Available online at www.sciencedirect.com

(2007) 1272–1276

Clinical Biochemistry 40

Effects of anticoagulants on the activity of gelatinases

Massimiliano Castellazzi a,⁎, Carmine Tamborino a, Enrico Fainardi b, Maria C. Manfrinato c,Enrico Granieri a, Franco Dallocchio c, Tiziana Bellini c

a Department of Medical and Surgical Sciences of the Communication and Behaviour, Section of Neurology, University of Ferrara,Corso della Giovecca 203, Ferrara I-44100, Italy

b Department of Neurosciences, Section of Neuroradiology, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna,Corso della Giovecca 203, Ferrara I-44100, Italy

c Department of Biochemistry and Molecular Biology, Section of Biochemistry and Clinical Biochemistry, University of Ferrara,Via Luigi Borsari 46, Ferrara I-44100, Italy

Received 31 January 2007; received in revised form 16 July 2007; accepted 9 August 2007Available online 24 August 2007

Abstract

Objectives: To identify the best procedure for preanalytical blood collection in the determination of matrix metalloproteinase (MMP)-2 and -9by testing the effects of anticoagulants on their activity.

Design and methods: Active forms of both gelatinases were measured by specific activity assay systems in serum, plasma EDTA, plasma-heparin and plasma-citrate obtained from 20 healthy volunteers, as well as in a pooled serum sample before and after anticoagulant treatment.

Results: Active MMP-2 and MMP-9 mean concentrations were similar in serum and in plasma-citrate, higher in plasma EDTA than in serum,in plasma-heparin and in plasma-citrate, and lower in plasma-heparin than in serum and plasma-citrate. A similar trend was observed in untreatedand treated pooled serum samples.

Conclusions: Our results indicate that MMP-2 and MMP-9 in their active forms are not released by platelets during blood clotting, whereas theuse of calcium chelating anticoagulants can profoundly alter the activity of endogenous gelatinases. This suggests that the determination of activeforms of MMP-2 and MMP-9 in serum samples represents a suitable procedure.© 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Keywords: Metalloproteinases; Activity assay; Anticoagulants

Introduction

Matrix metalloproteinases (MMPs) are the major group ofenzymes that regulate the extracellular matrix (ECM) composi-tion and turnover [1]. Twenty-three zinc-dependent endopepti-dases have been identified in humans and included in thisfamily of enzymes [2]. Most MMPs are secreted as zymogensthat require activation in order to cleave their substrates.Extracellular activation is a complex mechanism involving thedisruption of the interaction between the conserved cysteine ofthe prodomain and the zinc from the catalytic site [3].

⁎ Corresponding author. Fax: +39 532 205525.E-mail address: massimiliano.castellazzi@unife.it (M. Castellazzi).

0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemistsdoi:10.1016/j.clinbiochem.2007.08.011

There is emerging evidence indicating the role of theseenzymes in normal and pathological processes, includingembryogenesis, wound healing, inflammation, arthritis, cardi-ovascular diseases, cancer and neurological diseases [2,4].Based on these observations, some MMPs, such as MMP-2 andMMP-9 (also called Gelatinases-A and -B, respectively), havebeen proposed as relevant biomarkers in some pathologicalconditions like autoimmune diseases [5], cancer [6,7] and brainischemia [8].

Nevertheless, controversial results have been reported whendifferent preanalytical conditions used in sample collection andstorage before MMP-2 and MMP-9 measurements wereevaluated. In some studies, the best specimens to optimize theanalysis of circulating gelatinases were plasma samplesprepared in heparin treated tubes [9–11], whereas in others

. Published by Elsevier Inc. All rights reserved.

1273M. Castellazzi et al. / Clinical Biochemistry 40 (2007) 1272–1276

they were plasma samples collected in tubes with sodium citrate[12–14]. Moreover, similar findings were recently observed forlevels of these MMPs after blood collection in tubes withEDTA, citrate and heparin, suggesting that plasma is superior toserum in MMP analysis [15]. In fact, the release of pro-MMP-2[16] and pro-MMP-9 [17] contained within platelets duringclotting could enhance their concentrations in serum samples.However, at present, there is no experimental evidence to provea platelet shedding of active MMPs. In addition, in all previousstudies gelatinase amounts were detected by quantitativeELISA technique and qualitative/semiquantitative zymographywhich are not able to quantify only the active forms of MMPs[18]. Thus, as catalytic activity is exerted only by enzymes inactive form, the influence of preanalytical conditions in mea-surements of serum levels of active gelatinases still remains tobe clarified. The identification of the best procedure forpreanalytical blood collection in the determination of gelatinaseactivity is crucial for diagnostic and research purposes due tothe potential involvement of proteolytic activity of theseenzymes in regulating several physiological and pathologicalreactions [18].

