding of aQCM ceramic sensor chip and BMPR. In brief, anti-6xHis (3 ml,100 mg/ml) was placed on the gold electrode surface of the QCMceramic sensor chip and kept overnight at room temperature.After washing with distilled water, the sensor chip was soakedin a chamber containing 10 ml of PBS at 258C until frequencyequilibrium was attained. BMPR type I (100 mg/ml; volume 10 ml)and type II (100 mg/ml; volume 10 ml), each containing a6xHis-tagged Fc region, were added simultaneously into theequilibrated solution containing the anti-6xHis-immobilized sensorchip, after which heparin (1 mg/ml; volume 20 ml) was addedinto the equilibrated solution containing the BMPR type I- andtype II-binding sensorchip. The binding of heparin to BMPR wasdetermined by monitoring the alterations in frequency resultingfrom changes in mass on the electrode surface.

Statistical analysis

Statistical differences were determined using an unpaired Student’st-test with Bonferroni correction for multiple comparisons. Alldata are expressed as the means� standard deviation of threeexaminations, with similar results obtained in each experiment.

ResultsHeparin inhibits osteoblast differentiationinduced by BMP-2

To determine the effects of heparin on osteoblastdifferentiation induced by BMP-2, we assessed the ALP activity,a typical marker of osteoblast differentiation, and extracellularcalcium deposition during the formation of mineralized nodulesin the presence or absence of heparin. It is well known that ALPactivity and mineralization are dramatically enhanced, whenMC3T3-E1 cells are cultured with BMP-2. In the present study,heparin remarkably inhibited ALP activity induced by BMP-2after culturing for 3 days (Fig. 1) as well as mineralizationinduced by BMP-2 after culturing for 1 month (Fig. 2), both in adose-dependent manner. However, BMP-2 and heparin have nosignificant effect on cell proliferation rate and viability (data notshown).

Fig. 1. Heparin inhibits ALP activity induced by BMP-2 in a dose-dependent manner. MC3T3-E1 cells (2 T 105 cells/well) were stimulated withBMP-2 (100 ng/ml) in the presence or absence of several concentrations of heparin for 3 days. The specific activity of ALP (mM/mg protein) wasdeterminedasdescribedinMaterialsandMethodsSection.Valuesareexpressedasfold increaserelativetountreatedcontrols.Dataareexpressedas the mean W SD of triplicate cultures. The experiment was performed three times, with similar results obtained in each experiment. MP < 0.05(Student’s t-test).

846 K A N Z A K I E T A L .

Mechanism of binding of heparin to BMP-2 and BMPR

In the present study, we investigated the affinity betweenheparin and BMP-2 using a QCM technique, and confirmed thatheparin competitively inhibits the binding of BMP-2 and BMPR.When BMP-2 was injected into an equilibrated solutioncontaining a heparin-immobilized sensor chip at 258C, thefrequency was decreased by 160 Hz (Fig. 3A). Next, BMPR

Fig. 2. Heparin inhibits mineralization induced by BMP-2 in a dose-depeBMP-2(100ng/ml) inthepresenceorabsenceofseveralconcentrationsofhebonenodules as described in Materials andMethodsSection. Values areexpbe viewed in the online issue, which is available at www.interscience.wiley


type I and II, each containing a 6xHis-tagged Fc regionwere injected into the equilibrated solution containing ananti-6xHis-immobilized sensor chip at 258C. After the solutionwas equilibrated and heparin was injected into the solutioncontaining a BMPR type I- and II-binding sensor chip, thefrequency was decreased by 60 Hz (b). When BMP-2 wasinjected, the frequency was decreased by 90 Hz (c) (Fig. 3B).These results indicate that the binding of BMP-2 and BMPR is

ndent manner. MC3T3-E1 cells (4 T 105 cells/well) were cultured withparinfor4weeks.Thespecimenswerestainedtodetectminerals intheressedas fold increase relative tountreated controls. [Color figure can.com.]

Fig. 3. Bindingpatternsofheparin,BMP-2andBMPR.Heparin(3ml;100mg/ml)wasimmobilizeddirectlyonaQCMceramicsensorchipsoakedinPBS solution at 25-C as described in Materials and Methods Section. BMP-2 (250mg/ml; volume 20ml) was applied to the equilibrated solution (A).Next, anti-6xHis (3 ml; 100 mg/ml) was immobilized directly on a QCM ceramic sensor chip in PBS solution at 25-C as described in Materials andMethods Section. And then, BMPR type I (100mg/ml; volume 10ml) and BMPR type II (100mg/ml; volume 10ml), each containing a 6xHis-tagged Fcregion,wereappliedsimultaneously totheequilibratedsolution(B–E).ActivinA(a;40mg/ml;volume20ml),heparin (b;1mg/ml;volume20ml)andBMP-2 (c; 250 mg/ml; volume 20 ml) were applied separately to the equilibrated solution (B). Heparin was injected into the solution ( @), andBMP-2 was injected after the solution was equilibrated ( A) (C). BMP-2 was injected into the solution ( @), and heparin was injected after thesolution was equilibrated ( A) (D). Heparin and BMP-2 were injected into the solution simultaneously (E).


