www.elsevier.com/locate/thromres

Thrombosis Research (2008) 122, 743–752

REVIEW ARTICLE

The anti-inflammatory effects of heparin andrelated compounds

Edward Young ⁎

Department of Pathology and Molecular Medicine and Henderson Research Center, McMaster University, Hamilton,Ontario, Canada

Received 26 October 2006; received in revised form 26 October 2006; accepted 26 October 2006Available online 28 August 2007

⁎ Hamilton Regional LaboratoryMedicE-mail address: younged@hhsc.ca.

0049-3848/$ - see front matter © 200doi:10.1016/j.thromres.2006.10.026

Abstract

Heparin is a glycosaminoglycan well known for its anticoagulant properties. In addition,heparin possesses anti-inflammatory effects. Although the mechanisms responsible forthe anticoagulant effects of heparin are well understood, those underlying its anti-inflammatory effects are not. This review presents some of the evidence from clinicaland animal studies supporting an anti-inflammatory role for heparin and heparin-related derivatives. Potential mechanisms by which heparin can exert its anti-inflammatory effects are discussed. The clinical use of heparin as an anti-inflammatoryagent has been held back by the fear of bleeding. Development of nonanticoagulantheparins or heparin derivatives should mitigate this concern.© 2007 Elsevier Ltd. All rights reserved.

KEYWORDSHeparin;Inflammation;Cytokines;Selectins;Nuclear factor kappa B;Apoptosis

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744Clinical evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744

Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744Ulcerative colitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744Burns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744

Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745Ischemia-reperfusion injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745Heparin and myocardial ischemia-reperfusion injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745

Potential mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745Heparin-binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746Heparin and selectin-mediated cell adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746Heparin and nuclear factor kappa B (NF-κB). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747

ine Program,HendersonGeneral Site, 711 Concession Street, Hamilton,Ontario, Canada, L8V 1C3.

7 Elsevier Ltd. All rights reserved.

744 E. Young

Heparin and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748Nonanticoagulant heparins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749

Introduction

Heparin is a member of a family of polyanionicpolysaccharides called glycosaminoglycans. Thestructure of individual glycoaminoglycans is deter-mined by their repeating disaccharide sequences,which consist of alternating uronic acid and aminosugar residues. Heparin is a highly sulfated polysac-charide composed of hexuronic acid and D-glucos-amine residues joined by glycosidic linkages [1].

Heparin remains one of the most importantanticoagulant drugs in clinical practise. It is currentlyused for the prevention and treatment of venousthrombosis and pulmonary embolism, managementof arterial thrombosis in patients presenting withacute myocardial infarction and in the prevention ofrethrombosis after thrombolysis. Other uses includethe prevention of thrombosis in extracorporealcircuits and hemodialysis [2,3]. Although primarilyemployed for its anticoagulant properties, it has beenknown for many years that heparin possesses anti-inflammatory activity. The mechanisms responsiblefor the anticoagulant effects of heparin are wellunderstood; those underlying its anti-inflammatoryactivity are not. The mechanisms behind the antico-agulant effects of heparin have been the focus ofseveral excellent reviews [4,5]. This reviewwill try tohighlight some of the anti-inflammatory effects ofheparin but will not attempt to be inclusive.

Clinical evidence

The potential of heparin as an anti-inflammatorydrug is supported by several modestly sized clinicaltrials in various models of inflammatory disease.Heparin and related compounds have been shown tobenefit patients with bronchial asthma, ulcerativecolitis and burns.

