In Vivo Antithrombotic Synergy of Oral Heparin and Arginine: Endothelial

Endothelium andVascularDevelopment

In vivo antithromboticsynergyoforal heparin andarginine: Endothelial thromboresistancewithout changes in coagulation parameters

Bruce Daniels1 ,Robert J. Linhardt2 ,Fuming Zhang2 ,WenjunMao3 ,Sandra M. Wice 4 ,Linda M. Hiebert 4 1 4221 South Western, Suite 4045, OklahomaCity,Oklahoma,USA; 2 Department of Chemistryand Chemical Biology,Biology and Chemical and Biological Engineering, RensselaerPolytechnicInstitute,Troy, NewYork, USA; 3 Center forMarine Foodsand Drugs, Qingdao Ocean University,Qingdao,China; 4 Department ofVeterinaryBiomedical Sciences, University of Saskatchewan, Saskatoon,Saskatchewan, Canada

Summary On the basis of suggestedclinical efficacy in an uncontrolled able plasma anticoagulant activity.Anorally administeredlow- study in ninety-sevenpatientswith unstable angina,ananimal molecular-weight anticoagulant glycosaminoglycan mixture, su- study was conducted to investigate antithromboticsynergybe- lodexide(7.5 mg/kg),showed an 88%reductioninstable throm- tween orallyadministered heparin and arginine.A rat venous busformation when administeredalone but showed no synergy thrombosis modeltestedthe difference in thrombus formation with oral arginine.A 28-daystudy with oral sulodexide (2.9mg/ when heparin (7.5 mg/kg)and arginine (113 mg/kg)wereadmin- kg)and arginine (43.9 mg/kg), 20 rats/group, showed antithrom- istered,alone or in combination, by stomachtube withamini- botic activity withminimalanticoagulant activity indicating suit- mumof20rats/group.Oral heparin, arginine,and heparin plus ability forlong term treatment.Thesefindingssuggestthe en- arginine reducedthrombus formation by 50%,75%,and 90%,re- dotheliallocalization of heparin and asynergistic antithrombotic spectively, whencompared to salineadministration.Heparin was effect fororally administeredheparin and arginine. recoveredfromendothelium,yet there was little or no observ-

Keywords Heparin,thrombosis,endothelium,arginine,oral ThrombHaemost 2006; 95: 865–72

Introduction dothelial production of NO (5).NOreduces endothelialadhes- iveness, preventsplateletaggregation, and results in vasorelax- Previous investigations have demonstrated that heparin has anti- ation (6–8).Arginine is also reported to have anticoagulant prop- thromboticactivity when administered orally in rat thrombosis erties(9).Arginine groups on peptides aremajor determinants in models (1, 2). Several studiessuggest that heparins arefound on their interaction with endogenous heparan sulfates on the en- the endothelial surfacewithin minutesfollowing oraladminis- dothelialsurfaceofblood vessels (10).While arginine only tration with minimal observedevidenceofplasma distribution. weaklyinteracts with the O -sulfoesters of heparan sulfateand Endothelial concentrations of heparin arereported to be 1,000 heparin, this binding is stronger than that of other basic amino –10,000timesthe levels measurable in plasma followingoral ad- acids (11).Furthermore,heparin induces eNOS activity (12), ministration to rats (3, 4). If similareffectsoccur on humanen- and heparin as well as NO increase the biosynthesis of endothe- dothelium following oralheparin administration, potent anti- lialheparan sulfatewith increased amounts of ATIII binding thrombotic effectswould be anticipated without systemic antico- sites(13, 14). Evidencealso suggests both NO and heparan agulation. sulfatedeficienciesmay causeendothelial dysfunction, pro- Arginine and heparansulfate have important roles in the thromboticeffects, and plaqueformation (15–21).Thus, an im- functional and molecular organization of vascularhomeostasis portant relationship exists betweenheparin,heparan sulfate,argi- and hemostasis. Arginine is the substrate for eNOS in the en- nineand NO in promoting antithrombotic activityand vascular

Correspondence to: Financial support: RobertJ.Linhardt, PhD Thisworkwas supportedbyEndomatrix Inc. Santa Rosa,California,agrant to Linda Professor of Chemistry and Chemical Biology Hiebertfromthe Heart and Stroke Foundation of Saskatchewan and grants from the Biology and Chemical and Biological Engineering National InstitutesofHealth to RobertLinhardt (NIHHL52622 and GM38060). Rensselaer Polytechnic Institute BiotechnologyCenter 4005 Received December5,2005 1108th Street, Troy,NY, 12180–3590, USA Accepted after resubmission March 10, 2006 Te l.: +1 518 276 3404, Fax: +1 518 276 3405 E-mail: [email protected] Prepublished onlineApril 12, 2006 DOI: 10.1160/TH05–12–0786

