Heparin affects matrix metalloproteinases and tissue inhibitors of metalloproteinases circulating in peripheral blood

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Clinical Biochemistry 41 (2008) 1466 – 1473

Heparin affects matrix metalloproteinases and tissue inhibitors of metalloproteinases circulating in peripheral blood Ferdinando Mannello a,⁎, Klaus Jung b,c , Gaetana A. Tonti a , Franco Canestrari a a

b

Department of Biomolecular Sciences, Section of Clinical Biochemistry, University Carlo Bo, Urbino, Italy Department of Urology, Research Division, University Hospital Charité, Humboldt University, Berlin, Germany c Berlin Institute for Urologic Research, Berlin, Germany Received 6 May 2008; received in revised form 4 September 2008; accepted 5 September 2008 Available online 2 October 2008

Abstract Objectives: Blood sampling/handling alters matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) expression. The aim of this study is to evaluate the effects of high molecular weight heparin on MMP and TIMP expression in blood. Design and methods: We analyzed by gelatin zymography and ELISA assays the effects of different heparin salts, dose- and time-dependence of MMP and TIMP concentrations in plasma and sera collected with and without clot-accelerator in plastic tubes from 50 healthy donors. Results: The levels and zymography of MMP-2 did not show significant changes among all samples, and during time- and dose-dependent heparin treatments. MMP-9 and TIMP-2 expression were strongly affected by heparin, with significant increase of their content and gelatinolytic activity both in time- and in dose-dependent fashion. Addition of heparin allowed also the displacement of MMP-2 prodomain, favouring zymogen activation. Conclusions: Heparin has direct and indirect effects, altering MMP/TIMP complexes circulating in blood, and increasing the release of TIMP-2. To avoid misinterpretations due to MMP/TIMP complex alteration and MMP prodomain displacement, heparin should be cautiously used in blood collection procedures. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Matrix metalloproteinases; Tissue inhibitors of metalloproteinases; Peripheral blood; Serum; Plasma; Heparin; Preanalytical pitfalls

Introduction Matrix metalloproteinases (MMP) and Tissue inhibitors of metalloproteinases (TIMP) play key roles in the remodelling of extracellular matrix and cell signaling activities [1–4]. The expression of MMP and their activity are finely regulated by TIMPs [4]. Overexpression of numerous MMP in several cancer types has been found and their inhibition are currently being investigated in clinical studies and trials [5,6]. Following secretion Abbreviations: MMP, matrix metalloproteinases; TIMP, tissue inhibitors of metalloproteinases; APMA, p-amino phenyl-mercuric acetate; SDS, sodium dodecylsulphate; ELISA, enzyme-linked immunosorbent assay; LH, lithium heparin. ⁎ Corresponding author. Sezione di Biochimica Clinica, Dipartimento di Scienze Biomolecolari, Università “Carlo Bo”, Via O. Ubaldini 7, 61029 Urbino (PU), Italy. Fax: +39 0722 322370. E-mail address: [email protected] (F. Mannello).

into intercellular and extracellular space, several MMP and TIMP leak into the blood [7]; on these basis, plasma and serum concentrations of different MMP/TIMP have been analysed both in physiological and pathological conditions, as potential and accurate biological markers of several processes [8,9]. Among the twenty-three zinc-dependent endopeptidases belonging to the MMP family [2], MMP-2 (Gelatinase A, EC 3.4.24.24, 72 kDa) and MMP-9 (Gelatinase B, EC 3.4.24.35, of 92, 130 and 225 kDa) have been proposed as relevant biomarkers in several neoplastic conditions [10]. The balance between the proteolytic activity of MMP-2 and MMP-9 and the gelatinolytic inhibitory functions of TIMP-1 and TIMP-2 has been recognized as crucial determinant of the switch from physiologic tissue remodelling to neoplastic tissue transformation [11]. Gelatin zymography (for both gelatinases, MMP-2 and -9) and ELISA assay are the most commonly used tests to assess circulating MMP and TIMP levels. The evaluation of MMP and