Asthma

Several studies in the 1960s first described subjectiveimprovement in asthma symptoms using intravenousheparin [6,7]. This was followed by a trial of inhaledheparin involving 10 patients with mild-to-severeasthma who experienced subjective but no objectiveimprovement [8]. Further interest in using heparin inhuman studies did not appear until Ahmed et al.demonstrated in 1992 that inhaled heparin markedly

attenuated the increase in lung resistance induced inpreviously sensitized sheep by allergen reexposure [9].In follow-up studies [10,11], inhaled heparin was ableto inhibit the bronchconstrictive response in patientswith exercise-induced asthma. Inhaled enoxaparin (alow molecular weight heparin) demonstrated similarprotective effects and was superior to unfractionatedheparin at higher doses [12]. When tested as apretreatment for the early asthmatic response toinhaled allergen (dust mite extract), a small butstatistically significant protective effect was obtainedwith nebulized heparin [13]. In another study,multipledoses of inhaled heparin attenuated the early asth-matic response and significantly reduced the lateasthmatic response [14]. Trials of inhaled heparin onthe bronchoconstrictive response to bronchostimu-lants have yielded mixed results. In two studies[15,16], inhaled heparin inhibited methacholine-in-duced bronhoconstriction while another did notdemonstrate any effect [17]. Finally, two cases ofcorticosteroid-resistant asthma patients whoresponded to inhaled heparin during asthma exacer-bations have been reported [18].

Ulcerative colitis

Several uncontrolled studies conducted in the mid-to-late 1990's demonstrated that heparin reducessymptoms and improves healing in patients withsteroid-resistant ulcerative colitis [19–21]. This wasfollowed by two randomized controlled trials thatprovided conflicting results. Although Ang et al. [22]demonstrated both efficacy and safety; the study byPanes et al. [23] did not. Similarly, early case studiesusing low molecular weight heparin also found goodclinical response in patients with ulcerative colitis[24,25]. In contrast, a controlled, randomized trialcomparing lowmolecular weight heparin (tinzaparin)to placebo in patients with mild to moderately activeulcerative colitis showed no benefit of low molecularweight heparin [26].

Burns

Heparin has been used for many years to treat burnsalthough it has not become commonmedical practise.Heparin has been given to burn patients parenterally,topically, along with membranes used for skin graftingand inhaled as aerosols to hasten healing from burns

745The anti-inflammatory effects of heparin and related compounds

[27]. The evidence supporting the use of heparin inburns is weak and is based primarily on case reportsand series [Ref 28,29 for example]. These studiessuffer from serious methodological weaknesses andthe absence of well-designed randomized clinicaltrials. Despite these problems, the basic scientificliterature does provide a rationale that heparin canbe efficacious for the treatment of burn injury. Asystematic review of the literature on the effective-ness of heparin for burn treatment is being preparedfor the Agency for Healthcare Research and Quality(Evidence Report/Technology Assessment Number148, Publication No. 07-E004, December 2006).

Animal studies

Ischemia-reperfusion injury

Ischemia results in reduction or cessation of blood flowto a particular tissue. This deficiency in oxygen andnutrient supply can cause the build-up of metabolites,cellular and organ dysfunction and if prolonged,eventual tissue death. Reperfusion or restoration ofblood flow should theoretically alleviate the situationbut at the same time as the reperfusing blood halts theischemic process by supplying blood and nutrients, acascade of events with properties similar to theinflammatory response is rapidly initiated. The in-flammatory response begins with complement activa-tion followed by the release of oxygen free radicals,cytokines, and other proinflammatory mediators thatactivate both neutrophils and the vascular endotheli-um. Activation of these cell types promotes theexpression of adhesion molecules that recruit neutro-phils to the surface of the endothelium. Neutrophilsplay a central role in this inflammatory-like process toreperfusion by releasing oxidants and proteases thatdamage or kill tissues, and the release of chemoat-tractants that amplify the recruitment of greaternumbers of neutrophils into the affected tissue. Thisspecific series of events lead to reperfusion injury thatincludes endothelial dysfunction, microvascular col-lapse, blood flow defects, thrombosis, infarction andcell death. Ischemia-reperfusion injury is clinicallyrelevant to the heart because of myocardial infarctionand to other organs such as the liver and kidneysbecause of circulatory shock and to the brain becauseof stroke. Heparin has been reported to diminish orprotect against reperfusion injury in various animalmodels.