865 Daniels,etal.: Synergyoforal heparinand arginine health. Basedonthese reports, we speculated that administration Societies. The animals were fasted overnightprior to treatment of both heparinand arginine togetherwould simultaneouslyin- and were anaesthetized with barbitaland methoxyflurane for ex- creaseendothelial heparansulfate andNOand possiblyexhibit perimental procedures. antithrombotic synergy. Since oraladministration of heparinin rats results in its enhanced deposition on the endothelium relative Drug administration to plasma levels (3), we chosethis routefor our investigation. Forshorttermstudiesthe following treatment groups were used: We report herebeneficialcardiovasculareffectsinaseriesof saline,heparin, arginine, heparinplus arginine, sulodexide,and patients, with unstable angina in aclinical setting, treated for12 sulodexide plus arginine. Bovine heparin wasusedinthe animal monthswith oral heparinplus arginine. Dataare alsopresented studies in placeofthe porcine heparin used in the human study. confirming synergy of oral heparinplus arginine in inhibiting The antithromboticresponseissimilarfor the twoheparins when thrombus formation in an establishedrat venous thrombosis givenorallyinthe ratvenous thrombosis model(26). Heparin model of 4-hour duration. Oral sulodexide, aglycosaminoglycan and sulodexide were administered at 7.5mg/kg basedonpre- mixture(22), had antithromboticactivity in a4-hour and 28-day viousstudiesshowing a50% decrease in stable thrombus study when combined with arginine. formations with unfractionated heparin in this animal model (2). Arginine wasadministeredat112.5 mg/kg to approximate the Materials andmethods doseusedinthe human study.The dose of heparin or sulodexide wasassignedrandomly with6to8ratstreated per day. Astom- Patients ach tube wasfilledwith 0.2 ml saline followedby0.10 –0.18 ml Ninety-sevenconsecutive patients diagnosed with unstable angi- of heparin or sulodexide solution and 0.1 ml of arginine solution na were treated with the combination of 10,000 units of standard depending on ratweight. Thus, when the stomach tube was porcine intestinalheparin,sodium USP deliveredorallyin125 – placed in the stomach, the drugs were first introduced into the 200 ml of water,combinedwith arginine (JoMar Laboratories, stomach and flushed in by saline to give atotal volume of ap- Campbell, CA, USA) administeredastwo 825 mg capsules by proximately 0.4 ml. In the heparin onlygroup, heparin wasad- mouth twice daily. ThetotalL-argininedosewas 3.3 grams daily ministeredinavolume of 0.1 –0.2 ml followedby0.2 ml saline. in all patients.The patients were instructed to immediatelygoto Forlong-term studiessulodexide (2.9 mg/kg) plus arginine (42.9 the emergency room for admission if their symptoms recurred or mg/kg) were givendaily by stomachtube for 28 days. Control acceleratedduring the subsequent 48 hours. Patients were re- rats treatedfor four hours were givensaline (0.3ml) by stomach ferred for interventionaltreatmentifsymptoms failed to resolve tube.Control rats treatedfor 28 days were kept in metabolic within 72 hoursand if symptoms recurred at anytime within cages during that timeand were givenwater. 12 months.All other current medicationswerecontinued, patients remained on aspirin, beta blockers,statin drugsand other Thrombosistest treatments as were prescribedprior to the diagnosis.The resulting The thrombosistestwas performed in amodification of the pro- combined cardiac eventrates were compared to aseries of patient cedure by Blake et al.(24). Foranimals exposed to treatment for outcomesat12months postdiagnosisofunstableangina as re- 4hours, athrombus wasinitiatedinthe right jugular vein by ap- portedinthe recentFRISCIITrial(23). This uncontrolled ob- plication of 10% formalin in 65% methanol to the exposed ad- servationalstudywas conductedinaprivate medicalpractice. ventitial surface. Immediatelyfollowing, drugs were introduced Since this wasnot acontrolled trial,noinformedconsentwas ob- into the stomach by stomachtube. At four hours after thrombus tained. initiation, animals were again deeplyanaesthetizedand first examinedfor anyexternal signs of bleeding.The jugular vein Animal studies wasexamined for the presenceofaplug usingacotton pledget. Drugs The thrombus wasscored as +(stable thrombus) if the vesselwas Unfractionated bovine lung heparin (sodiumheparin 150 units/ blocked and remained blocked despite examination with acotton mg, LotNoZX320,Upjohn Ltd., Kalamazoo, MI, USA) was pledget. The thrombus wasscored as +/- (softorunstable throm- dissolvedinwater at aconcentration of 20 mg/ml. Sulodexide, a bus) if the vesselappearedcompletelyblockedonfirst examin- glycosaminoglycanmixturecontaining fast moving heparin ation and thenopenedasitwas examined. Thethrombus was 80% and dermatansulfate 20% (22),was obtainedfrom Keryx scored as –(negative)ifblood wasseen to flow freelyinthe Biopharmaceuticals(NewYork, NY,USA). Sulodexide wasdis- vessel. solved in waterataconcentration of 20 mg/ml for short-term Forthe long-term(28-day) study,athrombus wasinitiatedin studiesand 10 mg/mlfor long-termstudies. Gelatin capsules the jugularveinonthe final day, fourhours prior to killing the containing arginine, similar to those usedinthe human studies, rats. The last (28th)dose wasadministeredimmediately after were opened,and contents were dissolvedinwater at aconcen- thrombus initiation. Thethrombus wasevaluatedasdescribed tration of 300 mg/mlfor 4-hour studies and 150 mg/mlfor the forshort-termstudies. 28-daystudy. Collectionofblood andblood vessels Animals Immediately after examination of the jugularvein, alaparotomy One hundred eighty-twomaleWistarrats, weighing311 ±48g wasperformed and ablood sample of approximately 10 ml (± SD), were handled and housed according to the Principles of (9 parts blood to 1part3.8% sodium citrate) wastaken fromthe AnimalCare setout by the CanadianFederation of Biological abdominal aorta. Plasma wasprepared. As asourceofendothe-

