Low-Molecular-Weight Heparin andDermatan Sulfate End Group-Labeled withTyramine and Fluorescein. Biochemical and Biological Characterization of theFluorescent-Labeled Heparin DerivativeJob Harenberg, M.D.,1 Benito Casu, Ph.D.,2 Marco Guerrini, Ph.D.,2Reinhard Malsch, Ph.D.,1 Annamaria Naggi, Ph.D.,2 Lukas Piazolo, Ph.D.,1 andGiangiacomo Torri, Ph.D.2
ABSTRACT
To improve the understanding of the biological functions and pharmacologyof heparin and dermatan sulfate, low-molecular-weight heparin (LMWH) and low-molecular-weight dermatan sulfate (LMWDS) were labeled with tyramine (T) by cova-lently linking T to the terminal residue of 2,5-anhydromannose (or 2,5-anhydrotalose fordermatan sulfate). The covalent labeling was demonstrated by nuclear magnetic resonancespectroscopy. The tyramine-labeled LMWH (LMWH-T) was also labeled with fluores-cein (F) by further reacting it with fluorescein isothiocyanate. The fluoresceinatedLMWH-T (LMWH-T,F ) was used to analyze biological functions on blood coagulationand binding to leukocytes. The biological activities on factor Xa and thrombin inhibitionremained unchanged compared with the parent compound. Flow cytometric analysis ofleukocytes demonstrated binding of the modified heparin to granulocytes, monocytes, andlymphocytes, the half-live being twice as long as the antifactor Xa activity. F-labeled hepa-rin was displaced by unlabeled heparin from all three populations of leukocytes. Binding ofheparin to leukocytes may play an important role in inflammation and atherosclerosis.
Objectives: Upon completion of this article, the reader should be able to (1) describe some of the anticoagulant properties of fluo-rescein-labeled low-molecular-weight heparin and (2) explain the binding of this compound to leukocytes.Accreditation: Tufts University School of Medicine is accredited by the Accreditation Council for Continuing Medical Education toprovide continuing medical education for physicians. TUSM takes full responsibility for the content, quality, and scientific integrity ofthis continuing education activity.Credit: Tufts University School of Medicine designates this education activity for a maximum of 1.0 hours credit toward the AMAPhysicians Recognition Award in category one. Each physician should claim only those hours that he/she actually spent in the edu-cational activity.
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Glycosaminoglycans (GAGs) are naturally oc-curring polysaccharides, the one with the most clinicalrelevance being heparin, which consists of repeatingunits of variously sulfated hexuronic acid (d-glucuronicof l-iduronic acid) and d-glucosamine.1,2 The molecularmass of unfractionated heparins ranges from 3000 to30,000 daltons and of low-molecular-weight heparins(LMWH) from 1200 to 8000 daltons.3 Heparins exerttheir anticoagulant actions by enhancing the inactiva-tion of several serine proteases of the coagulation sys-tem and by potentiating the activity of antithrombin(AT).4 They also exhibit a variety of AT-independentsignificant anti-inflammatory and antimetastatic activi-ties.5,6 The antithrombotic potency is established inpostoperative7,8 and general medicine9,10 as well as forthe treatment of acute thromboembolic diseases.11,12
However, the AT-independent actions of GAGs are lessknown and currently under investigation. Other iduronicacid–containing GAGs and especially dermatan sulfateand its low-molecular-weight derivative (LMWDS) arealso being considered for clinical use as antithromboticagents with low anticoagulant activity.