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TIMP levels and zymographic profile in peripheral blood and biological fluids may be a diagnostic tool to characterize physio-pathological processes in healthy and diseased tissues [6,7]. However, the best source of MMP and TIMP between plasma and serum is a matter of recent debate that shows how the discrepancies between serum and plasma may be due to preanalytical artifacts, finally leading to pitfalls and misinterpretations [12,13]. While MMP-2 shows no significant difference between plasma and serum, concentrations and zymographic patterns of MMP-9 forms are strongly affected by anticoagulants and sample handling/processing [as reviewed in 13]. Serum was found to contain higher concentrations of MMP-9 and TIMPs than plasma, suggesting a possible release of both MMP and TIMP from leukocytes and platelets during both coagulation and fibrinolysis. Thus, plasma should be preferred in determining circulating MMP and TIMP levels as a reflection of extracellular matrix remodelling in tissue, even though preanalytical determinants during sample collections lead to controversial results due to the use of different anticoagulants (e.g., EDTA, heparin and citrate) [14–31]. Among the most common anticoagulants, heparin (a highlysulphated glycosaminoglycan) is widely used as an injectable anticoagulant with the highest negative charge density of any known biological molecule [32]. It can also be used to form an

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inner anticoagulant surface on various experimental and medical devices (such as blood collection tubes and renal dialysis machines). Although used principally in medicine for anticoagulation, the true physiological role in the body remains unclear, because blood anticoagulation is achieved mostly by endothelial cell-derived heparan sulphate proteoglycans [33]. There is a paucity of information about the effects of heparin on MMP-2, MMP-9, TIMP-1 and TIMP-2 circulating in peripheral blood and about the reason of the controversial results obtained by heparin [13]. Therefore, the aim of the present study was to evaluate through zymography and ELISA assays and using different approaches the preanalytical and direct analytical effects of heparin on MMP-2, MMP-9, TIMP-1 and TIMP-2 circulating in peripheral blood, in order to hypothesize a possible mechanism of heparin action. Materials and methods Sample collection and preparation We collected blood samples from 50 healthy volunteers (25 female and 25 male, median age 37 years; range 25–58 years). Serum samples (S) were obtained in plastic tubes with no additives. Sera obtained with coagulation accelerators (S+CA)

Fig. 1. ELISA quantitative evaluation of circulating gelatinase forms in serum and plasma in dependence of sample processing. The MMP-2 (A) and MMP-9 (C) concentrations were analysed in 50 healthy blood samples, (25 female and 25 male volunteers, median age 37 years; range 25–58 years). Serum samples (S) were obtained in plastic tubes with no additives; sera obtained with coagulation accelerators (S+CA) were collected in kaolin-coating plastic devices. Plasma samples were obtained in plastic tubes coated with lithium heparin (LH). All plasma and serum collection tubes were Monovette® from Sarstedt. All tubes were either centrifuged immediately at 1500 g for 12 min at 4 °C (considered as t0), after 0.5 and 2 h storage at room temperature (t0.5 and t2, respectively), and after 24 h storage at 4 °C (t24), and assayed for MMP-2 and MMP-9 concentrations in all samples (B and D, respectively) (empty: t0; vertical lines: t0.5; horizontal lines: t2; grey: t24). Values are given as arithmetic mean ± SD; the concentrations of gelatinases are expressed as μg/L, whereas the time-dependent MMP changes are expressed as percentage in respect of the initial value (t0). While in (B) all data show no significant differences, in (D) the time-dependent increase of MMP-9 of all conditions showed a significant difference respect to the initial t0 values (p b .0.0001).

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Fig. 2. Gelatin zymogram of serum obtained without and with clot activator and heparinized plasma. Peripheral blood samples were collected in empty plastic tubes (S), in plastic kaolin-coated device (S+CA) and tubes containing lithium heparin (LH). After sample centrifugation at 1500 g for 12 min at 4 °C, aliquots containing 150 μg of total protein were analyzed on 7.5% polyacrylamide gels containing 2 g/L gelatin 90 Bloom type A from porcine skin. Gelatinolytic bands were directly evaluated by densitometry using the Image Pro-Plus software. The gelatinase calibrators (lane Std) were generated from capillary whole-blood; proMMP-2 (72 kDa) and MMP-9 pro- and complexed forms (92, 130 and 225 kDa) were identified. Serum obtained with (S+CA) and without clot accelerator (S) were separated in lanes 1 and 2, respectively. Blood gelatinases circulating in LH plasma were visualized in lane 3. Molecular masses of proMMP-2 (72 kDa) and pro-form (92 kDa) and complexed forms (130 and 225 kDa) of MMP-9 are indicated. The zymogram is representative for the results obtained in all samples examined (n = 50).