866 Daniels,etal.: Synergyoforal heparinand arginine lium,the thoracic aorta wasremoved and placed in saline.Each Statisticalanalysis animalwas examinedfor signs of internal hemorrhage, and the Thrombosisdataisexpressed as apercentage with 95% con- timewhen blood clotted in the body cavity wasrecorded. fidenceintervals (2). Test fordifferences between proportions wasusedtocomparethe total thromboticincidence and inci- Harvesting of endothelium denceofhard thrombus between groups. Rat weight and surface Endotheliumwas removed fromblood vessels according to the area of endothelium are expressed as mean±standard deviation method of Hiebert and Jaques(25). The vessels were slit open, (SD).Otherdataisexpressed as mean±SE.Aone-wayANOVA pinned to dentalwax lumenside up, and rinsed in Locke's sol- withTukey’s post hoc test wasusedtocomparethe differences ution. Cellulose acetatepaper wasapplied to the luminal surface betweengroups when plasma coagulation tests and heparinor and when lifted, endothelium wasremoved.Thelength and width sulodexide concentrations in urine were examined. of the imprint were measured to the nearestmillimeter. Har- vested endothelialsurfacewas 2.28 ±0.74 (SD) and 0.57 ±0.25 Results cm2 for aorta and vena cava,respectively. Patients Determinationofheparin andsulodexidewith endothelium Results from the cohortofpatients on dailymaintenance of he- Celluloseacetatepaper wasremoved fromendothelium by dis- parin plus arginine, 12 monthspost diagnoses, areshown in solving in cold acetone followedbycentrifuging and discarding Table1.These data showthat the cardiacevent rates (death, the supernatant.The precipitates were thendigestedwith pro- myocardialinfarction, readmission and revascularization) were nase (Sigma,St. Louis, MO,USA;10 µ gof40mg/ml in Tris dramaticallyreduced compared to publisheddatausing other buffer). Sampleswere thencentrifuged at 8,000xgfor 10 min, treatment options. No othercomplications were observedinany supernatant wascollectedand the precipitate washedtwice with patient. These resultscomparefavorablytothose of the recent 100 µ l26.8% NaCl that wasadded to the supernatant. GAGs FRISC II trial (23) at 12 months and showadramatic reduction were precipitatedfrom the supernatant with five volumesofme- of 49 –93% seenindeath, myocardialinfarction or secondary thanol, and the precipitate wasdried. Agarosegel electrophore- cardiovascularevents without use of interventional treatment sis wasusedtoidentify and measure heparin in endothelial and areduction of 9–89% with interventional treatment. extractsbypreviouslypublished methods (1).The driedpowders were dissolvedinwater and appliedtoagarosegel slides along Animal studies withthe heparin or sulodexide used as references. Following On thebasis of these anecdotal results, showingeffectivenessof electrophoresis, gels were fixedin0.1% hexadecyltrimethyl- oralheparin and arginine on prothrombotic activity in humans, ammoniumbromide and air-dried. Slides were stained with an animalmodel wasusedtoinvestigatepossible mechanisms. 0.04% toluidine blue in 80% acetone,and background color was The ratjugular vein modelwas chosenasitiswellestablished removedwith 1% acetic acid. Heparinand sulodexide were and has been previously used to study the antithromboticactivity identifiedbycomparing migration to reference material. of heparins givenbythe oralroute (2).Sincemost of the previous Amounts contained in each sample were determinedbydensito- studies in this venous thrombosis model have beenconducted metry. using bovine lung heparin, this sourcewas chosentostudy its ef- fectswith arginine. Sulodexide,aglycosaminoglycan mixture Determinationofplasma anticoagulant activity wasalso choseninthe repeated dose long-termstudy of 28 days, The activatedpartialthromboplastin time(APTT) wasdeter- sinceitreportedlyhas loweranticoagulant activity and is suitable minedusing akit from Biopool(Ventura,CA, USA).Anti-factor for long-termoral administration (22). We measured antithrom- Xa and anti-factor IIa activity were measured using chromogenic assaykits,Accucolor TMHeparin ® (Sigma)and Spectrolyse ® He- parin,respectively. Ta ble1:Combinedcardiovascular eventrateswithoral heparin Heparin concentrationinplasma wasalso measured using and arginine as compared to FRISCIItrial (subcutaneous frag- the Heptest (Accuclot TM Heptest ® ,Sigma)wherethe clotting mintreatedpatients). timedeterminedwas converted to µg/ml by use of calibration curvesprepared at the same time with unfractionated bovine Oral Non- Difference Invasive* Difference heparin/ invasive (reduction) FRISC II (reduction) lung heparinorsulodexide. arginine FRISC II group (n =97) group (n =1222) Determinationofheparin andsulodexideinurine (n =1234) Rats were placed in metabolic cages,and urine wascollected Death 2(2.0%) 48 (3.9%)49%, P=0.58† 27 (2.2%)9%, P=1 over the 4-hourperiod,or24-hour urineswere collected on day1, Myocardialinfarction 3(3.0%) 143 (11.6%)75%, P= 0.006 105 (8.6%)65%, P=0.055 7, 14, 21 and 28 in the 28-daystudy.Urine wasdialyzed against Readmission 4(4.1%) 704 (57.1%)93%, P< 0.0001 451 (36.9%)93%, P< 0.0001 waterusing 1,000 molecularweightcut off(MWCO) dialysis Revascularization 3(3.0%) 383 (31.0%)90%, P< 0.0001 92 (7.5%)90%, P=0.15 tubing (SpectrumChemicaland LaboratoryProducts, Gardena, *The principal goal of theFragminand fast revascularization during instability in coronary artery CA,USA). Theresulting solution wasdried and analyzed by disease II (FRISCII) studywas to compare the effectiveness of invasivevs. non-invasivetreatment in terms of the incidence of death and myocardial infarction in patients with unstable coronaryartery agarose gelelectrophoresis as described above for endothelium. disease (CAD). The resultsindicatedthat an early invasiveapproach is preferable in the treatment of patients with unstable CAD. Invasiveprocedures were revascularization by percutaneous trans- luminalcoronaryangioplasty or bypassgrafting.†Fisher’sexact test.

