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Heparin, Low Molecular Weight Heparin,and Derivatives in Thrombosis, Angiogenesis,and Inflammation: Emerging LinksShaker A. Mousa, Ph.D., M.B.A., F.A.C.C., F.A.C.B.1

ABSTRACT

The key reason behind the success of heparin in thrombosis and beyond is itspolypharmacological sites of action for the prevention and treatment of multifactorialdiseases that will only benefit slightly with single pharmacological mechanism–basedagents. Thromboembolic disorders are driven by hypercoagulable, hyperactive platelet,proinflammatory, endothelial dysfunction, and proangiogenesis states. Heparin can effec-tively modulate all of those multifactorial components, as well as the interface among thosecomponents.

KEYWORDS: Q1

In recent years, clinical data and studies haveclarified the potential and shortcomings of anticoagulanttherapy in the prevention and treatment of thromboem-bolic disorders. The discovery and introduction of hep-arin derivatives such as low molecular weight heparins(LMWHs) have enhanced the clinical options for themanagement of thromboembolic disorders while en-hancing the safety of therapy. In the United States,LMWHs are currently approved for the prophylaxisand treatment of deep vein thrombosis (DVT).LMWH uses are also being expanded for additionalindications for the management of unstable angina andnon–Q-wave myocardial infarction.1,2 In addition to theapproved uses, LMWHs currently are being tested forseveral newer indications.3,4 Because they are polyphar-macological agents, these drugs are expected to find usesin several other clinical indications, such as inflammatorydiseases and cancer.3,4 Additional pharmacological stud-ies and well-designed clinical trials in which variouspharmacokinetic and pharmacodynamic parameters arestudied will provide additional evidence on the clinical

individuality of each member of this class of novelagents.1–4

Because heparin was discovered more than a halfcentury ago, our knowledge of the chemical structureand molecular interactions of this fascinating polycom-ponent was limited at the early stages of its development.Through the efforts of a major multidisciplinary groupof researchers and clinicians, it is now well recognizedthat heparin has multiple sites of action (Table 1) andcan be used in multiple indications. In the not-too-distant future, we may witness the impact of heparinderivatives in the management of various diseases.

EMERGING LINKS AMONG THROMBOSIS,ANGIOGENESIS, AND INFLAMMATION:POTENTIAL ROLE OF HEPARINQ4

Several lines of evidence demonstrated the interplaybetween the platelet/leukocyte in the activated stateand the coagulation cascade. That led to the exposure ofthe platelet glycoprotein IIb/IIIa receptors in its active

Q301The Pharmaceutical Research Institute and Albany College ofPharmacy, Albany, New YorkQ30.

Address for correspondence and reprint requests: Shaker Mousa,Ph.D., M.B.A., F.A.C.C., F.A.C.B., Pharmaceutical ResearchInstitute and Albany College of Pharmacy, 106 New ScotlandAvenue, Albany, NY 12208. E-mail: [email protected].

New Anticoagulants; Guest Editor, Job Harenberg, M.D.Semin Thromb Hemost 2007;33:524–533. Copyright# 2007 by

Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York,NY 10001, USA. Tel: +1(212) 584–4662.DOI 10.1055/s-2007-982084. ISSN 0094-6176.

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state, leading to platelet fibrinogen binding and ampli-fication of platelet aggregate formation. Activatedplatelets also interact with leukocytes, leading to pla-telet–leukocyte cohesion and leukocyte activation. Hy-peractive platelets also provide a surface for thrombingeneration; thrombin is a potent platelet and leukocyteactivatorQ5. In addition, there is significant interplayamong the coagulation cascade, platelets, and the vesselwall in the promotion of thromboembolic disorders.Depending upon the venous (low shear) versus arterial(high shear) shear level, platelet/fibrin proportions andcontributions vary.Q6