In a recent paper [19], we employed an activity assay toassess serum and cerebrospinal fluid concentrations of activeMMP-9 in patients affected by multiple sclerosis and otherneurological diseases. This technique is based on a previouslydeveloped method [18,20] using a modified pro-Urokinase asan artificial substrate to measure MMP activity in a simplecolorimetric assay. Without the addition of chemical activators,like organo-mercurials, for example, this assay only dosesendogenously active MMPs present in the analyzed samples,and not the zymogen forms.

In the light of these considerations, the aim of this study wasto clarify the influence of anticoagulants on gelatinase activity.To achieve this, we evaluated the concentrations of active formsof both MMP-2 and MMP-9 in serum and in plasma samplesobtained by using common anticoagulants for blood specimencollection. In addition, we investigated the effects of theseanticoagulants on the endogenous active forms of gelatinases ina pooled serum specimen.

Materials and methods

Blood specimens collection and storage

Venous blood samples from 20 healthy volunteers (8 femaleand 12 male; mean age±SD=30.7±6.4 years) were simulta-neously collected in different commercially availableVacutainerTM tubes and centrifuged within 30–60 min aftervenipuncture at 3000 rpm for 12 min at room temperature.Informed consent was given by all volunteers before inclusionand the study design was approved by the Local Committeefor Medical Ethics in Research. We obtained pure serum fromblood collected in tubes without additive, and plasma EDTA,plasma-heparin and plasma-citrate from blood collected intubes coated with dipotassium EDTA, lithium heparin andsodium citrate, respectively. After removal of the surnatants,serum and plasma samples were aliquoted and stored at

−70 °C until assay. Each sample was analyzed in duplicate inthe same session. In addition, to verify the actual effects ofanticoagulants on the activity of endogenous serum activegelatinases, a pooled serum sample, obtained by mixing dif-ferent sera from the healthy donors, was tested before andafter anticoagulant treatment which consisted of the additionof the pooled serum sample to the tubes, used for bloodsampling, containing EDTA, heparin and sodium citrate,respectively. After this preparation, untreated and treatedpooled serum samples were vortexed, aliquoted and frozen at−70 °C within a few minutes, stored under the same con-ditions as the serum and plasma samples, and then separatelyassayed in triplicate.

MMP-2 activity assay

Active forms of MMP-2 were measured in all the samplesusing a commercially available activity assay system (ActivityAssay System, Biotrak, Amersham Biosciences, UK; cod.RPN2631) following the manufacturer's instructions. All thereagents and standards were included in the kits. Briefly, weapplied 100 μL/well of each sample in duplicate into 96microwells microtiter plates precoated with anti-MMP-2antibody. Serum, plasma and anticoagulant treated-serumsamples, diluted to 1:100, were utilized. On each plate, wedispensed six serial dilutions of standard in duplicate with arange of 0.19–6 ng/mL, and dilution buffer in two wellsinstead of samples as negative control (blank). After anovernight incubation at 2–8 °C and four washing cycles,MMP-2 was bound to the wells, other components of thesample being removed by washing and aspiration. Thestandard is a human pro-MMP-2, which was activated byadding 50 μL/well of p-aminophenylmercuric acetate(APMA). To detect only endogenous levels of active MMP-2, 50 μL/well of dilution buffer was dispended into each wellinstead of APMA. At the same time, 50 μL of the detectionreagent was pipetted into each well. This detection reagentconsisted of detection enzyme and substrate. The detectionenzyme was the pro-form of a modified urokinase, an enzymethat can be activated by captured active MMP-2 into an activedetection enzyme, through a single proteolytic event. Thenatural activation sequence in the pro-detection enzyme wasreplaced using protein engineering, with an artificial sequencerecognized by specific MMP. Activated urokinase can then bemeasured using a specific chromogenic substrate (S-2444™).Active MMP-2 was detected through activation of themodified pro-detection enzyme and the subsequent cleavageof its chromogenic substrate. After 3 h incubation at 37 °C,color development was read at 405 nm in a microtiter platereader spectrophotometer (Microline Reader DV920, PoliIndustria Chimica, Milan, Italy). The amount of activeMMP-2 in all samples was determined by interpolation fromthe standard curve. According to the manufacturer's instruc-tions, the lower limit of quantification of the assay was0.19 ng/mL, the range of intra-assay coefficient of variations(CV) was 4.4–7.0%, whereas the range of inter-assay CV was16.9–18.5%.

1274 M. Castellazzi et al. / Clinical Biochemistry 40 (2007) 1272–1276

MMP-9 activity assay

Serum, plasma and anticoagulant treated-serum levels ofactive MMP-9 were determined as previously described [19]using a commercially available activity assay system (ActivityAssay System, Biotrak, Amersham Biosciences, UK; cod.RPN2634). All the samples were 1:20 diluted. Standard'sconcentration range was 0.125/4 ng/mL. As reported in themanufacturer's instructions, the lower limit of quantificationwas assumed at 0.125 ng/mL, the range of intra-assay CVwas 3.4–4.3%, whereas the range of inter-assay CV was20.2–21.7%.