867 Daniels,etal.: Synergyoforal heparinand arginine

trol or heparin treatedratsbut not different than sulodexide treatment alone. In the long-termstudy,control rats kept in metabolic cages for 28 days showeda75% incidenceofhard thrombi and a95% incidenceoftotal thromboticevents. Whenratswere treated with sulodexide (2.9mg/kg) plus arginine (42.9 mg/kg) for 28 days, incidenceofhard thrombi (10%) and totalthrombotic events (30%) were similartoratstreated fourhours with sulodexide alone or with sulodexide (7.5 mg/kg) plus arginine (112.5 mg/ kg). Rats treatedfor 28 days with sulodexide plus arginine showedsignificantly less thrombotic eventsthan 4-hour and 28-daytreated control rats,and 4-hour heparin (7.5 mg/kg) treatedrats.

Recoveryfromendothelium Extracts of endothelium applied to agarose gel electrophoresis showedthat heparin and sulodexide were detected in endothelial Figure1:Thrombosis incidence following administrationofhe- samples, as determinedbyboth their migration distanceand parin, sulodexide and/orarginineinarat model. Thrombosis was their staining characteristics (Fig. 2). The concentrations of he- initiated by applicationofformalininmethanoltothe exposedright parin and sulodexide found with endothelium areshown in Table jugular vein. In the4-hourstudies, heparin (7.5 mg/kg) or sulodexide 2. As expected,endogenous glycosaminoglycans, migrating (7.5 mg/kg) and/or arginine (112.5 mg/kg) were administered,immedi- similar to sulodexide, were observedincontrol endothelial atelyafter thrombus initiation,bystomach tube.Incidence of thrombosis wasevaluatedfourhours later. In the28-daystudy,sulodexide (2.9 mg/ samples while little if anyendogenous heparin wasdetected in kg)and arginine (42.9 mg/kg) were administered dailybystomach tube. these samples.Theendothelium from rats treatedwith oral hepa- Athrombus was initiated four hoursprior to theend of theexperiment. rinclearlyshowedthe presenceofheparin. Concentrations of he- Upwarddeflecting errorbarsshow95% confidence intervalsfor total parin in both the aortic and vena cavalendothelium of rats were thromboticevents, downwarddeflecting barsshow95% confidence in- greatestwhen treatedwith heparinalone,followedbythose tervals forhardthrombi. Significantly less than: 4-hour controlincidence treatedwith heparinplus arginine and thenbycontrols. The of hardthrombi (A); 4-hour controltotalthromboticincidence (B); heparin alone incidence of hardthrombi (C); heparin alone totalthrom- meanconcentrations of heparin with endothelium of rats treated boticincidence (D); 28-daycontrol incidence of hardthrombi (E); withheparin alonewere morethan an orderofmagnitude greater 28-daytotalthromboticincidence (F). (Chi2 -test forgoodness of fit.). thanthat found with endothelium of control rats, while endothelium from rats treated with heparinplus arginine were fourto five timesgreater thancontrol endothelium.Ingeneral,more he- botic activity,compounds recovered from the endothelium and parin and sulodexide were recovered from vena cavalendothe- urine,and anticoagulant activity following administration of he- lium than from aortic endothelium. parin and sulodexide with and without arginine. The recovery of sulodexide from the vena cavalendothelium wasgreatestfor rats treatedfor 28 days with sulodexide plus ar- Thrombosisincidence ginine. Sulodexide concentrations on aortic endothelium,for su- In the 4-hourstudies, rats treated with saline (control) showedan lodexide plus arginine treatedgroups,was morethan twice that 80% incidenceofhard thrombi and an 88%incidence of total thromboticevents (hard plus soft thrombi) (Fig. 1). Whenoral heparin (7.5mg/kg) wasadministered, the incidenceofhard Ta ble2:Sulodexide or heparin with endothelium following ad- thrombi wassignificantly reduced to 42% although the total ministrationwithand withoutarginine. thrombotic incidencewas minimallyaffected. When arginine (112.5 mg/kg)was administered alone, the incidenceofhard Treatment Durationof Aortic Vena Caval thrombi wasreduced to 20%,significantly different from control treatment concentration concentration butnot from heparin treatedrats, while totalthrombotic events µg/cm2 µg/cm2 were 50%, significantly less thanboth control and heparin Control 4h 0.02 ±0.007 0.05 ±0.03 treatedrats. When arginine plus heparin(7.5 mg/kg) were ad- Control** 4h 0.04 ±0.01 0.05 ±0.04 ministered orally,the incidenceofhard thrombi wasreduced to Heparin 4h 0.28 ±0.13 1.27 ±0.49 5% and the totalthrombotic eventswere reduced to 30%,both Arginine** 4h 0.04 ±0.01 0.11 ±0.05 significantly less thancontrol or heparin treatedrats. When Heparin +Arginine 4h 0.08 ±0.02 0.25 ±0.14 using sulodexide (7.5mg/kg) alone in the 4-hour study,the inci- Sulodexide** 4h 0.03 ±0.01 0.15 ±0.08 denceofhard thrombi was10% and the total thromboticinci- Sulodexide+Arginine** 4h 0.09 ±0.03 0.12 ±0.03 dencewas 30%, both significantly less thanobservedfor control Sulodexide+Arginine* , ** 28 day0.06 ±0.01 0.21 ±0.04 or heparin treatedrats. When arginine plus sulodexide were ad- *Dosesfor the 28 daystudy were sulodexide2.9 mg/kg/day;arginine 42.9mg/kg/day. All other ministered,the incidenceofhard thrombi was15%, and total groups were given single dosesofheparin or sulodexide at 7.5 mg/kg; arginine 112.5 mg/kg. thromboticincidence was45%, both significantly less thancon- ** comparedtosulodexide standards, all othergroups comparedtoheparin standards.