Emerging links are shown among thrombosis,angiogenesis, and inflammation in vascular, cardiovas-cular, and inflammatory disorders (Fig. 1). For example,infection leading to the initiation of proinflammatorystimuli could be a major predisposing factor in prop-agation of thromboembolic disorders. Endotoxin that

can be liberated from Escherichia coli and other bacteriacan induce proinflammatory state, with the increase oftissue necrosis factor alpha (TNF-a) and other cyto-kines. That would lead to the activation of leukocytes,with increased expression of membrane L-selectin andthe shedding of soluble L-selectin, which can serve as asurrogate marker of leukocyte activation. Activation ofleukocytes leads to the propagation and generation oftissue factor, which initiates and amplifies a hypercoagu-lable state as well as the upregulation of TNF-a pro-duction. A hypercoagulable state with the generation ofthrombin activates the platelets, leading to the over-expression of platelet membrane P-selectinQ7 and theshedding of soluble P-selectin, which can act as asurrogate marker of platelet activation.5 In addition,the proinflammatory state can induce endothelial cell(EC) insult, leading to increased EC membrane expres-sion and shedding of soluble vascular adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin.

Heparin is a glycosaminoglycan formed fromsulfated oligosaccharides; it varies in the length ofpolymeric units and therefore has different molecularweights. LMWH is made by partial hydrolysis or enzy-matic degradation of unfractionated heparin (UFH).Heparin and LMWH prevent the process of bloodcoagulation and have a natural antithrombin effect. Inrecent years, several studies have shown that heparin andLMWH have an obvious anti-inflammatory activity inaddition to their traditional anticoagulant effects.6,7 Inanimal models, heparin disaccharides inhibited TNF-aproduction by macrophages and decreased immune in-flammation.8 Heparin accelerated the healing process ofmucosa in colitis in several clinical studies and had anti-inflammatory effects.9–14 Therefore, administration of

Table 1Q2 Heparin Mechanisms of Action

AT dependent (FXa, FIIa, FIXa, etc.)

Anticoagulant, antithrombotic

AT independent (TFPI)

FXa, TF/FVIIa: antithrombotic

Angiogenesis, metastasis, and inflammation

AT and TFPI-independent:

Inhibition of matrix-degrading enzymes, proteases,

heparinasesQ3; inflammatory and cancer cell invasion

Heparin and selectin modulation: metastasis,

inflammation, VTE

Other mechanisms?

AT, antithrombin; F, factor; TFPI, tissue factor pathway inhibitor; TF,tissue factor; VTE, venous thromboembolism.

Figure 1 Emerging links among thrombosis, angiogenesis, and inflammation in vascular, cardiovascular, and inflammatory diseases.

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HEPARIN AND LMWH IN MULTIFACTORIAL DISEASES/MOUSA 525

heparin can afford both non-anticoagulant (anti-inflam-matory and antiangiogenesis) and anticoagulant effects(Fig. 2).

Heparin and Venous Thromboembolism

The large majority of patients with venous throm-boembolism (VTE) currently are treated with full dosesof UFH or LMWH, followed by at least 3 months oforal anticoagulant therapy.15 Selected patients withcritical manifestations of pulmonary embolism (PE) areadministered thrombolytic drugs, whereas intravenalcava filtersQ9 are confined to patients with either DVTor PE who present with contraindications to conven-tional anticoagulation.15

Although considerable progress has been made inthe treatment of venous thromboembolic disorders, manyunanswered questions remain, which are awaiting propersolution. Furthermore, new opportunities are emerging,which have the potential to modify the therapeuticscenario substantially in the near future. Among thetopics that are worth exploring are home treatment ofselected patients with DVT, the treatment of cancerpatients with venous thrombosis, the renewed interestfor thrombolytic drugs in patients with PE, the optimalduration of oral anticoagulant therapy, and the potential

of new drug categories for the initial treatment andsecondary prevention of VTE.

TREATMENT OF CANCER PATIENTS WITHVENOUS THROMBOSISPatients with DVT who also have cancer have a higherrisk of recurrent thromboembolism and major bleedingduring anticoagulation.16,17 In a recent prospective co-hort study in a wide series of patients with venousthrombosis with or without cancer, the 12-month cu-mulative incidence of both recurrent thromboembolismand major bleeding during anticoagulation was signifi-cantly higher in patients with cancer than in thosewithout cancer.18 Recurrence and bleeding were bothrelated to cancer severity, occurred predominantly dur-ing the first month of anticoagulant therapy, but couldnot be explained by sub- or over-anticoagulation.18

Possibilities for improvement using the current para-digms of anticoagulation, therefore, seem limited, andnew treatment strategies should be developed.