Labeling of heparins and dermatan sulfates byradioactivity and fluorescence is a major problem be-cause the biological activity and receptor-mediatedbinding of the GAGs may be substantially modified bythe labeling reactions. Therefore, we have developed amethod for labeling GAGs through a tyramine residue,which was linked to the anhydrohexose group of LMWHand LMWDS by endpoint attachment,13 thus notmodifying the structure of the GAG chains and permit-ting them to interact with biological receptors as for un-labeled GAGs. Further labeling of the tyramine moietyof LMW-T with iodine has been used to develop a sen-sitive binding assay14 and to study the renal and livermetabolism in animals.15 Fluorescein-5-isothiocyanate(FITC) has been specifically tagged to the tyraminegroup of LMWH without modifying the biological ac-tivity as compared with LMWH-T.13,16 The binding ofthe fluoresceinated product (LMWH-T,F ) to humanleukocytes presented evidence of specific binding.17
MATERIALS AND METHODS
Materials
Unfractionated porcine intestinal mucosa heparin wasobtained from Medac, Hamburg, Germany. LMWHwith terminal 2,5-anhydromannose residues (batch20006000), average molecular mass in daltons (Mw =5275 Da, range 1200–10215 Da), was generously pro-vided by Novartis Pharma GmbH, Nürenberg, Germany.Reference low-molecular-weight dermatan sulfate, hepa-rin pentasaccharide, and LMWH dalteparin were fromAlfa Wassermann, Bologna, Italy; Sanofi-SynthelaboRecherche, Toulouse, France; and Pharmacia GmbH,
Erlangen, Germany, respectively. Rabbit phycoerythrinconjugated antimouse CD11c was obtained from Daco(Hamburg, Germany). Tyramine (no. T 7255), fluores-cein-5-isothiocyanate (no. F 7250), sodium cyanobor-hydride (no. S 8628), and protamine (no. P 3880) werefrom Sigma GmbH (Deisenhofen, Germany). Ace-tonitrile was from Fison (Loughbourough, England). An-tithrombin was from Behringwerke (Marburg, Germany).The chromogenic substrates S2222 (N-benzoyl-iso-leucyl-L-glutamyl-(OR)-glycyl-L-arginine-p-nitroanilinehydrochloride) and its methyl ester and S2238 (H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihy-drochloride) were from Chromogenix (Mölndal, Sweden)and Pharmacia AB (Stockholm, Sweden), respectively.
Preparation of Tyramine-Conjugated and
Fluorescent-Labeled Glycosaminoglycans
Tyramine (T) was bound to LMWH by reductive ami-nation in the presence of sodium cyanoborohydride, aspreviously described.18 The product (LMWH-T, Mw =6,361 Da, range 660–10,340 Da) was purified by high-performance size exclusion chromatography (HPSEC).and reacted with FITC in solution buffered with so-dium hydrogen carbonate (pH 8.5) to afford the fluo-resceinated derivative LMWH-T,F. The product wasprecipitated by ethanol, purified by HPSEC, dialyzedagainst distilled water, and freeze-dried (Mw = 5,775Da, range 660–7600 Da). It was excited at 480 nm andemitted fluorescence at 515 nm. Nuclear magnetic reso-nance (NMR) spectra indicated that tyramine was end-point attached to LMWH and FITC to the secondaryamino group of LMWH-T. Averages of 1 tyraminemolecule and of 0.8 FITC molecule per 20 disaccharideunits of heparin were calculated from quantitative 1H-NMR analysis.16 Tyramine-labeled LMWDS was pre-pared from partially N-deacetylated DS. N-Deacety-lation was performed with hydrazine as previouslydescribed for the preparation of intermediates of deu-terium-labeled GAGs. The resulting LMWDSs termi-nating with 2,5-anhydrotalose residues were reactedwith tyramine in the presence of sodium cyanoborohy-dride under conditions similar to those used for prepa-ration of LMWH-T to afford LMWDS-T. The prod-uct had a mean molecular weight of 3600 daltons andcontained 80 antifactor Xa units/mg and 5 AT units/mgusing the first international low-molecular-weight hep-arin standard.18
Coagulation Assays
Antifactor Xa and AT activities were determined usinga pool of human (Behringwerke, Marburg, Germany) orrat plasma, respectively, loaded with the first interna-tional standard for LMWH and the specific chromo-genic substrates S2222 and S2238, respectively. The
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tests were performed according to standard laboratoryprocedures.19,20 The specific activities were for LMWH-T110 aXa and 41 aIIa and for LMWH-T,F 66 aXa and 5aIIa units mg-1 with human plasma.
Neutralization of Endpoint-Attached LMWH
The neutralization of the LMWH preparations wasperformed using protamine as antagonist. The methodwas described before.21 Briefly, heparin and LMWHs ata concentration of 1 mg mL�1 were incubated withprotamine ranging from 1 to 10 mg mL�1. The antifac-tor Xa activity of these mixtures was assayed with thechromogenic antifactor Xa assay, as described earlier.
Animal Experiments
The pharmacokinetic and pharmacodynamic propertiesof LMWH-T,F and LMWH-T (150 aXa IU per ratcorresponding to 2.1 and 1.5 mg, respectively) werecompared with those of the parent compound (LMWH)and of heparin. Eight male Sprague Dawley rats (350–550 g) were anesthetized by intramuscular administra-tion of 0.3 mg kg�1 ketamine hydrochloride and 0.04mg kg�1 diazepam (Hoffmann La Roche, Basle, Switzer-land). Thereafter, ether narcosis was performed for bloodsampling. Blood (0.45 mL) was taken by puncturingthe retro-orbital sinus of the rats. Blood samples weredrawn at 0, 10, 30, 60, 120, 240, and 360 minutes. Theywere collected into syringes containing 0.05 mL of 0.13M sodium citrate solution and mixed immediately.Within 30 minutes, the samples were centrifuged at3000 g, 20 minutes, and the plasma was shock frozenand stored at �70°C until assayed.