Statistic analysis

The normality of each variable was checked by using theKolmogorov–Smirnov test. As normality of data distributionwas rejected in several variables, statistical analysis wasperformed by a non-parametric approach. Kruskal–Wallis testwas used to compare serum and plasma mean levels of activeMMP-2 and MMP-9 among the various groups examined. Ifsignificant differences were found, Mann–Whitney U-test wasthen used to compare mean values between two differentgroups. A value of pb0.05 was considered as statisticallysignificant.

Results

Active MMP-2 and MMP-9 concentrations in serum andplasma samples

Measurable levels of active MMP-2 and MMP-9 weredetected in 100% of samples analyzed. Mean levels of activeMMP-2 and MMP-9 were significantly different in the variousgroups (Kruskal–Wallis; pb0.0001). As indicated in Figs. 1Aand B, mean concentrations of active MMP-2 and MMP-9 were

Fig. 1. Active MMP-2 and MMP-9 mean levels in serum and plasma samples colleactive MMP-2 were higher in plasma-EDTA than in serum, in plasma-heparin and inthan in serum and in plasma-citrate (Mann–Whitney; ^pb0.0001). Panel B shows t(Mann–Whitney; ∗pb0.01), in plasma-heparin (Mann–Whitney; ^pb0.0001) and inthan in serum (Mann–Whitney; °pb0.05) and in plasma-citrate (Mann–Whitney; †

within the box indicates the median. The whiskers above and below the box corresp

higher in plasma-EDTA than in serum (pb0.0001 and pb0.01,respectively), in plasma-heparin (pb0.0001) and in plasma-citrate (pb0.0001 and pb0.01, respectively). In addition, activeMMP-2 and MMP-9 mean levels were lower in plasma-heparinthan in serum (pb0.0001 and pb0.05, respectively) and inplasma-citrate (pb0.0001 and pb0.01, respectively). Therewere no significant differences between serum and plasma-citrate for mean values of both active MMP-2 and MMP-9.

Concentrations of active MMP-2 and MMP-9 in pooled treatedserum

After the comparative analysis between serum and plasma, toprovide an indication of the effects of anticoagulants, not onlyin the preanalytical but also in the analytical phases, activeMMP-2 and MMP-9 levels were determined in a pooled serumsample before and after treatment with anticoagulants obtainedby adding it to the tubes containing EDTA, heparin and sodiumcitrate. As shown in the Table 1, when changes in MMP-2 andMMP-9 activity after treatment with anticoagulants wereexpressed as percentage of values measured in untreatedserum, active MMP-2 and MMP-9 levels increased anddecreased following EDTA and heparin treatments, respec-tively. Differences in active MMP-2 and MMP-9 concentrationsbetween citrate treated and untreated sera were small with atrend toward a decrease in active MMP-2 and a tendency towardan increase in active MMP-9.

Discussion

In this study, we investigated, for the first time, active MMP-2 and MMP-9 concentrations in serum and in plasma samplestreated with widely used anticoagulants in order to identify thebest procedure for blood specimen collection. For this purpose,we employed specific activity assay systems [18] whichallowed us to detect only the bioactive forms of these enzymes

cted from 20 healthy volunteers. Panel A indicates that mean concentrations ofplasma-citrate (Mann–Whitney; ∗pb0.0001), and were lower in plasma-heparinhat mean values of active MMP-9 were higher in plasma-EDTA than in serumplasma-citrate (Mann–Whitney; ∗pb0.01), and were lower in plasma-heparin

pb0.01). The boundaries of the box represent the 25th–75th quartile. The lineond to the highest and lowest values, excluding outliers.

Table 1Active MMP-2 and MMP-9 concentrations (mean±standard deviation, range) in pooled serum untreated and treated with various anticoagulants

MMPs Pooled serum

Untreated EDTA-treated Heparin-treated Citrate-treated

(ng/mL) (ng/mL) Change (%) (ng/mL) Change (%) (ng/mL) Change (%)

Active MMP-2 142.7±9.3, 132.9−151.4 1056.0±87.5, 957−1123 +640.2 69.3±6.7, 62.2−75.4 −51.4 122.6±7.2, 116.4−130.5 −14.0Active MMP-9 3.49±0.15, 3.39−3.36 8.75±0.65, 8.19−9.44 +150.6 1.31±0.12, 1.21−1.44 −62.6 3.86±0.26, 3.58−4.10 +10.4

Each determination was performed in triplicate. Changes in active MMP-2 and MMP-9 mean values after treatment with anticoagulants were expressed as percentageof those measured in untreated serum.