The long-term use of LMWH recently has beencompared with warfarin for the initial treatment andsecondary prevention of VTE in cancer patients withvenous thrombosis, favoring LMWH.19 No significantdifference between the dalteparin group and the oralanticoagulant group was detected in the rate of majorbleeding or any bleeding. These results are supported byother investigation.20 Taken together, these two studiesshow clearly that in patients with cancer and acute VTE,LMWH is more effective than an oral anticoagulant inreducing the risk of recurrent thromboembolism withoutincreasing the risk of bleeding.

TREATMENT OF PEPatients with noncritical manifestations of PE havelong been treated with UFH in therapeutic doses. Totest the hypothesis that LMWH treatment can beextended to cover the entire spectrum of patientspresenting with acute VTE (thus including also pa-tients with noncritical PE), two multicenter clinicaltrials have been performed in the second half of the1990s. In the first investigation,21 all consecutive

Figure 2 Heparin and its anticoagulant and nonanticoagulanteffects, as well as the mediators involved. TFPI, tissue factorpathway inhibitor; TF, tissue factor; VIIa, factor VIIa.

Figure 3 Q8Antiangiogenic effect of low molecular weight heparin in the basic fibroblast growth factor-2 (b-FGF2) -stimulated chickchorioallantoic membrane model. LMWH, low molecular weight heparin.

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526 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 33, NUMBER 5 2007

patients with acute thromboembolism were enrolled inthe study irrespective of the modality of clinical pre-sentation; in contrast, in the second investigation,22

only patients with symptomatic PE were eligible forthe study. In both studies, the investigated LMWH(reviparin and tinzaparin, respectively) proved to be atleast as effective and safe as UFH, suggesting thatunder some circumstances also noncritical patientswith symptomatic PE might be treated at home withLMWHs. In addition, a recent meta-analysis of allavailable comparative clinical trials has confirmedfirmly that for the treatment of PE, LMWH is aseffective and safe as UFH.23

Recent studies have put into question the sys-tematic use of anticoagulants alone in the initial treat-ment of patients with submassive PE. Given that therisk of an unfavorable outcome definitely seems higherin patients with right ventricular dysfunction, as shownby echocardiography,24,25 the use of thrombolyticdrugs (ie, drugs that have the potential to promptlyrestore the patency of the pulmonary arterial vessels),might improve the outcome of patients with PE.Recently, two meta-analyses of comparative studiesbetween thrombolysis and heparin in the treatmentof acute PE have been published.26,27 The results ofthese meta-analyses consistently show that patientstreated with thrombolytic drugs have a more favorableoutcome in terms of prevention of short-term recurrentepisodes of PE than those treated with heparin alone.The difference becomes statistically significant when acomposite end point consisting of death/recurrence iscalculated.26 However, patients treated with thrombo-lytic drugs definitely exhibit a higher hemorrhagicrisk.26,27

In a recent prospective controlled study, 256patients with submassive PE and a contemporary rightventricular dysfunction were randomly assigned to re-ceive heparin plus alteplase or heparin plus placebo.28

The results of this study have the potential to expand theuse of thrombolysis in patients with acute PE, at least inthose with right ventricular dysfunction. In addition,whether the prognostic value of echocardiographicallyassessed right ventricular dysfunction applies to non-critical patients with PE still remains to be demonstratedconvincingly.29

THE OPTIMAL DURATION OFANTICOAGULANT TREATMENT

Available Information from Recent Clinical

Trials

Prospective cohort30–32 and population-based studies33

performed in recent years have shown that the inci-dence of recurrent VTE in patients receiving a short(3- to 6-month) course of anticoagulation is higher

than previously believed. Schulman et al34 performed amulticenter trial comparing 6 weeks of oral anticoagu-lant treatment with 6 months of such therapy in897 patients who had had a first episode of VTE. After2 years of follow-up, there were 80 recurrences amongthe 443 patients randomly assigned to the 6-weekgroup (18.1%), and 43 recurrences among the 454patients randomly assigned to the 6-month group(9.5%). Therefore, this trial showed a substantial re-duction in the risk for recurrent thromboembolismamong patients who were administered 6 months ofanticoagulation. Other recent studies have addressedthe potential for prolonged anticoagulation in selectedcategories of patients. Kearon et al35 randomly assignedconsecutive patients to receive 3 months or 2 years oforal anticoagulant therapy following an episode of acuteVTE. A prespecified interim analysis led to earlytermination of the trial after 162 patients had beenenrolled for an average of 10 months. Of 83 patientsassigned to continue to receive placebo, 17 had arecurrent episode of VTE (27% per patient-year),compared with one of 79 patients assigned to receivewarfarin (1.3% patient-year). There was a nonsignifi-cant trend toward a higher risk of (nonfatal) majorbleeding in patients assigned to warfarin compared withthose assigned to the placebo group.