Heparin and myocardial ischemia-reperfusioninjury

Myocardial ischemia-reperfusion injury has beenparticularly well studied. There is considerable

interest in developingmedical therapies tominimizemyocardial ischemia-reperfusion injury followingmyocardial infarction. Heparin and heparin deriva-tives have shown benefit in reducing myocardialischemia-reperfusion injury. Black et al. [30] eval-uated the effects of heparin and an analog of heparin(N-acetyl heparin) administered just before reper-fusion in a canine model and reported a significantreduction in infarct size while Kouretas et al foundan improvement in coronary endothelial function[31]. Friedrichs et al. [32] also using heparin and N-acetyl heparin observed a reduction in myocardialdysfunction and infarct size in the rabbit isolatedheart subject to global ischemia and reoxygenation.Gralinski et al. [33] demonstrated the cardioprotec-tive effects of a low molecular weight polysulfatedheparin against complement-mediated (human plas-ma) myocardial injury. A low molecular weightheparin in the form of an ion-pair complex hasbeen shown to reduce arrhythmias following region-al ischemia and reperfusion in isolated rat hearts[34]. Thourani et al. [35] studied ischemia-reperfu-sion in a canine infarct model using an O-desulfatedheparin and found decreased neutrophil influx to theischemic myocardium and reduced infarct size.Heparin added to cardiopelgia solution improvedthe recovery of left ventricular function in anisolated ischemic rat heart model [36].

There is some evidence that heparin may bebeneficial when used in a clinical setting. In twoclinical studies [37,38], patients undergoing pro-spective or emergent cardiopulmonary bypass sur-gery using heparin-bonded circuits had a lowerincidence of myocardial infarction, less need ofionotropic support, a lower incidence of prolongedventilation and fewer postoperative complications.In another small study, forty patients requiringelective cardiopulmonary bypass surgery wererandomized to receive a standard dose or a higherthan usual dose of heparin. Although not statisti-cally significant, there was a trend for the proin-flammatory cytokines interleukin-6 and tumournecrosis factor-α to be lower in the group receivingthe higher heparin dose [39].

Potential mechanisms

Heparin is a highly acidic polymer and its biologicaleffects depend on both specific and nonspecificionic interactions that are mediated by sequencecomposition, charge density, charge distributionand molecular size. Except for high affinity bindingof antithrombin to heparin via a unique pentasac-charide sequence [40], no hard evidence for otherexamples of “specific binding” has been described.Heparin binds “nonspecifically” to many proteins.

746 E. Young

The mechanisms responsible for the anti-inflamma-tory effects of heparin are complex and incom-pletely understood. Some of the potentialmechanisms are:

Heparin-binding proteins

More than 100 heparin-binding proteins have beenidentified and the list is growing [41]. It is notsurprising that binding to heparin modulates thefunction and activity of a variety of proteins. Theseproteins fall into diverse groups, including proteinsinvolved in coagulation and fibrinolysis, manygrowth factors, several proteins of the extracellularmatrix, a number of proteins involved in lipidmetabolism and finally, mediators of the immuneresponse. Heparin has been shown to bind acutephase [42] and complement proteins [43] and thisproperty may contribute to the anti-inflammatoryactivity of heparin.

Heparin and related glycosaminoglycans such asheparan sulfate play an important role in theimmune system because of their ability to interactwith pro-inflammatory cytokines and chemokines. Itis important to separate the physiological role ofendogeneous heparin-like glycoaminoglycans fromthe pharmacological effects of heparin. In vivo,heparin-like molecules such as heparan sulfate arefound on the surface of the vascular endothelium. Itis thought that the binding of a cytokine to theseheparin-like molecules can protect the cytokinefrom proteolytic degradation. For example, heparinbinding to the C-terminal motif of interferon γ (INF-γ) blocks proteolytic cleavage at this site thuspreventing its rapid clearance from the circulationresulting in increased bioavailability of this cytokine[44]. It has been suggested that another form ofprotection may be the ability of heparin-likecompounds to stabilize and to localize cytokines/chemokines at the site of inflammation. Basicfibroblast growth factor (bFGF) is a cationicpolypeptide and its conformation is changed afterbinding to heparin. This binding protects bFGF fromheat and acid-mediated inactivation [45] as well asnonenzymic glycosylation [44]. This led to theconcept whereby FGFs are bound on the vascularendothelium or secreted into the extracellularmatrix where it would be sequestered. This concepthas been extended to cytokines/chemokines onendothelial cell surfaces or the extracellular matrixwhere they direct and promote leukocyte migrationand activation. Thus, cell surface glycosaminogly-cans sequester local concentrations of cytokine/chemokine in the vicinity of its receptor therebyenhancing binding to its receptor. The significanceof endogenous glycosaminoglycan–cytokine inter-