stronger than that of heparin and BMPR. On the contrary,activin A, a negative control, has nonbinding property withBMPR type I and II (a). In addition, we examined the competitiveability of heparin toward BMP-2 through binding to BMPR.Following the binding of heparin to BMPR, BMP-2 bound to theBMPR and heparin complex with high affinity (Fig. 3C). Further,after BMP-2 was bound to BMPR, heparin also bound to theBMPR and BMP-2 complex with high affinity (Fig. 3D). Theseresults indicate that heparin binds to both BMP-2 and BMPRwith high affinity. Next, when heparin and BMP-2 were injectedsimultaneously into the solution containing a BMPR type I- andtype II-binding sensor chip, the complex of heparin and BMP-2bound to BMPR with high affinity (Fig. 3E). Similar results were


obtained, when the experiments were performed using lowconcentrations of heparin (data not shown).

Heparin interferes with BMP-2-mediated signaling

We also examined the effects of heparin on BMP-2 signaling inMC3T3-E1 cells. Runx2 and osterix are transcription factorsessential for osteoblast differentiation and bone formationinduced by BMP-2. When the cells were cultured with BMP-2(100 ng/ml) and heparin (100 mg/ml), heparin suppressed thegene expressions of Runx2, osterix, ALP and osteocalcin(Fig. 4A–D). Further, we examined the effects of heparin on thephosphorylation of Smad1/5/8 and p38 MAPK in MC3T3-E1

Fig. 4. Heparin inhibits the gene expressions of Runx2, osterix, ALP and osteocalcin in MC3T3-E1 cells stimulated with BMP-2. MC3T3-E1 cells(4 T 105 cells/well) were incubated with BMP-2 (100 ng/ml) in the presence or absence of heparin (100 mg/ml) for 10 h. Total RNA from each cellculturewasreverse-transcribedwithrandomprimers.PCRamplificationwasperformedusingprimersspecificforRunx2(A),osterix(B),ALP(C),osteocalcin (D)andGAPDH.Eachof thePCRproductswasresolved in2%agarosegelandstainedwithethidiumbromide.Thescanneddensities ofeach lane are shown in the graphs.


cells stimulated with BMP-2 for 30 min. Heparin also inhibitedthe phosphorylated levels of Smad1/5/8 and p38 MAPK inMC3T3-E1 cells stimulated with BMP-2 in the presence ofheparin (Fig. 5).

Discussion

It is well known that a long-term administration of heparin isassociated with an increasing risk for the development ofosteoporosis (Jones and Sambrook, 1994; Wolinsky-Friedland,1995), and that some patients who undergo long-term heparintreatment experience a subclinical reduction in bone density(Walton et al., 2002). Pikul et al. reported that the heparintended to increase the formation of osteoclasts at lowerconcentrations, whereas at the highest concentrations ittended to decrease the numbers of osteoclasts in rat bonemarrow cell cultures (Folwarczna et al., 2005). However, thereare no informations on the effect of a short-term heparintreatment on osteoblastic cells. In the present study, weexamined the effects of heparin on bone formation induced byBMP-2 in vitro, and clarified the precise mechanism by whichheparin regulates osteoblast differentiation and function duringbone metabolism.

Many studies have reported that the binding of heparin topeptides (Chevanne et al., 1999) and chemokines (Witt andLander, 1994; Ariyoshi et al., 2008) may be critical for their


activities that are dependent on immobilization. However,there are few reports indicating that heparin inhibits bonemetabolism through the regulation of osteoblast functions. Inthe present study, we found that heparin remarkably bound toBMP-2 and BMPR. To our knowledge, it is the first reportconcerning the ability of heparin to bind to these osteogenicmolecules. Our findings clearly indicate that such binding abilityis involved in the inhibition of osteoblast differentiation inducedby BMP-2 (Figs. 1 and 2). In addition, we found that treatmentwith heparin remarkably suppressed the mRNA expressions ofosterix and Runx2 (Fig. 4A,B), which are well known as essentialfactors in osteoblast differentiation and bone formation(Komori et al., 1997; Nakashima et al., 2002). Taken together,these findings suggest that treatment of heparin suppress thecellular response in BMP-2-stimulated osteoblastic cellsthrough osterix and Runx2.