In a recent Italian multicenter study, 267 patientswith a first episode of idiopathic proximal DVT who hadcompleted 3 months of anticoagulant treatment wererandomly assigned either to withdraw anticoagulation orto continue for an additional 9 months.36 Of the134 patients assigned to extended anticoagulation, 21had a recurrence of VTE (5.0% per patient-year; averagefollow-up, 37.8 months), compared with 21 of the133 patients randomized to withdrawal of anticoagula-tion (5.1% per patient-year; average follow-up,37.2 months). Four patients had nonfatal major bleedingduring extended anticoagulant treatment (3.0%). Theresults of this study suggest that extending to the courseof anticoagulant treatment in patients with idiopathicproximal DVT from 3 months to 1 year is not associatedwith long-term clinical benefit. These conclusions havebeen supported by those of a French multicenter clinicaltrial, which addressed the comparison between 3 and6 months of anticoagulation in patients with proximalDVT, and that between 6 and 12 weeks in patients withisolated calf DVT.37 The benefit of extending theduration of oral anticoagulation beyond the currentlyrecommended 3-month period in selected patients withclinically symptomatic PE recently has been addressedby another Italian multicenter trial.38

In a multicenter trial addressing the optimalduration of oral anticoagulant therapy after a secondepisode of VTE, Schulman et al39 found a considerablereduction in the risk for recurrent thromboembolism(from 21 to 3%) in patients allocated to receive 4 years


compared with 6 months of warfarin. Surprisingly, thisbenefit was offset by a remarkably higher incidence ofmajor bleeding (8.6 versus 2.7%).

In a recent multicenter, double-blind, random-ized trial, Ridker et al40 demonstrated convincingly thatlow-intensity warfarin prophylaxis, using a targetedinternational normalized ratio (INR) of 1.5 to 2.0, issuperior to placebo in preventing recurrent VTE inpatients with idiopathic VTE who have been treatedpreviously for at least 3 months with warfarin at theconventional level of intensity. Kearon et al41 reportedsimultaneously the results of a different randomized,double-blind trial of similar size that found that low-intensity warfarin (INR, 1.5 to 1.9) was significantly lesseffective than conventional-intensity warfarin (INR, 2.0to 3.0) for extended prevention of recurrent VTE, with-out significant differences in the rate of bleeding com-plications.41

HEPARIN AND ASTHMAThe significant reduction in symptoms at 10 minutesmight be related to the ability of heparin to prevent therelease of histamine from mast cells. Heparin mayinterfere with stimulation of mast cell mediator secre-tion by blocking internal calcium release. Later, hep-arin may reduce eosinophil recruitment throughdifferent mechanisms: by preventing mast cell media-tor release, heparin could indirectly downregulateadhesion molecules on ECs, and thus limit eosinophilmigration into the nasal mucosa.42–44 Furthermore, theheavily anionic heparin may inactivate platelet-activat-ing factor, a cationic protein with a potent chemotacticactivity for human eosinophils. Intranasal heparinattenuated the nasal response to an allergic challengein atopic rhinitic subjects, and no adverse reaction wasnoted.42–44 More studies are needed to explain com-pletely the mechanisms by which heparin produces itsanti-inflammatory activity; this will allow us to opti-mize heparin use in allergic diseases, such as rhinitisand asthma.