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exerting catalytic activity, and not total amounts of gelatinases,as measured in previous studies [9–15].

Our principal finding was that measurements of activeMMP-2 and MMP-9 in serum and plasma-citrate samples weresubstantially equivalent. These data were concordant with ourrecent preliminary findings obtained with the same procedure[22] but differed from some studies performed with othertechniques, such as quantitative ELISA technique and qualita-tive/semiquantitative zymography [12–14,21]. These resultssuggest that platelet activation and the subsequent release ofMMPs do not influence levels of active enzyme in serumsamples since, as previously demonstrated [16,17], duringclotting, MMPs are not secreted in active forms but only aszymogens.

Another relevant observation was that plasma-EDTApromoted a strong increase in MMP-2 and MMP-9 activityin comparison to serum, plasma-heparin and plasma-citrate.This finding is difficult to interpret. However, it is tempting tospeculate that this increment could be ascribed to the zinc-chelating properties of EDTA [23]. In fact, the odd cysteinelocated in the propeptide region forms an intramolecularcomplex with the zinc positioned in the catalytic domainblocking off the active site [24]. The removal of zinc atom byEDTA could result in the displacement of the odd cysteineproducing a partial unfolding of the propeptide. A potentialconsequence of the dissociation of this cysteine may be thegeneration of zymogens which are able to exert proteolyticactivity when zinc and calcium ions are rehabilitated.Although the chelating action of EDTA leads to an inhibitionof catalytic activity by subtraction of the zinc atom present inthe active site [25], the presence of the zinc atoms in the assaybuffer could contribute to the incorrect refolding of the pro-peptide and the appearance of proteolytic activity of zymo-gens. Such a phenomenon might also be seen in zymographywhere, after the denaturation of proteins and chelation of thezinc atoms produced by the presence of sodium dodecylsulfate (SDS) [23], the removal of SDS and the addition ofzinc and calcium ions during renaturing step trigger theactivation of both pro-MMP-2 and pro-MMP-9. Therefore,since EDTA shares the same zinc-chelating properties as SDS,and zinc atoms were present in our assay buffer, a misfoldingof the propeptide may be a possible explanation for theenhancement in MMP-2 and MMP-9 activity observed inplasma-EDTA. Further studies are warranted to verify thereliability of this hypothesis and the actual interactionsoccurring between EDTA and gelatinases.

The lower levels of both active MMP-2 and MMP-9 wefound in plasma-heparin compared to other samples argue for aninhibition role of this agent on MMPs. It is well-known thatheparin inhibits the induction of some MMPs at the transcrip-tional level [26], but the demonstration of its effects on theactivity of these enzymes once they are secreted is currentlylacking.

Data coming from untreated and treated pooled sera sampleslargely reproduced those obtained with the comparative analysisbetween serum and plasma. In particular, it was confirmed thatcitrate does not induce any significant alteration in gelatinaseactivity which is enhanced by EDTA and inhibited by heparin.Furthermore, these findings show that some anticoagulants,such as EDTA and heparin, influence the activity of MMP-2 andMMP-9, regardless of their release by platelets which occursduring the coagulation process, by exerting a direct effect on theendogenous forms of MMPs.

In summary, we found that measurements of active MMP-2and MMP-9 levels in serum and plasma-citrate samples arecomparable since gelatinases in active forms are not released byplatelets during clotting. In addition, we demonstrated that theuse of calcium chelating anticoagulants, such as EDTA andheparin, should be avoided in the analysis of circulating activeMMP-2 and MMP-9 because these molecules profoundlyinterfere with the activity of endogenous MMPs. Takentogether, these findings suggest that the use of serum sampleswould seem to represent a suitable procedure for preanalyticalblood collection in the determination of active MMP-2 andMMP-9. Our results may have important clinical implications.In fact, the detection of circulating MMP-2 and MMP-9 inactive forms is useful to directly evaluate the systemicproteolytic activity of these enzymes and to avoid misleadinginformation derived from the analysis of blood total MMPlevels. In addition, the employment of a commercially availabletechnique makes these measurements rapid, readily accessibleand easily feasible. Therefore, the quantification of serum activeMMP-2 and MMP-9 concentrations by means of activity assaysystems can be considered a promising tool to clarify whethergelatinases effectively modulate and can be used as biomarkersfor monitoring regenerative and inflammatory responses whichtake place in normal and pathological conditions [2,4]. Thesame method could also be utilized for measuring MMP-2 andMMP-9 activity in some compartmentalized body fluids such assynovial and cerebrospinal fluids [19,20].

Future studies in a larger number of samples are needed toestablish the consistency of these observations.

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Acknowledgments

The study was supported by Fondazione Cassa di Risparmiodi Ferrara, Italy. The authors thank Dr. Elizabeth Jenkins forhelpful corrections on the manuscript.

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