actions may be to support cellular mechanismsleading to inflammation.

Targeting glycosaminoglycan–cytokine interac-tions have potential therapeutic benefit. Whengiven in soluble, pharmacological doses, exogenousheparin and heparinoids attenuate ongoing tissuedamage because heparin can bind to and neutralize awide variety of mediators released from inflamma-tory cells [46,47]. Many chemokines, cytokines andcomplement factors are bound by heparin therebypreventing these pro-inflammatory molecules frominteracting with their respective receptors.

Heparin and selectin-mediated cell adhesion

Leukocyte adhesion and activation play a centralrole in the inflammatory response [48]. Excessiveleukocyte activation leads to their intravascularaggregation and to the release of toxic oxygenradicals and proteolytic enzymes that in turncontribute to vascular and tissue damage.

There is accumulating evidence that heparininterferes with the adhesion of leukocytes to theendothelium.Heparin has been shown to inhibit f-met-leu-phe-activated (fMLP) neutrophil adherence toresting endothelial cells [49] while another studydemonstrated that heparin and partially desulphatedderivatives were able to inhibit nonactivated neutro-phils to platelet activating factor (PAF) stimulatedendothelial cells [50]. Low molecular weight heparinhas also been reported to inhibit adhesion ofneutrophils to endothelial cells [51].

The selectins are responsible for the loose interac-tion between the endothelium and the neutrophils,tethering the neutrophil to the endothelial surface.Three glycoprotein adhesion molecules belong to theselectin family (P-selectin, L-selectin and E-selectin).L-selectin is expressed constitutively on the surface ofneutrophils andmonocytes, whereas E- and P-selectinare mainly expressed on activated endothelial cells.P-selectin is also found on activated platelets. Thecounter-ligands for the selectins are the carbohydratemoieties, sialy Lewis X (sLex), siayl Lewis A (sLea) orthe sialomucin P-selectin glycoprotein ligand-1 (PSGL-1) [52–54]. Under static conditions, heparin and lowmolecular weight heparin oligosaccharides inhibit thebinding of L-selectin and P-selectin chimeras toadsorbed sLex [55,56]. Similarly, platelet P-selectinalso binds to heparin-Sepharose [57]. Heparin alsoeffectively inhibits the binding of P-selectin to eitherneutrophils or to a promyelocyte cell line (HL-60) [58].When heparin or a number of low anticoagulantderivatives were incubated with neutrophils, thesecells were inhibited from adhering to endothelial cellmonolayers stimulated with PAF or thrombin, stimu-lants that induce the expression of P-selectin [50].

747The anti-inflammatory effects of heparin and related compounds

Under similar conditions, increased binding of unfrac-tionated heparin and lowmolecular weight heparin tothrombin-activated endothelial cells has been dem-onstrated [59]. These findings lend further support tothe hypothesis that heparin and its low molecularweight derivatives can inhibit neutrophil adherenceto activated endothelial cells by binding to P-selectin,a known heparin-binding protein.