Calculation of the Pharmacodynamic and
Pharmacokinetic Parameters
The following parameters were calculated from theplasma antifactor Xa, AT activity, and the concentrationof LMWH-T,F . The maximal concentration (Cmax)after intravenous application was calculated by extrapo-lation of the beta-elimination phase to the y-axis andthe instantaneous volume of distribution was estimatedas Cmax/dose. The time versus concentration curve wascalculated by a linear trapezoidal method with extrapo-lation to infinity. The mean total clearance was obtainedby the ratio of the injected dose to the area under thecurve (AUC). The volume of distribution Vd was esti-mated as the ratio of the clearance to the slope of theterminal phase of elimination.22 The relative bioavail-ability was the ratio of the intravenous AUC ofLMWH-T and LMWH-T,F to the intravenous AUCof LMWH. Statistical significance of the differencesbetween the drugs was analyzed using the Mann-Whitney U test. The level of significance was set at p =
.01 and is referred to in the text as significantly differentor as significant.
Reversed Phase High-Performance Liquid
Chromatography (RP-HPLC)
An aliquot of the plasma samples (100 �L) was pre-cipitated by bentonite (Roche Diagnostics, Mannheim,Germany). Twenty microliters of the supernatant wasused for RP-HPLC analysis in duplicates. A systemconsisting of a Waters multisolvent delivery system witha Waters 600 E system controller, an injector, a 20-�Lloop, a column resolve 5 �m spherical C18 3,9 .150 mmand guard-pak module with HPLC precolumn insertsC18 from Millipore Waters (Milford, MA)23 was con-nected between the injector and the pump. A fluores-cence detector model Waters 420 AC was used, and thefluorescence signals with emission at 510 nm were re-corded and integrated by PAD software from MilliporeWaters. For the elution, a linear isocratic 25/75% ace-tonitrile-water mixture and a flow rate of 0.5 mL/minwere used. The area under the absorption time curve(AUC) was integrated with the linear trapezoidal methodand extrapolated to infinity. Plotting the area under theconcentration time curve (AUC) versus the concentra-tion of LMWH-T,F, a linear correlation was found. Theconcentration of LMWH-T,F, which is directly avail-able for the anticoagulation of the blood, was measuredby emission of fluorescence. A small amount of back-ground fluorescence was due to the autofluorescence ofthe supernatant. The lower detection limit of thismethod was 50 �g mL�1 LMWH-T,F and the recoveryof LMWH-T,F was 89%.
Blood Sampling
Venous blood was obtained from healthy volunteers,who did not take any medications for the preceding 10days. All volunteers had given conformed consent priorto blood sampling. Blood was obtained by puncturingan antecubital vein with an 18-gauge butterfly withouttourniquet to minimize platelet activation during bloodcollection. After the first 2 mL of blood was discarded,5 mL was collected in plastic tubes containing EDTAfor anticoagulation.
Preparation of Samples for Flow Cytometry
One hundred microliters of anticoagulated whole bloodwas incubated with 10 �L of LMWH-T,F at concen-trations ranging from 0.01 to 100 �g/mL for 15 min-utes in the dark and at room temperature. Then 200 �Lof lysis reagent (Becton Dickinson, Heidelberg, Ger-many) was added and incubated for 10 minutes atroom temperature and in the dark, at which time eryth-rocytes were lysed. Samples were centrifuged at 500 g
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for 5 minutes. The pellet was washed twice with 2 mLof 0.05 m tris-NaCl buffer, pH 7.4.
Flow Cytometry Analysis
The fluorescence on the leukocytes was analyzed by aFAC-Scan cytometer (Becton Dickinson) equipped witha 15-mW air-cooled 488-nm argon laser. Forward andside scatters as well as green (FITC) signals were ac-quired by logarithmic amplification with a 585 � 21nm filter for collection of FITC signals. Acquisitionprocessing of data from 10,000 cells was carried outwith the Consort 30 software (Becton Dickinson) on aHewlett Packard 300 computer. The percentage of pos-itive cells and the mean fluorescence intensity were cal-culated as median, mean, and standard deviation. Meanvalues and standard deviations were calculated from thedata given in the tables and figures.