868 Daniels,etal.: Synergyoforal heparinand arginine

Figure3:Changes in coagulation parametersfollowing admin- istrationofsulodexide or heparin plus arginine to rats. In the 4-hour groups, heparin (7.5 mg/kg) or sulodexide (7.5 mg/kg) and/or arginine (112.5 mg/kg) were administered by stomachtube four hours Figure2:Agarosegel electrophoresis slidesshowing heparin priortoblood collection. In the 28-daystudy,sulodexide (2.9 mg/kg) and sulodexide with endothelium following oral administration. and arginine (42.9 mg/kg) were administered dailybystomach tube,the Lane1,sulodexide standard; lane 2, heparin standard; lane 3, raten- lastdosegiven four hoursprior to blood collection. One-wayANOVA, dothelium extract in the absence of administered agent (control); lane 4, Tu key‘s post hoc test. Results aresignificantlygreater than control(a); ratendothelium extract four hoursafter 7.5 mg/kg oralheparin; lane 5, heparin (b); heparin plus arginine (c); sulodexide plus arginine (d); argi- ratendothelium extract four hoursafter 7.5 mg/kg oralsulodexide;lane nine (e); sulodexide plus arginine 28 days (f). P<0.05. 6, ratendothelium extract four hoursafter 7.5 mg/kg oralsulodexide + arginine 112.5 mg/kg; lane 7, ratendothelium extract 4hours after 7.5 mg/kg oralheparin +arginine; and lane 8, ratendothelium extract 28 days after2.9 mg/kg/dayoralsulodexide +arginine 42.9 mg/kg/day.

of the control group. Endothelium from rats treatedwith arginine aloneshowedtwice theconcentrations of sulodexide on aortic and vena cavalendothelium as that from control rats.

Anticoagulant activity APTT,Heptestand anti-factor Xa activity wasassessedtodeter- mine if sulodexide or heparinand/or arginine modified coagulation following oraladministration (Fig. 3). Although only small changes were observedinAPTTinthe 28-daystudy of the sulodexide plus arginine group, the APTT in the 28-daygroup wassignificantly greater thanthe 4-hour treatedsulodexide plus arginine, the heparin plus arginine or the arginine alone treated Figure4:Sulodexide-likecompounds in 24-hoururine samples groups. Interestingly, anti- factor Xa activity wassignificantly at differentdaysfollowing long-term administration of sulod- greater in controls thaninthe groups treatedwith heparinplus ar- exide plusarginine. Sulodexide 2.9 mg/kg plus arginine 42.9 mg/kg ginine and arginine alone.Inaddition, the 28-daysulodexide were given by stomachtube dailyfor 28 days.Mean±SE areshown. plus arginine treatedgrouphad noticeably loweranti-factor Xa One-wayANOVA.Tukey‘s post hoctest; significantlygreater than con- activity,and this activity wassignificantly lowerthan the heparin trol (a); day1(b);day 21 (c); day28(d).P<0.05. plus arginine treated, sulodexide treated and arginine alone treated groups. Little or no anticoagulant wasfound in plasma as determined Recoveryfromurine by the anti-IIa assay.Noanti-IIaactivity wasdetected in plasma When changes in the amount and concentration of urinary sulod- of the 4-hoursulodexide plus arginine, heparin plus arginine and exide with time were determinedbyelectrophoresis with time the 28-daylong-term sulodexide plus arginine groups. Asmall for animals administered sulodexide plus arginine for28days, amount of anti-IIa activity wasdeterminedinthe plasma of ani- maximum increases in totalµgand µg/ml were seen at day7(Fig. mals administered sulodexide alone and arginine alone (values 4). While the total amount of sulodexide remained elevated equivalent to 1.43±0.97, and 4.36 ±5.04 [SE] g/ml,respec- throughoutthe 28-dayperiod,its concentration in the urine de- tively). clinedafter day7.With the exception of alarge spikeonday 1,