The anti-inflammatory activity of heparin hasbeen reinforced by positive, although small, clinicaltrials in patients suffering from a range of inflamma-tory diseases, including rheumatoid arthritis and bron-chial asthma.45,46 In addition, several clinical studieshave recently demonstrated the anti-inflammatoryactivity of heparin in the treatment of inflammatorybowel disease (IBD) at doses that do not produceantihemorrhagic complications.47 Given that it isnow well recognized that different portions of theheparin molecule exhibit anti-inflammatory activity,and that a pentasaccharide sequence retains the abilityto inhibit antithrombin III,48 it is possible that theanti-inflammatory actions of heparin are distinct fromits anticoagulant activity.49

HEPARIN/LMWH AND INFLAMMATORYBOWEL DISEASESUnder various experimental and clinical conditions,heparin was found to reduce actively the process ofleukocyte recruitment into the site of injury or ofapplication of inflammatory stimuli. Salas et al50 provideevidence for the first in vivo mechanism responsible forthe antimigratory action of heparin. In fact, intravitalmicroscopy techniques have allowed direct observationof inflamed microvascular beds with definition of theparadigm of white blood cell extravasation. Leukocyteinteraction with the endothelium of an inflamed post-capillary venule is initially intermittent and dynamic(cell rolling); it then becomes static (firm adhesion),and is finally followed by diapedesis.51 Using the potentcytokine TNF-a to promote this cascade of events invivo, Salas et al50 reported that heparin downregulatedTNF-a–induced leukocyte rolling, adhesion, and mi-gration into gut tissue without affecting changes invascular permeability. These data extend and confirmprevious studies in which heparin reduced leukocyteadhesion to vascular ECs in vitro52 and recruitment ofinflammatory cells into other tissues during an exper-imental inflammatory reaction.49

Several uncontrolled studies have suggested thatheparin may be potentially therapeutic in the clinicalmanagement of both ulcerative colitis and Crohn disease.Although these studies included only a limited number ofpatients, they demonstrated apparent beneficial effects ofheparin with no associated hemorrhagic complica-tions.53–60 Heparin has been shown to suppress selectedneutrophil functions, such as superoxide generation andchemotaxis in vitro, to reduce eosinophil migration,and to diminish vascular permeability.61,62 Among themechanisms that may account for the anti-inflammatoryactions of heparin, binding of this glycosaminoglycan toadhesion molecules expressed on the surface of activatedECs and/or leukocytes has been proposed. Recent in vitrostudies have demonstrated the ability of heparin to bindeffectively to endothelial P-selectin (but not E-selec-tin),63 as well as L-selectin and CD11b/CD18 expressedon neutrophils.Q1064,65 Taken together, these reportssuggest that the therapeutic actions of heparin observedin patients with IBD may involve attenuation of inflam-matory processes as well as a hypercoagulable stateassociated with clinical exacerbation of IBD; these effectsmay promote mucosal repair.

Available uncontrolled dataQ11 show that hep-arin may be effective in steroid-resistant ulcerativecolitis, with a percentage of complete clinical remissionof more than 70% after an average of 4 to 6 weeks oftherapy. The administration of heparin currently is notjustified by the very limited available data. LMWHwasused in a single trial in patients with steroid-refractoryulcerative colitis, with results similar to those observedwith heparin. Given that a prothrombotic state has

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been described in IBD, and microvascular intestinalocclusion seems to play a role in the pathogenesis ofIBD, it is reasonable that part of the beneficial effects ofheparin in IBD may result from its anticoagulantproperties. However, beyond its well-known anticoa-gulant activity, heparin also exhibits a broad spectrumof immune-modulating and anti-inflammatory proper-ties by inhibiting the recruitment of neutrophils and byreducing proinflammatory cytokines. In conclusion,heparin or heparin derivatives may represent a safetherapeutic option for severe, steroid-resistant ulcer-ative colitis and other inflammatory disorders, althoughrandomized, controlled trials are needed to confirmthese data.

HEPARIN VERSUS LMWHIn contrast to UFH, LMWHs have a lower affinity tobind to plasma proteins, ECs, and macrophages. Thisdifference in binding profile explains the pharmacoki-netic differences observed between LMWHs and UFH.The binding of UFH to plasma proteins reduces itsanticoagulant activity, which combined with the varia-tions in plasma concentrations of heparin-bindingproteins, is reflected in its unpredictable anticoagulantresponse.