MAPK family members are proline-directed serine/threonine kinases that are important for cell growth,differentiation, and apoptosis (Nishida and Gotoh, 1993;Avruch et al., 1994; Davis, 1994; Iwasaki et al., 1996), and areactivated by phosphorylation of threonine and tyrosine inresponse to external stimuli. MAPKs, such as extracellularsignal-regulated kinase, c-Jun N-terminal kinase (JNK), andp38 MAPK, have been shown to induce osteoblastic celldifferentiation (Xiao et al., 2000; Sowa et al., 2002; Ziros et al.,2002; Kanno et al., 2007). Among them, p38 MAPK is well

Fig. 5. Heparin interferes with BMP-2-mediated Smad-1/5/8 andp38 MAPK phosphorylation. MC3T3-E1 cells (4 T 105 cells/well) werestimulated with BMP-2 (100 ng/ml) in the presence or absence ofheparin (100 mg/ml) for 30 min. Whole cell lysates were subjected toimmunoblotting analyses. A: Expression of Smad5 andphosphorylated Smad1/5/8. B: Expression of p38 MAPK andphosphorylated p38 MAPK. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]


known to participate in many cellular process including cellcycle arrest, apoptosis and differentiation of osteoblasts (Katzet al., 2006; Kanno et al., 2007). Guicheux et al. (2003) havereported that p38 MAPK and JNK as new signaling pathwaysinvolved in enhancement of ALP and osteocalcin expression inMC3T3-E1 cells. We found that heparin strongly suppressedthe gene expression of Runx2, osterix, ALP and osteocalcin(Fig. 4), and the phosphorylation of p38 MAPK in MC3T3-E1cells stimulated with BMP-2 (Fig. 5). These findings suggest thatthe inhibitory effect of heparin on osteoblastic differentiation isresponsible for the regulation of p38 MAPK.

Members of the Smad protein family are known to playpivotal roles in signaling by the intracellular TGF-b family, suchas TGF-b, BMP-2 and activins. Among them, Smad1, Smad2,Smad3, and Smad5 become phosphorylated by specificactivated type I serin/threonine kinase receptors and thus act ina pathway-restricted fashion (Heldin et al., 1997). We clearlydemonstrated that heparin bound to BMP-2 and BMPR.In addition, the treatment of heparin inhibited thephosphorylation of Smad family proteins and p38 MAPK (Fig. 5).

It is well known that Smads play critical roles in stimulation byBMP-2 of MC3T3-E1 cells. In the present study, heparin wasfound to inhibit the signaling pathways of Smads throughinhibition of BMP-2 and BMPR binding. This is consistent withprevious reports demonstrating that heparin inhibits BMP-2and BMP-7 signaling pathways (Irie et al., 2003; Jiao et al., 2007).On the other hands, it is reported that heparin enhancesBMP-2-induced osteoblast differentiation in C2C12 myoblasts,demonstrating that its expression level was changed when beingtreated for 72 h (Zhao et al., 2006). They suggest that heparinenhances BMP-indicated osteoblast differentiation by BMPfrom degradation and inhibition by BMP antagonists. Onepossible explanation for this discrepancy is the difference of theconcentration and the action time. Further, it appears likely thatthe differences might be due to the assay systems and themicroenvironments used in the experiments.

In addition, we confirmed that a low molecular weightheparin (3 kDa) also bound to BMP-2 and BMPR using a QCMtechnique (data not shown). Based on this result, we speculatedthat a low molecular weight heparin might inhibit BMP-2osteogenic properties by binding to BMP-2 and BMPR. Since it


would be of more clinical significance, further analyses areplanned to clarify this point to determine the functional role of alow molecular weight heparin in bone metabolism.

In conclusion, the present results demonstrated that heparininhibits the binding of BMP-2 to BMPR and subsequent mRNAexpression of osterix and Runx2, as well as phosphorylation ofSmad1/5/8 and MAPK signal transduction. Further, heparin wasfound to suppress the differentiation of osteoblastic MC3T3-E1cells treated with BMP-2. These findings suggest that heparinplays a crucial role in bone tissue in both physiological andpathological conditions. Recently, a biochemical studydemonstrated that heparin bound to BMP-7 and its bindingproperty was specific for heparin structures, such as N- and6-O-sulfates (Irie et al., 2003). In addition, heparin-bindingdeterminants were found to be located in the N-terminalsegments of BMP-2 (Ruppert et al., 1996). Additionalbiochemical research is needed to further define the bindingproperties of heparin to BMP-2 and BMPR.

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