were collected in kaolin-coated plastic devices. Plasma samples were obtained in plastic tubes coated with lithium heparin (LH). All plasma and serum collection tubes were Monovette® from Sarstedt [34]. The tubes were centrifuged immediately at 1500 g for 12 min at 4 °C (considered as t0), after 0.5 and 2 h storage at room temperature (t0.5 and t2, respectively), and after 24 h storage at 4 °C (t24). The samples were stored at − 80 °C until analysis and analysed immediately after thawing. To study the effects of heparin before blood sampling processes, we added increasing amounts of heparin into S, S+CA and LH tubes prior to blood collection. Thus, blood was collected into: a) serum tubes without additives, b) plastic serum tubes supplied with increasing heparin, c) plastic kaolincoated tubes without and with additionally supplied increasing heparin, and d) plastic devices already containing LH and additionally supplied with increasing amount of heparin. To study the effects of heparin after blood collection, we added increasing amounts of heparin to previously separated serum, serum plus clot activators and heparinized plasma. To test the effects of heparin on MMP, we evaluated three different heparin salts (lithium, sodium and ammonium heparin) (Sigma), at increasing concentrations. MMP-2, MMP-9, TIMP-1 and TIMP-2 ELISA assays All specimens of plasma and serum with and without added lithium heparin were analyzed for MMP-2 and MMP-9 quantitative Biotrak™ assays (RPN 2614 and 2617, respectively; Amersham-Pharmacia) [16,20]. These assays recognize the progelatinase forms with the detection limit of 0.37 and 0.25 μg/L for MMP-2 and -9, respectively. No cross-reactions

occur with other MMP and the assays show cross-reactivity for MMP-tissue inhibitor of metalloproteinase complexes. The within- and between-assay precision (as % coefficient of variation) for duplicate determinations were 3.5% and 4.7%, respectively. TIMPs were measured with the Biotrak™ TIMP-1 and TIMP2 ELISA assays (RPN 2611 and 2618, respectively; AmershamPharmacia) according to the manufacturer's instructions [21,27]. The TIMP-1 assay recognizes total human TIMP-1 (i.e., fully cross-react with free TIMP-1 and that complexed with pro and active MMP-9) and does not show cross-reactions with TIMP-2. The TIMP-2 assay recognizes free TIMP-2 and that complexed with the active forms of MMP-2, but not TIMP-2 complexed with the zymogen precursor of MMP-2 (proMMP-2). The reproducibility of assays (%CV) for duplicate determinations were calculated (6.5% and 10.1% for within- and between assay, respectively). Gelatin zymography Aliquots of all plasma and serum samples (containing 150 μg of total protein) were analysed by gelatin zymography carried out on 7.5% polyacrylamide gels copolymerized with 2 g/L 90 Bloom Type A gelatin from porcine skin (from Sigma) [19]. After electrophoresis, gels were washed in Triton X-100 (25 mL/L) and incubated for 24 h at 37 °C in enzyme incubation buffer (containing 50 mM Tris–HCl, pH 7.5; 5 mM CaCl2; 100 mM NaCl; 1 mM ZnCl2; 0.3 mM NaN3, 0.2 g/L of Brij®-35; and 2.5 mL/L of Triton X-100). Zymograms were incubated in the presence of 5 mM EDTA and 2 mM 1,10-phenanthroline (o-Phe) for inhibition studies and with 2 mM p-amino phenyl-mercuric acetate (APMA) for the activation of zymogens [20]. Staining was performed in Coomassie brilliant blue R-250 (2 g/L), and gels were destained in methanol-acetic acid standard solution. Clear gelatinolytic bands on uniform blue background were densitometrically measured with the image analyzer Image Pro-Plus (from Cybernetic). Gelatinase calibrators prepared by diluting healthy capillary blood with 15 volumes of nonreducing Laemmli sample buffer [12] were used as previously described [14]. In whole capillary blood, used as calibrator [14], we identified proMMP-2 at 72 kDa, proMMP-9 and MMP-9 complexed forms at 92, 130 and 225 kDa (Fig. 2, lane Std), which were recognized by monoclonal anti-MMP-9 and anti-MMP-2 antibodies and characterized as latent pro-enzymes, activated by APMA and inhibited by both calcium and zinc chelators (data not shown). Statistical analysis Differences in the densitometric analyses of gelatinolytic activity in zymograms and between the mean of MMPs among all samples were determined by Student paired t-test, using Prism 3 software (GraphPad). pb 0.05 was considered statistically significant. Results Using ELISA quantitative assay we found that the concentrations of MMP-2 detected in serum, serum plus clot-activators