Displacement Experiments
Dilutions of the unfractionated heparin, dalteparin,dermatan sulfate, and pentasaccharide were made fromto 0.1 �g to 1 mg/mL. Samples were mixed with 10 �gLMWH-T,F/mL and incubated with leukocytes from 1mL of EDTA-anticoagulated blood in the dark for 30minutes. Fixation of cells was performed with 5% para-formaldehyde as described.24 All experiments were car-ried out threefold and in duplicate.
RESULTS
Synthesis and Structural Characterization of
Labeled LMWH and LMWDS
In order to prepare tyramine- and fluorescent-labeledLMW GAGs, the strategy of end-group derivatizationwas followed in order to preserve the structure of theoriginal polysaccharide chains. LMW heparin and der-matan sulfate (LMWH and LMWDS) to be conju-gated were prepared by partial nitrous acid depolymer-ization according to established methods, but avoidingthe final step of reduction commonly used to stabilizethe terminal 2,5-anhydrohexose residues. To prepareLMWDS, dermatan sulfate was partially N-deacety-lated with hydrazine. Different times of the hydrazinol-
ysis reaction were used to afford dermatan sulfate frag-ments of different molecular weights (Table 1), and afragment with Mw 3100 was chosen for conjugationwith tyramine, which was performed as previously de-scribed for LMWH. The reaction schemes for prepara-tion of LMWH-T, LMWDS-T, and LMWH-T,F areshown in Figure 1.
In order to characterize the structure of LMWDSand LMWDS-T by NMR spectroscopy, model disaccha-rides were prepared by exhaustive nitrous acid depoly-merization of both heparin and dermatan sulfate. The 1Hand 13C signals of these models and of the correspondingtyramine conjugates, as assigned by two-dimensionalhomo- and heterocorrelation methods, are shown inTable 2a to 2c. Signals are fully compatible with thestructure of L-iduronic acid 2-sulfate �-linked to 2,5-an-hydromannose 6-sulfate for the heparin derivative and ofL-iduronic acid �-linked to 2,5-anhydrotalose 4-sulfatefor the dermatan sulfate derivative. For LMWDS-T,conjugation of LMWDS with tyramine is revealed by ashift of the C-1 signal of 2,5-anhydrotalose 4-sulfatefrom 91.8 to 52.5 ppm and of the tyramine C-8 signalfrom 44.0 to 51.7 ppm, confirming covalent attachmentof tyramine to the reducing anhydrohexose residue.
Anticoagulant Activities
The antifactor Xa and IIa activities of heparin andLMW heparins were determined in plasma. All lipo-philically substituted heparins could be determinedwith these test systems because they were water soluble.LMWH-T exhibited antifactor Xa and IIa activity inplasma and in aqueous solution similar to that of LMWH(Table 3). The antifactor Xa and antifactor IIa activityof LMWH-T,F, however, was slightly decreased be-cause of the further substitution.
Neutralization of Endpoint-Attached LMWH
Heparin was neutralized by protamine on an equigravi-metric level. Neutralization of LMWH and its derivativeswas achieved with a 1.6- to 4.1-fold excess of protamine.Substituted low-molecular-weight heparins required dif-ferent amounts of protamine for neutralization indepen-dent of the lipophilicity of the substitution (Table 4).
Pharmacokinetic and Pharmacodynamic
Parameters of Endpoint-Labeled LMWH
The endpoint-labeled low-molecular-mass heparins,LMWH, and heparin were injected into eight rats each.Table 5 summarizes the pharmacokinetic and pharma-codynamic data after intravenous administration. Theantifactor Xa dose administered showed only minordifferences (Fig. 2). The AT doses, however, varied be-tween 19 to 150 units/kg�1 for all LMWH species but
Table 1 Molecular Weight (dalton)* of Four Samples of
LMW Dermatan Sulfate
Samples Mw Pd
1 3100 1.212 2400 1.133 1800 1.134 1450 1.20
*Determined by GPC-HPLC.
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Fig
ure
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showed only minor variation for the single LMWH(Fig. 3).
The respective data for the extrapolated Cmaxare given in Table 4. The Cmax values of LMWH andLMWH-T showed only minor differences. LMWH-tyr-FITC was about twofold higher. In contrast, theCmax did not show significant differences for theLMWH. The concentration of LMWH-tyr-FITC wasalso measured by RP-HPLC using fluorescence detec-tion. The Cmax was somewhat lower (25 vs. 39 g/mL)when compared with the concentration obtained by theantifactor Xa determination (Table 5 and Fig. 4). Com-paring the in vitro with the ex vivo data, it is noteworthythat LMWH-tyr-FITC displayed lower anticoagulantactivities in vitro than ex vivo. Some explanations ofthese phenomena are mentioned later (see discussion).