869 Daniels,etal.: Synergyoforal heparinand arginine urinary anti- factor Xa activity levels closelyparalleledthe con- ing that arginine upregulates the heparansulfate on the endothe- centrations found by chemical determination. No anti-factor IIa lialcellsurface. The highest recovery from endothelium wasob- activity wasfound in urine samples from control or experimen- tained in the group treatedwith heparinalone.Moreover,the tal animals. Heparin and sulodexide were found only in small vena cavalendothelium generallyshowedsubstantiallyhigher amountsinthe urine of rats treatedfor four hours with heparin or concentrations of heparinand glycosaminoglycanthan the aortic sulodexide, with heparin onlytreated animals showinglevels endothelium,agreeing with our previous work on orally admin- significantlygreater thancontrol rats (data not shown). istered low-molecular-weightheparins (4, 27). Heparin, however,was notrecovered from the endothelium of allanimals that Discussion showedantithrombotic activity.This is probablythe result of both the limited sensitivity of our assayand the sampling of only The results of the study of oral administrationofheparin plus ar- asmall amount of the totalendothelium, an averageof2.9 cm2 of ginine in unstable angina patients, suggest that this therapeutic approximately 2m2 of total endothelial surface. Since the mi- approachcompares favorably to both strategiesinpatients re- gration of sulodexide under electrophoresis is similartoendo- ceiving subcutaneous LMWHinboth invasively and non-invas- genous endothelial glycosaminoglycans, it wasdifficulttoestab- ivelytreated groups (23).Controlled human studies, using a lish the presence of the exogenously administered sulodexide on larger numberofpatients, are required to establish the signifi- the endothelium. cance of heparinplus arginine on the variousendpoints,particu- No evidenceofgastrointestinalirritation or bleeding wasob- larlypatient death. Basedonthese anecdotalresults, the same servedineitherhumans or rats during these investigations.There combination dosage of oral heparinplus arginine, as well as oral were only minor changes in the plasma anticoagulant activity sulodexide plus arginine, and each agentalone were investigated followingoral administration of sulodexide or heparinascom- in arat jugularveinthrombosis model. The resultsofthis animal pared to controls. Noteworthy is the observedincrease in APTT study clearly demonstrate that orallyadministeredarginine has in the group receiving long-termsulodexide plus arginine. A an antithrombotic effect in this rat thrombosis modelasdoes or- similar small butsignificant increase in APTTwas seen in hu- ally administered heparin.Inaddition, the administration of he- mans receiving repeated oral heparin at 20,000 units twice parin plus arginine has additional antithromboticeffectswhen weeklyfor twotoeight months(28). Our ownwork with dextran comparedtoheparin or arginine alone. Arginine didnot have the sulfateshowedasimilarincrease in APTT in humans treated same synergistic effect when combined with sulodexide,al- 29–335 days with 1gram fourtimesper dayofdextran sulfate. though sulodexide alonehad antithrombotic activity.These re- This occurred despite dextran sulfatehaving one hundreththe sults suggest thatsulodexide and arginine mayact by asimilar anticoagulant activity of heparin (29).Inthe present study,anti- mechanism whereas heparinhas additionalantithrombotic ef- factor Xa activity wassignificantly elevatedinthe arginine fects.Sulodexide alone wasmore effective thanheparin alonein alone,and the heparin plus arginine treatedgroups,leading one the short-termstudy.This maybedue to the presenceofderma- to speculatethat arginine mayhavesome effect on anti-factor Xa tansulfate or possible shorter chain length heparin (Mr (avg) activity.Incontrast, anti-factor Xa activity waslow in the long- ~8,000) present in this glycosaminoglycanmixture(22).These term 28-dayarginine plus sulodexide treated rats. Apossible ex- components of sulodexide mayexhibit afasterrateoforal ab- planation for this lowanti-factor Xa activity is that long-termad- sorption and slowerrateofclearance thanobservedwith the un- ministration of sulodexide and arginine mayresultinchangesin fractionated heparin (Mr (avg)~12,000) used here. This is con- production or consumption of coagulation factorstobalance the sistent with our previous resultsinwhich lowmolecular weight increasing anticoagulant effect of sulodexide or arginine. heparins were effective at muchlower dosesthan unfractionated Consistent withheparin binding to endothelium, little hepa- heparins (2). rinorsulodexide wasrecovered from urine in the 4-hourstudy Adecision to do the long-term, 28-daystudy with onlysulod- with onlythe group receiving heparinalone showingasignifi- exide and arginine wasbased on the assumption thatsulodexide cant increase in urinary heparin concentration comparedtocon- would cause less bleeding in long-termstudiesbecauseofits re- trols.This result might be explained by afasterrateofabsorption duced anticoagulant activity compared to heparin. It is interest- or excretion for heparin thanfor sulodexide, or for heparin or su- ing to note that28-dayadministration of sulodexide and arginine lodexide in combination with arginine. Small amounts of hepa- waseffectiveinreducing thrombosis incidencebut not signifi- rinorsulodexide were found in the urine of allrats, as expected. cantly different than 4-hour administration. However, the dose In contrast to 4-hour administration, 28-dayadministration of administered per dayoverthe 28-dayperiod,was less thaninthe sulodexide and arginine showedsignificant increases in glyco- 4-hour study.This suggests that long-termadministration results saminoglycans in the urine,beginningonday 1when anti-factor in an accumulation of the drug or its beneficial effects. Further Xa activity wasmeasured,and on day7when measuredchemi- studiesare warranted to examine this phenomenon,perhaps be- cally. This supports the observations thatsulodexide is absorbed, ginning with usingthe lowerdose in ashort-termexperiment and thatsome accumulation occurs throughout the 28-dayperi- (4 hours). od,resulting in an increase in excreted material. As in previous experiments (1–3, 26), heparin wasrecovered Since this studyshows that oralarginine plus heparinhas sig- from aortic and vena cavalendothelium following its oraladmin- nificantlyincreased antithromboticactivity compared to oral he- istration. In the present studies, arginine alone appearstoin- parin alone, we speculatethat arginine enhancesthe absorption crease the concentration of endogenous glycosaminoglycans on of heparin, and/or arginine enhancesheparin’santithrombotic the vena cavalendothelium, consistent with the studies indicat- activity.This is supported by the observation thatanti-factor Xa