The firm attachment of the neutrophil to theendothelial surface involves the β2-integrins (CD11/CD18 complex). The β2-integrins are a family ofheterodimeric glycoproteins that are constitutivelyexpressed on the surface of neutrophils. There arethree distinct α-chains (CD11a, CD11b, CD11c) and acommon β subunit. The CD11/CD18 complex isstored in secondary granules in neutrophils. Uponactivation by a number of proinflammatory media-tors such as platelet-activating factor, C5a and IL-8,there is an increased surface expression of CD11/CD18 complexes (CD11b/CD18, CD11c/CD18). Thisis achieved by rapid translocation from granules tothe membrane surface followed by a conformation-al change and conversion from a low affinity to ahigh affinity binding state. Endothelium-derivedadhesion molecules known as intercellular adhesionmolecules (ICAMs, part of the IgG superfamily) actas the counter ligands for the CD11/CD18 complexto mediate firm adhesion to the endothelium. Oncethis has occurred, the neutrophils can proceed bytransendothelial migration into the interstitialspace. This transmigration is primarily stimulatedby PECAM-1, another member of the immunoglob-ulin superfamily [60,61]. It has been reported thatimmobilized heparin can mediate neutrophil adhe-sion via interactions with Mac-1 (CD11b/CD18).Structural comparisons of heparin derivatives forMac-1 indicated a requirement for either N- or O-sulfation [62]. Further studies have shown that thatheparin can bind directly to Mac-1 and mediate thebinding of the soluble ligands fibrinogen, factor Xand iC3. Low molecular weight heparin also inhibitsbinding of fibrinogen to Mac-1. These results in-dicate that heparin binding to Mac-1 may directlyalter neutrophil function in coagulation, inflamma-tion and cell proliferation [63]. A number of studieshave now confirmed that heparin and relatedoligosaccharides inhibit selectin-mediated cell ad-hesion and block acute inflammation using a varietyof experimental procedures including cell adhesionassays, flow cytometry, intravital microscopy andthioglycollate-induced peritonitis [56,64–67].

Heparin and nuclear factor kappa B (NF-κB)

At the molecular level, there is evidence thatheparin and related compounds may exert its anti-

inflammatory effects through the transcriptionfactor NF-κB. Inflammation is associated with thecoordinated action of a series of cytokine andadhesion molecule genes. The regulation of thesegenes involves nuclear factor κB (NF-κB), a ubiqui-tous inducible transcription factor. NF-κB is acti-vated by a vast number of agents includingcytokines, lipopolysaccharide (LPS), UV irradiation,growth factors, free radicals, oxidative stress andviral infection [68]. The genes regulated by NF-κBare diverse but include those that transcriptionallypromote expression of many inflammatory andimmune response genes including ICAM-1, L- andP-selectins, and interleukin-6 and 8 [69,70].

In nonstimulated cells, NF-κB is sequestered withinthe cytosol by an inhibitory protein called I-κB thatmasks the nuclear localization sequence presentwithin the NF-κB protein sequence [71]. Whenstimulated, the NF-κB-I-κB complex is phosphorylatedand the I-κB protein is dissociated and inactivated[72,73]. Release from I-κB exposes the NF-κB nuclearlocalization sequence, a highly cationic domain ofnine amino acids (VQRDRQKLM) that targets nucleartranslocation [74]. The degradation of I-κB allows NF-κB to translocate into the nucleus where it acts as atranscription factor. NF-κB activation has been shownin various models of experimental myocardial ische-mia and reperfusion [75,76]. NF-κB is activated byischemia alone or by ischemia-reperfusion withsubsequent upregulation of adhesion molecules onthe myocyte surface [77] and production of pro-inflammatory cytokines such as TNF-α [78] and IL-6[79]. A number of studies have shown that heparin isreadily bound and internalized into the cytosoliccompartement of endothelium [59], vascular smoothmuscle cells [80], liver cells [81] and cardiacmyocytes[82]. Once bound and/or internalized into thecytoplasm, heparin and related molecules can bindelectrostatically to the positively charged nuclearlocalization sequence of NF-κB and prevent it fromtranslocating to the nucleus. Blocking of thistranscriptional factor can potentially reduce inflam-matory gene activation and regulate the geneexpression and production of proinflammatory cyto-kines, chemokines and adhesion molecules. Heparinand o-desulphated heparin have been reported toinhibit NF-κB activation in a tumour necrosis factor(TNF-α)-stimulated human endothelial cell line and inischemic-reperfused rat myocardium [35]. Low mo-lecular weight heparin also appeared to inhibittranslocation of NF-κB in high glucose-stimulatedhuman endothelial cells [83] and in T cells [84] whileboth unfractionated heparin and low molecularweight heparin downregulated proinflammatory cyto-kines and NF-κB in LPS-stimulated human monocytes[85].