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and heparinized plasma (collected in empty, kaolin-coated and LH tubes, respectively) did not show significant differences among samples (Fig. 1A). Moreover, increasing the time between sampling and centrifugation did not significantly alter MMP-2 levels in all serum and plasma samples (Fig. 1B). In sharp contrast to MMP-2, the concentrations of immunoreactive MMP-9 were significantly higher in serum obtained with clot accelerator than in LH plasma and pure serum (Fig. 1C) (p b 0.001); moreover, LH plasma showed the lowest concentrations of MMP-9. In all samples examined, MMP-9 levels increased with the time between sampling and centrifugation (Fig. 1D) (p b 0.0001 in all conditions). These changes were also confirmed by gelatin zymography, showing negligible differences in the gelatinolytic band of MMP-2 and more intense gelatinolytic bands of all MMP-9 forms in serum obtained with clot accelerators in respect to serum and heparinized plasma (Fig. 2). TIMP-1 concentrations were significantly higher in serum collected with clot-accelerators than in pure serum and LH plasma (Fig. 3A) (p b 0.01 and p b 0.001, respectively); LH plasma showed the lowest circulating concentrations of TIMP-1. These results were unaffected increasing the time of processing between sampling and centrifugation (Fig. 3B). On the contrary, heparinized plasma contained the highest levels of TIMP-2,

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significantly higher than those found in serum with and without clot accelerators (Fig. 3C) (p b 0.001); however, no difference was detected between serum with or without clot accelerator. In all samples examined, a significant time-dependent increase of TIMP-2 concentrations was found (Fig. 3D); in particular, heparinized plasma samples showed the highest rates of TIMP-2 increase according to the increasing time between sampling and centrifugation (p b 0.0001). In order to find an explanation of the effects of heparin on MMP and TIMP circulating in blood, we evaluated if different heparin salts and increasing amounts of heparin added to serum and plasma samples (before and after blood collection) may alter the gelatinolytic activity/zymographic pattern of MMP and the concentrations of MMP and TIMP measured by ELISA. Among the high molecular weight heparins (lithium, sodium and ammonium salts), in all conditions tested (time- and dosedependence, before and after blood collection) we did not find any significant difference in both MMP and TIMP levels, as well as in MMP-2 and MMP-9 gelatinolytic activity (data not shown). For this reason, we report all the data obtained using lithium heparin, the most widely used anticoagulant present in blood collection devices. Either increasing the amounts of heparin to plastic tube collection devices (from 1.5 to 30 IU/mL) before the addition of

Fig. 3. Circulating TIMP forms and their time-dependence in plasma, serum with and without clot accelerators. The TIMP-1 (A) and TIMP-2 (C) concentrations were analysed in 50 healthy blood samples. Serum samples (S) were obtained in plastic tubes with no additives; sera obtained with coagulation accelerators (S+CA) were collected in kaolin-coating plastic devices. Plasma samples were obtained in plastic tubes coated with lithium heparin (LH), all collected in tubes Monovette® from Sarstedt. The samples were analysed after centrifugation at 1500 g for 12 min at 4 °C (considered as t0), after 0.5 and 2 h storage at room temperature (t0.5 and t2, respectively) and after 24 h storage at 4 °C (t24), and assayed for TIMP-1 and -2 concentrations (B and D, respectively) (empty: t0; vertical lines: t0.5; horizontal lines: t2; grey: t24). The total amounts of TIMPs are expressed as μg/L (Mean ± SD), whereas the time-dependent TIMP levels are expressed as percentage respect to initial value (t0). While in (B) all data show no significant differences, in (D) the time-dependent increase of TIMP-2 of all conditions showed a significant difference respect to the initial t0 values (p b .0.001).