LMWHs exhibit improved subcutaneous bioa-vailability; lower protein binding; longer half-life; vari-able number of antithrombin III binding sites; variableglycosaminoglycan contents; variable anti–serine pro-tease activities (anti–factor [F]Xa, anti-FIIa, anti-FXa/anti-FIIaQ12 ratio, and anti-other coagulation factors);variable potency in releasing TPFPIQ13; and variablelevels of vascular EC binding kinetics.66–69 For thesereasons, during the last decade, LMWHs have increas-ingly replaced UFH in the prevention and treatment ofVTE disorders. Randomized clinical trials have demon-strated that individual LMWHs used at optimizeddosages are at least as effective as and probably saferthan UFH. The convenient once- or twice-daily sub-cutaneous (SC) dosing regimen without the need formonitoring has encouraged the wide use of LMWHs. Itis well established that different LMWHs vary in theirphysical and chemical properties due to the differences intheir methods of manufacturing. These differencestranslate into differences in their pharmacodynamicand pharmacokinetic characteristic.67 The WorldHealth Organization and U.S. Food and Drug Admin-istration regard LMWHs as individual drugs that cannotbe used interchangeably.67

Bioavailability of LMWHs after intravenous (IV)or SC administration is greater than for UFH and wasdetermined to be between �87 and 98%. UFH, incontrast, has a bioavailability of 15 to 25% after SCadministration. LMWHs have biological t½

Q14 (basedon anti-FXa clearance) nearly double that of UFH. The

t½ of LMWHs enoxaparin, dalteparin, tinzaparin, andothers has been documented to be between �100 and360 minutes, depending on whether the administrationof LMWH was IV or SC. The anti-FXa activity persistslonger than antithrombin activity, which reflects thefaster clearance of longer heparin chains.70

LMWH, in doses based on patient weight, needsno monitoring, possibly because of the better bioavail-ability, longer plasma t½, and more predictable anti-coagulant response of LMWHs compared with UFH,when administered SC. Though LMWHs are moreexpensive than UFH, a pilot study in pediatric patientsfound SC LMWH administration to reduce the numberof necessary laboratory assays, nursing hours, and phle-botomy time.71

LMWHs are expected to continue to erode UFHuse, through development programs for new indicationsand increased clinician comfort with use of the drugs. Inaddition, as both patients and health care providersrecognize the relative simplicity of administration withan SC injection, together with real cost savings andquality-of-life benefits by reducing hospital stays, thetrend toward outpatient use will continue.

HEPARIN AS AN ANTI-INFLAMMATORYMOLECULE: POTENTIAL MECHANISMSHeparin is used in the treatment and prevention ofthrombotic and thromboembolic conditions such asDVT, PE, and crescendo angina.72–74 Heparin activatesantithrombin III to prevent conversion of fibrinogen tofibrin; it accelerates inhibition of FXIIa, FXIa, FIXa,and FXa. Heparin also possesses non-anticoagulantproperties, including modulation of various proteases,anticomplement activity, and anti-inflammatory actions(Table 1). Inhaled heparin has been shown to reduce theearly phase of asthmatic reaction and suppress allergen-induced increase in bronchial hyper-reactivity. Heparinalso inhibits the acute cutaneous reaction due to aller-gens. The ubiquitous distribution of heparin in tissuespaces may serve to limit inflammatory responses invarious tissues where leukocytes accumulate followingan inflammatory challenge. It is interesting that heparinis found in high concentrations in the gastrointestinaltract and the lung,72,73 the two organs exposed to theexternal environment.

Few studies have reported an effect of heparin onreactive oxygen species (ROS) generation75 and cytokinesecretion76 by leukocytes in vitro. It has been demon-strated recently that heparin, when injected intrave-nously, into normal subjects at a dose of 10,000 IUinhibits ROS generation by mononuclear cells andpolymorphonucleocytes.77 Heparin has been also shown,in a series of experiments using N-acetyl heparin, toprotect the heart from ischemia-reperfusion injury bothin vivo and in vitro, independently of its antithrombin

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mechanism.78,79 It was suggested that this protectionmay be due to a reduction in complement activation–mediated injury to the heart.79 Given that ischemia-reperfusion injury may also be mediated by oxidativedamage,80 it is therefore possible that the protectiveeffect of heparin may be due to an inhibition of ROSgeneration. A reduction in superoxide radical formationby heparin is likely to allow a greater bioavailability ofnitric oxide (NO) for vasodilation. In fact, heparin hasbeen shown to exert a vasodilatory effect in normalsubjects in vivo.81 Increased bioavailability of NO mayhave an additional beneficial effect: inhibition of leuko-cyte adhesion to the endothelium, which would, in turn,inhibit or retard inflammation.82 In addition, NO in-hibits the proinflammatory transcription factor nuclearfactor kappa-BQ15, which plays a central role in thetriggering and coordination of both innate and adaptiveimmune responses.