870 Daniels,etal.: Synergyoforal heparinand arginine activity is significantly higher in heparin plus arginine treated Suchendogenous heparansulfateshavebeen shown to mediate rats thaninheparin onlytreated rats.(It should be notedthat flow-induced eNOS signaltransduction (33).Thecellularuptake these changes in anti-factor Xa activity areminor although sig- of heparansulfate proteoglycans has also been shown to be re- nificant,Fig. 3).Results from the analysis of the endothelium in latedtoNOcatabolism (20). There has alsobeen arecent report this studyalso showlessheparin on the endothelium in the pre- implicating oral heparininenhanced NO production, whichac- senceofarginine thanwhen heparin is givenalone. celerates healing of gastric ulcers in animals (34). Decreased ex- The clinicalresults reported suggest thatheparin is absorbed cretion of heparininthe presenceofarginine hasnot beenex- in humans followingoral administration and is distributedpri- cludedasanoperative mechanism for their synergistic anti- marily to endothelium. Thesestudiesare supportedbythe work thrombotic effect.Moreover,itisinteresting that oralsulodexide of Engelbergwho showedincreases in APTT in ahuman study also has antithromboticactivity thatisnot enhancedbyits co-ad- with oralheparin (28)and by our ownstudiesshowing an in- ministration with arginine. crease in anti-factor Xa activity and heparin in urine following a Both endothelialheparan sulfateand NO effects areknown single dose of 7.5 mg/kg of heparin to humansubjects (30).Are- to be reducedinthe atherothrombotic processesaswellasin cent studyalso confirmssimilarendothelial deposition of hepa- aging in humans (35,36). Cholesterol plaque accumulation oc- rinaswellasantithrombotic efficacywhen administered orally cursatsitesofreduced eNOS activity.Rupture sites of culprit in arat modelofcarotid artery thrombosis (31). In the current plaque lesions, responsible for unstable angina and myocardial study,the observation thatarginine enhances the activity of or- infarction, showmarkedlydepleted levels of endogenous pro- ally administered heparin in both humans and animals, despite teoglycans compared to normalarterysegments (37). The little change in plasma anticoagulant activity,isanew finding marked decrease in expected cardiovascularevents rates seenin and suggests that arginine and heparin have asynergistic anti- suchpatients over a12-month period of using daily, continuous thromboticeffect primarily on the endothelium. oral heparinand arginine treatment suggests sustained pass- The exact mechanism by whicharginine enhances the anti- ivation of plaque surfaces in these treated patients. Further thrombotic effect of oral heparinremains unknown, although en- studiesare required to assess the roles of NO production, endo- hanced absorption or enhancedendothelial antithromboticactiv- genousendothelial heparansulfate effects, coagulation factor ity,may contribute to these effects. Arginine onlyweaklybinds complex assembly,thrombolytic processes, and other questions heparin(11), so that it is unlikelythat arginine interacts with he- raised by the observations reported in the currentstudy.These re- parin in the body due to the presenceofsalts and competing, sults suggest the suitability of oral heparinorrelated glycosami- higher-affinity heparin-binding proteins. We suggest insteadthat noglycans with arginine, with no demonstrated adversereactions it is likelythat heparin and arginine exerttheir antithrombotic and absence of systemic anticoagulation effects, forlong-term throughdifferent pathwaysbut in asynergistic fashion. Since ar- antithrombotic prophylaxis and treatment in larger humanand ginine is asubstrate for NO (5),these resultssuggest that NO animal trials. mayplayarole in this phenomenon. Heparin increases NO activity through effects on eNOS (32).NOenhances the production of Acknowledgements endogenous endothelial anticoagulant heparansulfate (14). Theauthorsthank TillyPing fortechnical assistance.