The AUC of the antifactor Xa activity wasabout three- to fourfold higher after administration ofLMWH-tyr-FITC (82 �g/mL/h) compared with
LMWH (27 �g/mL/h). The AT activity expressed in�g/mL was rather high for the low-molecular-massheparin because of the low aIIa activity in vitro. Whencalculated from the AT activity, the AUCs were similarfor LMWH and LMWH-tyr (35 and 39 �g/mL/h)and 10-fold higher for LMWH-tyr-FITC (340 �g/mL/h). The AUC of the concentration measured byRP-HPLC (40 �g/mL/h) was similar to the antifactorXa activity and 10% of the AUC of the AT activity ex-pressed in �g/mL/h (Table 4).
The plasma clearances differed after bolus injec-tion of heparin, LMWH, and modified LMWHs.Based on the antifactor Xa assay; the clearances of theendpoint-attached heparins were 59 to 64% lower com-pared with LMWH. The respective clearances based onthe AT assay were 20 to 87% lower. The clearance ofLMWH-tyr-FITC measured by RP-HPLC was 2-foldhigher compared with the antifactor Xa clearance and10-fold higher than for the AT clearance (Table 4).
Table 2 Chemical Shifts of (A) IdoA-At4SO3
and IdoA-Atol4SO3
Disaccharides (ppm referred to internal
standard 13C: MeOH 51.7 ppm; 1H TSP: 0 ppm), (B) IdoA-Atol4SO3-Tyrm Disaccharide (ppm referred to
internal standard 13C: MeOH 51.7 ppm; 1H TSP: 0 ppm); and (C) IdoA2SO3-Amol6SO
3-Tyrm Disaccharide
(ppm referred to internal standard 13C: MeOH 51.7 ppm; 1H TSP: 0 ppm)
Excitation — n.d. 480Emission wavelength (nm) 515Color White White Yellow
*The LMWH s have a comparable average molecular mass (Mm equivalent to Mw) and anticoag-ulant activity. Neutralization of the antifactor Xa activity was performed by different amounts ofprotamine. The absorbance of heparin and LMWH was the same and differed from the modifiedendpoint-attached LMWH. The sensitivity of the LMWH-T,F and its maximum wavelengths forexcitation and emission are shown. The color of the compounds was different.†n.d., not done.
The distribution volumes (Vd) did not differ tothe same extent as the clearances. When calculated fromthe antifactor Xa activity, Vd did not differ substantiallybetween LMWH and the N’-alkylamine LMWHs. Incontrast, when calculated from the AT activity, Vd waslower for LMWH-tyr (41%) and LMWH-tyr-FITC(94%) compared with LMWH , respectively. The Vdmeasured by the concentration (RP-HPLC), however,showed a value similar to the Vd of the AT activity (274vs. 259 mL/kg) and was about 100% higher than the an-tifactor Xa activity Vd (Table 5).
The elimination half-lives of the endpoint-attached heparins were calculated. Two eliminationphases were identified for LMWH and the modifiedendpoint-attached LMWH preparations. The alphahalf-life of LMWH-tyr was significantly longer com-pared with the other LMWH preparations. The betahalf-life of both endpoint-attached LMWH prepara-tions was significantly longer compared with LMWH.
When calculated from the AT activity, the alphahalf-lives of the LMWHs did not differ. The beta-elimination did not differ significantly between theLMWH preparations (Table 5). Based on the RP-HPLC method, two elimination phases were calculatedfor LMWH-tyr-FITC. The alpha phase of eliminationwas 55 minutes and the beta phase was 132 minutes.
The relative bioavailabilities of the endpoint-attached LMWH preparations were calculated versusLMWH. Calculated from the antifactor Xa activity,LMWH and LMWH-tyr displayed the same andLMWH-tyr-FITC a twofold relative bioavailability.Calculated from the AT activity, the relative bioavail-abilities of the LMWH preparations did not differ.
Dose-Dependent Binding of LMWH-T,F
to Leukocytes
LMWH-T,F was incubated in increasing amounts from0.01 to 100 �g in 1 mL of human blood containing 6 �106 leukocytes. The relative fluorescence intensity ofLMWH-T,F bound to lymphocytes, monocytes, andgranulocytes was analyzed by flow cytometry. The num-ber of cells was plotted against the relative fluorescenceintensity. Figure 5 shows the binding of LMWH-T,F tohuman granulocytes, lymphocytes, and monocytes. Ascan be seen, the relative fluorescence intensity was high-est on granulocytes.