HEPARIN DERIVATIVES ANDANGIOGENESISAngiogenesis is an essential feature of normal biologicprocesses such as growth, development, reproduction, andrepair of damaged tissue.83–85 Endogenous promotersand inhibitors regulate the complex process of angio-genesis.84 The later stages involve proliferation and or-ganization of ECs into tube-like structures. Vascularendothelial growth factor and fibroblast growth factor-2(FGF2) are important promoters of this process; thesepromoters exert their effects by binding to cell surfacereceptors. Human ECs in culture can form tube-likestructures with lumens and represent an in vitro modelsystem for the study of the angiogenesis process. Patho-logic angiogenesis, which may occur when a normalcontrol mechanism is defective, contributes to the growthandmetastasis of tumors,85 as well as to inflammatory andcertain ocular diseases.

Drugs that inhibit angiogenesis may be effectivein the treatment of these human disorders.85 The mech-anisms by which antiangiogenic drugs exert their effectscan vary widely, acting on different points in the complexprocess of tumor angiogenesis. Potential points of con-trol include blocking the action of endogenous stimula-tors; inhibiting the growth, migration, and tubeformation of EC; and inhibiting the turnover of thecapillary basement membrane.83

The role of heparin in angiogenesis modulationand its potential anticancer effect has been describedpreviously, but without clear delineation of its pro-versus antiangiogenic effect as well as its mechanism ofactions.86,87 The LMWH tinzaparin demonstrated po-tent inhibition of angiogenesis.88

Tinzaparin, a LMWH (average molecularweight, 6.5 kd), produced by enzymatic degradationof heparin, has proven efficacy in the treatment of

DVT and PE.89,90 The antithrombotic activity oftinzaparin is mediated by binding to microvasculartissues and activation of antithrombin III, a potentanticoagulant.91 Tinzaparin also causes release of tissuefactor pathway inhibitor (TFPI)Q16, an importantendogenous inhibitor of tissue factor (TF)/FVIIaQ17.92

Several clinical trials have shown improved survival ofcancer patients following heparin therapy.93–95 In onedouble-blind, multicenter clinical trial, tinzaparin wasshown to be effective in the treatment of proximalDVT in a patient population that included a largepercentage of cancer patients.89 These clinical datasuggest that tinzaparin may have some benefit in thetreatment of cancer patients. The present study wasundertaken to elucidate the mechanisms throughwhich tinzaparin may affect tumor angiogenesis andto assess its efficacy in inhibiting the angiogenesisprocesses.

LMWH AND TFPI INHIBIT EC TUBEFORMATIONA pivotal stage of angiogenesis is the formation of tube-like structures from ECs,83–88 a process that is medi-ated by cytokines binding to EC surface receptors.96

In addition, Mousa and Mohamed88 reported thatthe inhibitory efficacy of tinzaparin or r-TFPI onFGF2-induced EC tube formation was totally reversedby a specific monoclonal TFPI antibody directed to-ward TF/FVIIa but not FXaQ18 binding domain. Sim-ilar data were shown for this antibody in reversingthe inhibitory effect of tinzaparin or r-TFPI onTF/FVIIaQ19-induced EC tube formation. Further-more, additional reports demonstrated that the anti-angiogenesis and antimetastatic activity of LMWH tobe independent of anticoagulant activity.97

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55. Evans RC, Rhodes JM. Treatment of corticosteroid-resistantulcerative colitis with heparin. A report of nine cases. Gut1995;37:A49 (abst)

56. Brazier F, Yzet T, Duchmann JC, et al. Effect of heparintreatment on extra intestinal manifestations associated withinflammatory bowel diseases. Gastroenterology 1996;110:A872 (abst)

57. Brazier F, Yzet T, Boruchowicz A, et al. Treatment ofulcerative colitis with heparin. Gastroenterology 1996;110:A872 (abst)

58. Dupas JL, Brazier F, Yzet T, et al. Treatment of activeCrohn’s disease with heparin. Gastroenterology 1996;110:A900 (abst)

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