References 1. Jaques LB,HiebertLM, Wice SM.Evidence from 9. StiefTW,WeippertM,KretschmerV, et al.Arginine 16. Stevens RL, Colombo M,Gonzales JJ, et al. The endotheliumofgastric absorption of heparin and of inhibits hemostasisactivation. Thromb Res 2001; 104: glycosaminoglycans of thehuman artery and their dextran sulfates 8000. JLab Clin Med 1991;117: 265–74. changesinatherosclerosis.JClin Invest 1976;58: 122–30. 10. Hileman R, Fromm J, Wieler J, et al. Glycosami- 470–81. 2. HiebertLM. Oral heparins. Clin Lab2002;48: noglycan-proteininterations:definitionofconsensus 17. BingleyJA, HaywardIP, GirjesAA, et al. Ex- 111–6. sitesinglycosaminoglycanbinding proteins.Bioessays pressionofheparan sulphate N-deacetylase/N-sulpho- 3. HiebertLM, Wice SM,McDuffieNM, et al.The 1998; 20: 156–67. transferasebyvascular smooth musclecells.Histo- heparin target organ–theendothelium. Studies in arat 11. Fromm JR, Hileman RE, Caldwell EE, et al. Differ- chemJ2002; 34: 131–7. model. QuartJMed 1993;86: 341–8. ences in theinteractionofheparin witharginineand ly- 18. BombeliT,KarsanA,Tait JF,etal.Apoptotic vascu- 4. HiebertLM, Ping T, Wice SM.Antithrombotic ac- sine and theimportance of thesebasic amino acids in larendothelial cellsbecome procoagulant. Blood 1997; tivityoforallyadministered lowmolecular weight he- thebinding of heparin to acidicfibroblast growth fac- 89: 2429–42. parin (logiparin)inarat model. Haemostasis2000;30: tor. Arch Biochem Biophys 1995; 323:279–87. 19. Constantinescu AA,VinkH,SpaanJA. Endothelial 196–203. 12. Florian JA,Kosky JR, Ainslie K, et al. Heparan cell glycocalyx modulates immobilization of leuko- 5. Napoli C, Ignarro LJ.Nitric oxideand atherosclero- sulfate proteoglycanisamechanosensor on endothelial cytesatthe endothelialsurface. Arterioscler Thromb sis.Nitric Oxide2001;5:88–97. cells.CircRes 2003; 93: 136–42. Vasc Biol2003;23: 1541–7. 6. Armstead VE,MinchenkoAG, Schuhl RA,etal. 13. Pinhal MA, WalengaJM, JeskeW,etal.Antithrom- 20. DingK,Sandgren S, Mani K, et al. Modulations of RegulationofP-selectin expressioninhuman endothe- boticagents stimulate thesynthesis and modify thesul- glypican-1heparansulfate structure by inhibitionof lialcellsbynitric oxide. Am JPhysiol 1997;273: fationpatternofaheparansulfate proteoglycanfrom endogenous polyamine synthesis.Mappingofsper- 740–6. endothelial cells.Thromb Res 1994; 74: 143–53. mine-binding sitesand heparanase, heparin lyase, and 7. Ignarro LJ,CirinoG,CasiniA,etal. Nitric oxideas 14. Irokawa M, NishinagaM,Ikeda U, et al. Endothe- nitric oxide/nitrite cleavage sites. JBiolChem 2001; asignaling molecule in thevascular system: an over- lial-derived nitric oxidepreservesanticoagulant he- 276:46779–91. view. JCardiovasc Pharmacol 1999; 34: 879–86. paran sulfate expressionincultured porcine aortic en- 21. RubergFL, Leopold JA,Loscalzo J. Atherothrom- 8. Keha D, Thieme A, Kurer I, et al.Inactivationof dothelial cells.Atherosclerosis 1997; 135:9–17. bosis:plaque instability and thrombogenesis. ProgCar- platelet glycoproteinIIb/IIIa receptorbynitric oxide 15. Lloyd-JonesDM, BlochKD. Thevascular biology diovasc Dis 2002; 44: 381–94. donor 3-morpholino-sydnonimine. Blood Coagul Fi- of nitric oxideand its roleinatherogenesis.Annu Rev 22. OfosuFA. Pharmacological actions of sulodexide. brinolysis 2003; 14: 327–34. Med 1996;47: 365–75. SeminThromb Hemost 1998;24: 127–38.

871 Daniels,etal.: Synergyoforal heparinand arginine

23. Wallentin L, Lagerqvist B, Husted S, et al.Out- 28. EngelbergH.Orally ingested heparin is absorbed in 34. LiY, Wang WP,Wang HY,etal. Intragastricadmin- comeat1year after an invasive compared with anon- humans. Clin Appl ThrombHemost 1995; 1: 283–5. istrationofheparin enhances gastriculcer healing invasive strategyinunstable coronary-artery disease: 29. HiebertLM, Wice SM, Jaques LB,etal. Orally ad- through anitric oxide-dependent mechanism in rats. theFRISC II invasive randomised trial. FRISC II Inves- ministered dextran sulfate is absorbed in HIV-positive Eur JPharmacol 2000; 399: 205–14. tigators.Fast Revascularisationduring Instability in individuals. JLab Clin Med 1999;133: 161–70. 35. Hoffmann J, Haendeler J, Aicher A, et al.Aging en- Coronaryarterydisease. Lancet 2000; 356: 9–16. 30. HiebertLM, Wice SM, Ping T. Increased plasma hances thesensitivity of endothelialcells toward apop- 24. BlakeOR, AshwinJG, JaquesLB. An assay for the anti-Xa activity and recovery of heparin from urine totic stimuli: important role of nitric oxide. Circ Res antithrombotic activity of anticoagulants . JClin Path suggestabsorptionoforally administered unfraction- 2001; 89: 709–15. 1959; 12: 118–22. ated heparin in human subjects. JLab Clin Med.2005; 36. MurataK,Murata A, Yo shida K. Heparan sulfate 25. HiebertLM, JaquesLB. Heparin uptakeonen- 145: 151–5. isomers in cerebral arteries of Japanese womenwith dothelium. Artery 1976; 2: 26–37. 31. Pinel C, Wice SM, HiebertLM. Orally adminis- aging and with atherosclerosis--heparitinase and high- 26. HiebertLM, Wice SM, PingT, et al.Tissue distribu- tered heparins prevent arterial thrombosisinarat performance liquidchromatography determinations. tionand antithrombotic activity of unlabeled or 14C-la- model.Thromb Haemost 2004; 91: 919–26. Atherosclerosis 1997; 132: 9–17. beled porcine intestinal mucosal heparin followingad- 32. Kouretas PC, Hannan RL,Kapur NK, et al.Non- 37. KolodgieFD, Burke AP,Farb A, et al.Differential ministrationtoratsbythe oral route. Can JPhysiol anticoagulant heparin increases endothelialnitric oxide accumulationofproteoglycansand hyaluronan in cul- Pharm2000; 78: 307–20. synthase activity:Roleofinhibitory guanine nucleo- prit lesions: insights into plaque erosion. Arterioscler 27. HiebertLM, Wice SM, PingT, et al.Antithrombotic tideproteins. JMol CellCardiol 1998; 30: 2669–82. Thromb Vasc Biol 2002;22: 1642–8. efficacyinarat model of thelow molecular weight he- 33. Florian JA,KoskyJR, AinslieK,etal. Heparan parin, reviparin sodium,administered by theoral route. sulfateproteoglycan is amechanosensor on endothelial Thromb Haemost2001; 85: 114–8. cells.CircRes 2003; 93: 136–42.

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