Displacement of LMWH-T,F by Different GAGs
The displacement of fluorescent-labeled LMM-heparinhas been studied using unfractionated heparin, LMM-heparin, dermatan sulfate, and pentasaccharide. Increas-ing concentrations of these compounds were incubatedwith 10 �g of LMWH-T,F and thereafter incubated with1 mL of human blood. The displacement of LMWH-T,Fwas calculated from the mean of the relative fluorescenceintensity and expressed as a percentage of the relative flu-orescence intensity of 10 �g of LMWH-T,F (Table 6).
The percent displacements of 10 �g of LMWH-T,F plotted against the various amounts of unfraction-ated heparin and LMM-heparin are given in Figure 6.As can be seen, there are minor differences for unfrac-tionated and LMM-heparin. Dermatan sulfate displacesto a lower extent LMWH-T,F from all three leukocytepopulations (data not shown).
The pentasaccharide was less effective in displac-ing LMM-heparin-tyr-FITC. The dependence of the
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Table 4 Pharmacokinetic Parameters (n = 8, mean � SD) Calculated after
Intravenous Administration of 150 aXa Units of Heparin, LMWH, LMWH-
*Cmax is the maximal extrapolated plasma level; AUC is the area under the activity and concen-tration time curve extrapolated to infinity. The clearance is obtained by the dose Cmax�1. The Vdis the distribution volume of the beta-phase. RP-HPLC is the fluorescence measurement usingreversal phase chromatography, nd: not done.
Table 5 Ratios by which 50% and 20% Displacement of LMWH-Tyr-FITC Were Obtained by Different
Glycosaminoglycans on Lymphocytes, Monocytes, and Granulocytes
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Figure 2 The ex vivo antifactor Xa activity of low-molecular-weight endpoint-attached heparins expressed in U mL�1. Theyexhibit different pharmacodynamic profiles from heparin andLMWH. A bolus of 150 aXa units was administered to eight ratsand the mean for eight rats plotted against time up to 360 min-utes. Lipophilically, LMWH and LMWH-T,F exhibit higher an-tifactor Xa levels and area under the activity time curve thanLMWH.
Figure 4 The concentration of LMWH-T,F measured with RP-HPLC is shown in comparison with the concentration calcu-lated from the antifactor Xa and antithrombin activity after an in-travenous bolus of 150 aXa units in rats (n = 8, mean values).
molecular weight of di-, tetra-, penta-, hexa-, octa-, anddecasaccharides, LMM, and LMH-heparin from thedisplacement of LMWH-tyr-FITC from granulocytesis shown in Figure 6.
DISCUSSIONThis study compared the pharmacokinetic and pharma-codynamic properties of endpoint-attached heparins(LMWH-tyr and LMWH-tyr-FITC) with LMWH .As heparin is a heterogeneous compound concerningsize, structure, and activity,25 which might have an im-pact on its pharmacokinetic properties, standardizeddoses of heparin, low-molecular-mass heparin, and end-point-attached low-molecular-mass heparins (LMWH-tyr and LMWH-try-FITC) 150 U kg�1 were adminis-tered to rats in our study.
Modifications of heparin by endpoint attach-ment are considered to differ from those obtained bysubstitution at the saccharide backbone.26,27 The selec-
tively tagged N�-alkylamine low-molecular-mass he-parins were anticoagulantly active in vitro and wereused for metabolic and pharmacokinetic investigations.
The pharmacokinetic-pharmacodynamic find-ings for LMWH-tyr-FITC resulted from the cleavageof heparin, forming heparin fragments with lower an-tifactor IIa activity. A minimal binding chain length of17 or 18 saccharide units is required for the AT activ-ity.28 The molecular mass distribution of LMWH, ana-lyzed by polyacrylamide gel electrophoresis, suggestedonly a small proportion of material above 6000 daltons.A critical molecular mass and affinity toward thrombinmay be required for the prolongation of the antifactorXa activity because LMWHs, which are covalently
Figure 3 The antithrombin activity of low-molecular-weightendpoint-attached heparins expressed as U mL�1. They exhibitpharmacodynamic profiles similar to those of heparin andLMWH. A bolus of 150 aXa units was given to eight rats andthe mean of eight rats plotted against time (360 minutes).