748 E. Young

Heparin and apoptosis

Tumour necrosis factor (TNF-α) and NF-κB are bothintimately involved in apoptotic cell death. Sinceheparin and related compounds have been shown tomodulate the activity of these important membersof the apoptosis cascade, it is interesting tospeculate that heparin my have therapeutic poten-tial in this area. The role of heparin in apoptosis hasbeen largely unexplored.

Activation of apoptotic pathways and programmedcell death can be initiated by the binding of a specificprotein ligand to the cell surface. One of the moreimportant receptors is a member of the tumour ne-crosis receptor family, TNFR1 (also called p55). WhenTNF-α attaches to TNFR1, the receptor trimerizesand binds a series of other proteins called TRADD(TNFR-associated death domain), TRAF-2 (TNFR-associated protein-2) and FADD (Fas-associateddeath domain). FADD couples the TNFR1–TRADDcomplex to activate caspase-8 thereby initiating theentire cascade of other caspases that effect apopto-sis. The caspases are a group of cysteine proteasesthat play a crucial role in initiating and executingapoptosis [86].

TNF-α plays an important role in ischemia-reperfusion injury. TNF-α mediates contractile de-pression of myocardium following ischemia and re-perfusion during myocardial infarction [87]. TNF-αalso plays a significant role during hemorrhagicshock [88]. Apoptosis from TNF-α produced endog-enously by overloaded myocardium has been shownto contribute to initiation and progression of con-gestive heart failure [89,90]. There is evidence thatheparin can inhibit gene expression and productionof TNF-α in ischemic rat heart [36]. Secondly, hep-arin blocks P-selectin- and integrin-mediated recruit-ment of neutrophils. These cells are rich sources ofTNF-α production and myocardial cell apoptosis ishighly correlated with inflammatory cell influxfollowing ischemia-reperfusion [91]. Through thesemechanisms, heparin and related compounds havethe potential to reduce TNF-α available to induceTNFR1 death domain mediated apoptosis.

At a second site, apoptosis is initiated by eventsthat disturb mitochondria. Either disruption of elec-tron transport and aerobic oxidative phosphorylationor opening of pores in the mitochondria membrane bypro-apoptotic cytoplasmic proteins will allow leakageout of the mitochondria of the respiratory chaincomponent cytochrome c. Upon entering the cyto-plasm, cytochrome c binds to a cytosolic protein calledapoptotic protease activating factor-1 (Apaf-1). In thepresence of ATP, the complex of cytochrome c andApaf-1 activate procaspase 9 that initiates subsequentactivation of the caspase cascade. The TNRF1 or death

receptor-mediated and the mitochondria-mediatedpathways converge on a common downstream path-way at effector caspases such as caspase-3 [86].

Cytochrome c plays a prominent early role in thesignal transduction of caspase activation and cardi-omyocyte apoptosis induced by reactive oxygenspecies [92]. Production of reactive oxygen speciesis greatly enhanced by ischemia-reperfusion injuryto the myocardium and the oxidant stress produceleads to myocardial apoptosis [93]. Thus, theactivity of cytochrome c when it is released intothe cytoplasm plays a pivotal role in activating pro-apoptotic pathways making cytochrome c an attrac-tive target for therapeutic intervention.