Figure 5 Binding of increasing amounts of LMWH-T,F tohuman granulocytes, monocytes, and lymphocytes. The rela-tive fluorescence intensity is plotted against the concentrationsof LMWH-T,F (�g).
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Figure 6 Relation of the molecular weight of different LMM-heparin preparations (daltons) plotted against the molar excessto displace 50% of LMWH-tyr-FITC from granulocytes. The low-est molecular weight preparation is a disaccharide.
bound to proteins of larger molecular size, showed aprolongation of the antifactor IIa activity.29
The binding of heparin and LMWH to plasmaproteins was analyzed by Young et al.30 Low-molecular-mass heparins bind less to plasma proteins than unfrac-tionated heparin.31 A butyryl derivative of heparin wasreported to bind more tightly to albumin and in thisway to reduce the catalytic activity of the antifactor Xaand the AT activity.
The interaction of modified polyanionic com-pounds with protamine as a polycationic agent may pro-vide information on structural changes during the syn-thesis. Therefore, neutralization experiments with themodified LMM-heparins were performed with thepolycationic agent protamine. Protamine, which is ex-tracted from fish sperm, possesses free arginine residues,giving a positive net charge to the protein surface. Neu-tralization of heparin by protamine occurred at a ratioof 1.1. A minimal size of 20 saccharide units is requiredfor optimal binding. Thus, low-molecular-weight com-pounds show decreased binding to protamine and alarger amount is needed for neutralization.32 Lipophiliccompounds can enforce the binding to proteins (prot-amine) by hydrophobic interactions. The compoundsmeasured needed different amounts of protamine, rang-ing from a 2.1- to 4.4-fold excess of protamine for neu-tralization, which might be due to steric hindrance byFITC in the LMWH-tyr-FITC molecule.
In the present studies, the pharmacodynamic prop-erties of UFH and LMWH were compared with thoseof the N’-alkylamine derivatives of LMWH. In addi-
tion, the pharmacokinetics of the LMWH-tyr-FITCderivative were analyzed by RP-HPLC with fluores-cence detection. The pharmacodynamic properties ofLMWH differed from those of unfractionated heparinin many respects, such as area under the concentrationtime curve, half-life, and bioavailability.33 For LMWHa biphasic and longer elimination was measured for theantifactor Xa and AT activity. The half-life of the an-tifactor Xa activity of LMWH was longer than that ofheparin, in accordance with the decreased molecularmass. The results may also reflect more rapid clearanceof longer chain molecules with antifactor Xa activity inrat, as suggested previously. The clearance of the AT ac-tivity of LMWH was smaller than that of the antifactorXa activity, which may result from the differences in thebinding to albumin and the fact that the AT doses dif-fered between the heparins administered.
After bolus injection of the endpoint-attachedmodified LMWH, the antifactor Xa profile showedsimilar elimination phases compared with LMWH.The half-lives of the AT activity, however, were alike forall LMWH preparations. The area under the antifactorXa activity-time curve of endpoint-attached heparinsincreased, the clearances decreased, and the half-lifevalues for the antifactor Xa activity were prolonged.This might be explained by a blocking of the scavengerreceptor in the liver by lipophilically labeled heparins.When calculated from the AT activity, the eliminationdid not differ between LMWH and the endpoint-attached heparins. This finding is in accordance withdata from the literature.
The relative bioavailability allows comparison ofthe pharmacokinetic profile of an agent with that ofa reference or parent substance.34 Thus, LMWH wascompared with LMWH-T and LMWH-t,F. LMWH-T and LMWH displayed equivalent relative bioavail-ability of the antifactor Xa and AT activity. LMWH-T,F showed twofold higher bioavailability of antifactorXa activity and similar bioavailability of AT activitycompared with the parent compound. These results in-dicate that antifactor Xa activity might be liberated invivo, which cannot be measured by in vitro test systems.These results may also be correlated with the liberationof endogenous “heparin-like” substances.35 Another ex-planation for the increased pharmacological response ofthe ex vivo antifactor Xa activity is stronger binding toAT. However, the present data suggest that the bindingof modified LMWHs to albumin may also contributeto the enhanced antifactor Xa activity.