Mitochondrial cytochrome c is a basic protein witha positive charge of +9.5 at neutral pH [94,95]. Withinthe cell, cytochrome c forms complexes with itsnatural electron transport partners such as cyto-chrome-bcl complex and cytochrome c oxidase. Thesecomplexes are electrostatic in nature and involvecharge dependent binding of the positive lysineresidues surrounding the exposed edge of the haememoiety to the negatively charged amino acids on itsrespiratory chain partners. Because of its positivecharge, cytochrome c likely binds to Apaf-1 on anegatively charged region of the Apaf-1 moleculecharacterized by 12 WD (trytophan-aspartic acid)repeats [96]. Binding of the positively chargedcytochrome c to the negatively charged region ofthe Apaf-1 molecule induces conformational changesthat allow Apaf-1 to bind and activate caspase-9[86,97]. As a strong polyanion, heparin would beexpected to compete with Apaf-1 for binding topositively charged cytochrome c thus protecting thecell from apoptosis. Electrostatic interaction withproteins has been shown to be responsible for theinhibitory effect of heparin and other sulfatedpolysaccharides on the activity of positively chargedgranular neutrophil proteases such as human leuko-cyte elastase and cathepsin G [98]. There is evidencethat heparin is able to modulate the activity ofcytochromec. For example, heparin greatly decreasesits reactivity in redox reactions. There was a 200-foldreduction in the reaction of cytochrome c withascorbic acid in the presence of heparin [95]. Thecomplex of heparin and cytochrome c occurs whethercytochrome c is in the reduced or oxidized state [99].

Finally, heparin and other polyanions may actdirectly at the nuclear level to control the activity ofpro-apoptotic proteins. Recently, heparin was shownto inhibit a major apoptotic nuclease responsible forDNA fragmentation in cells undergoing apoptosis[100]. Another study reported that high affinitybinding sites for heparin are generated on leukocytesduring apoptosis [101]. The heparin binding domainsoriginated from the nucleus and corresponds to

749The anti-inflammatory effects of heparin and related compounds

ribonucleoprotein structures. Ribonucleoproteinsplay diverse roles in the post-transcriptional proces-sing and subsequent packaging, transport and trans-lation of mRNA. Binding to nucleoproteins is anotherpotential mechanism whereby heparin and relatedpolysaccharides can modulate the expression of pro-inflammatory and apoptotic genes.

Nonanticoagulant heparins

The use of heparin as an anti-inflammatory agent hasbeen largely hindered by the fear of bleeding. Theanti-inflammatory effects of heparin are problem-atic because these activities are only expressed withhigh heparin concentrations where the anticoagu-lant effects predominate. Because of the importanttherapeutic potential, considerable effort has beendirected towards the discovery of nonanticoagulantheparin or “heparin-like” derivatives or heparin-mimicking polyanions (heparinoids).

It is possible to chemically modify heparin toproduce a derivative that has minimal antifactor Xaand antifactor IIa activity [102]. Other nonanticoa-gulant heparins or derivatives have shown promisein animal models of myocardial ischemia-reperfu-sion injury [35] and in the inhibition of selectin-mediated cell adhesion [56,65–67]. More recently,methods have been described to prepare charge-reduced heparin derivatives that possess increasedselectivity for binding to heparin-binding proteins[103]. Other workers have focused on preparingsemisynthetic glucan sulfates with optimal struc-tures that target P-selectin binding [104] or cyto-kine gene expression [105]. Finally, small peptidesthat mimic the heparin-binding domains of cyto-kines have been developed to attenuate the in-flammatory response [106].

Conclusions

There is significant clinical and basic scienceliterature to support the hypothesis that heparinpossesses anti-inflammatory effects. The use ofheparin as an anti-inflammatory agent though, hasbeen handicapped by the fear of bleeding. This fearshould be mitigated by the development of non-anticoagulant heparins, heparin derivatives andheparinoids. Ongoing investigations must focus onelucidating the mechanisms of action of thesenonanticoagulant heparins in cellular and animalmodels of acute and chronic inflammation. Most ofthe published clinical studies are insufficientlypowered and suffer from serious methodologicalweaknesses. Future clinical trials of these com-pounds should be carried out using well-designedrandomized clinical trials. Because of the links

between inflammation, atherogenesis, thrombo-genesis and cell proliferation, the pleiotropiceffects of heparin and related compounds mayhave greater therapeutic potential than compoundsthat are directed against a single target.

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