The measurement of the pharmacokinetics ofGAGs depends on labeling these materials with eitherradioactivity or fluorescence. Labeling with fluores-cence did not alter the biological activity of heparan sul-fates.36 The measurement of the concentration of fluo-rescent-labeled heparins by the emission of fluorescencein aqueous solution and in plasma enables comparison
TYRAMINE AND FLUORESCENT LABELED GLYCOSAMINOGLYCANS/HARENBERG ET AL 353
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of the pharmacokinetic and pharmacodynamic proper-ties of LMWH. The concentration of LMWH-T,F inplasma and the pharmacokinetic profile after intra-venous administration were measured by RP-HPLCcombined with a fluorescence detector. The Cmax,AUC, and the half-lives were comparable to those ofthe antifactor Xa activity of LMWH-T,F. The clearanceand distribution volume were similar to those of theparent agent. Thus, the pharmacokinetic data of end-point-attached fluorescent LMW-heparin supports theassumption that the long-lasting antifactor Xa activityis mainly due to unspecific binding to plasma proteinsor to AT. So far, this explanation seems to be more likelythan that of the release of endogenous substances withantifactor Xa activity.
Several studies have shown that LMWH is avaluable anticoagulant and antithrombotic drug withhigher bioavailability than heparin. The answer to thesefindings is not yet fully explained. The endpoint-attached heparins may be helpful in solving some aspectsof the pharmacodynamic and pharmacokinetic differ-ences between heparin and LMWH and their interac-tions with the endothelium, blood cells, and plasma pro-teins. They might also be of therapeutic interest becauseof their improved anticoagulant properties.
Nonanticoagulant activities of heparins havebeen reemphasized37 on the basis of reports of a reduc-tion of exercise-induced asthma with inhaled heparin.38
The mechanisms of action of this effect are unknown sofar. Heparin is assumed to be phagocytized by mastcells, and this releases endogenous heparin. Direct inhi-bition of the stimulation of the complement system orthe leukotriene pathway is also discussed. When admin-istered by inhalation, heparin comes in direct contactwith cell surfaces of the bronchoalveolar system and ofmast cells. However, binding of heparin to these cellshas not been demonstrated. The effect of systemicallyadministered heparin on inflammatory and other anti-coagulant-related properties of heparin are being inves-tigated. In this respect, the present study demonstratesfor the first time that heparin, low-molecular-mass hep-arin, and the pentasaccharide as well as the fluorescent-labeled modified low-molecular-mass heparin are boundto lymphocytes, monocytes, and granulocytes.
The clinical relevance of lymphocytes, mono-cytes, and granulocytes in the pathogenesis of autoim-mune diseases and the development of arteriosclerosisand inflammation is well documented. The beneficialeffects of these cells in arteriosclerosis, inflammatory,and autoimmune diseases are well documented. Theconsecutive healing process leads to the formation ofcollagen and fibrin fibers and tissue repair. These pro-cesses destroy the original cell system and lead to sec-ondary destruction of the organ. Inhibition of theseprocesses may be beneficial. It can be speculated fromthe present results that heparin may be beneficial in
these senses by inhibiting the fibrous repair and pro-moting angiogenesis.39
Equimolar amounts of unfractionated heparinand LMW-heparin were required to displace theLMWH-T,F from the surface of leukocytes. This indi-cated specific binding of the negatively charged poly-saccharide backbone to positively charged groups on thesurface of leukocytes. Alternatively binding may alsohave been mediated by the lipophilic fluorescein-5-isothiocyanate bound to LMWH-T,F. However, thesedata clearly indicate that the lipophilic substitute doesnot influence the binding of the synthesized LMWH tothe surface of leukocytes.
Dermatan sulfate has a lower degree of sulfationthan heparin. Much higher amounts of dermatan sul-fate are needed to displace LMWH-T,F from leuko-cytes. To interpret this finding, it must be consideredthat FITC-labeled dermatan sulfate binds about 100-fold less to leukocytes than LMWH-T,F (data notshown). Therefore, it can be concluded from these re-sults that GAGs with a low degree of sulfation interactless with the binding sites on the surface of leukocytes.
The rather low rate of displacement of LMWH-T,F by the synthetic pentasaccharide may be explainedin two ways. First, the lower molecular mass comparedwith heparin results in a higher number of cellular bind-ing sites. Second, the molecular structure of the bindingsites is different from that of the AT binding sites of thepentasaccharide. Therefore, the positively charged pep-tide sequences differ from those of the AT binding sitefor the pentasaccharide. Further studies are under wayto identify the sequences of binding of GAGs to the cellsurfaces of lymphocytes, monocytes, and granulocytes.
ACKNOWLEDGMENTSThis study was supported by the Deutsche Forschungs-gemeinschaft (DFG), grant Ha 1164/3-1 and 3-2,Fakultät für Klinische Medizin Mannheim, D. HoppStiftung, Rosiny Stiftung, Ministerium für Wisssen-schaft und Kunst, Baden-Württemberg, Germany.
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