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Fig. 4. The influence of heparin on MMP-9 and TIMP-2 concentrations in peripheral blood (n = 50). The MMP-9 and TIMP-2 levels (A and B, respectively) in serum without and with clot accelerators, and in heparinized plasma were measured after the addition of different concentrations of lithium heparin. (empty: serum without clot accelerator; grey: serum collected with clot activator; black: LH plasma) Data are given as percentages of the arithmetic means ± standard deviation of the samples without heparin, indicated as 100%.

peripheral blood, or adding heparin after the centrifugation of serum and LH plasma, showed no significant variation when compared to serum or LH plasma obtained without heparin addition (no difference in gelatinolytic activity/zymographic pattern, in MMP-2 concentrations as well as for TIMP-1) (data not shown). On the contrary, the zymographic profile and the concentrations of MMP-9 by ELISA, as well as the concentrations of TIMP-2, were found strongly affected by heparin. The addition

of heparin to heparinized plasma samples caused a significant increase in MMP-9 levels with a greater extent to that occurred in all serum samples. In fact, as shown in Fig. 4A, increasing the amount of heparin leads to over two-fold increase of MMP-9 levels in LH plasma (p b 0.01); also in sera samples the increased amounts of heparin caused an increase of MMP-9 concentration, even if the differences did not reach a significance level (Fig. 4A). The two-fold increase of heparin added to serum (from 7 to 15 and 30 IU/mL) correlated with the evident increase in gelatinolytic activity of MMP-9 bands, as shown by gelatin zymography (Fig. 5A) and confirmed by ELISA (Fig. 4A). Interestingly, the addition in test tubes of increasing heparin amounts to all serum samples (independently of clot-activator presence) revealed a two-fold increase of TIMP-2 levels (Fig. 4B); moreover, we also found a highly significant increased TIMP-2 concentration (up-to 5-fold) in LH plasma (Fig. 4B). A significant positive correlation between heparin amounts and TIMP-2 levels was found (y = 108x − 34, r2 = 0.957, p b 0.0001). The increase of TIMP-2 concentrations in plasma was greater than that occurred in all serum samples (data not shown). Finally, in order to achieve informations about the possible interaction of heparin on the zymogen structure of gelatinases, we analysed the effect of lithium heparin before and after the APMA activation of circulating MMP. Heparin alone is not able to activate either MMP-2 or MMP-9 (Fig. 5B, lane 2) since the addition of 30 IU of heparin does not modify the electrophoretic migration of MMP-2 and -9, suggesting a lack of molecular weight modification of zymogens. After the treatment with APMA, zymogens of MMP-9 forms (but not MMP-2) were peculiarly activated (as shown in Fig. 5B, lane 3). However, after a combined treatment with APMA, heparin led to the unexpected activation of also MMP-2, as evidenced by its molecular weight lowering shift (Fig. 5B, lane 4). Discussion About twenty years ago, it was firstly described that peripheral blood contains several forms of MMP and TIMP (i.e., soluble constitutive form in plasma, and intracellular

Fig. 5. Modification of serum MMP zymographic profiles in dependence of heparin. (A) Serum samples collected without clot accelerator were centrifuged at 1500 g for 12 min at 4 °C; aliquots were treated with increasing amounts of heparin (7, 15 and 30 IU/mL in lanes 1, 2 and 3, respectively) for 30 min at 37 °C. (B) Serum obtained in plastic empty tubes were treated with 30 IU/mL heparin and 2 mM APMA alone or in combination for 1 h at 37 °C, and loaded as following: lane 1, serum with no treatment; lane 2, serum supplied with heparin; lane 3, serum treated with APMA; lane 4, serum pre-treated with heparin and then incubated with APMA. Representative zymograms were chosen to show the results obtained in all samples examined (n = 50). MMP calibrators from capillary whole blood (lane Std).

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zymogens in platelets and leukocytes) [7,35,36]. In particular, MMP-2 was constitutively found in plasma and defined as fibroblast-like gelatinase [35]; MMP-9 forms (225, 130 and 92 kDa) were characterized as gelatinolytic enzymes derived mainly from leukocytes and platelets [7,36], involved as effectors in the orchestration of leukocyte biology and platelet functions [37,38]. Moreover, TIMP-1 and TIMP-2 are intracellular proteins highly expressed in both leukocytes and platelets [37,38] that may be found circulating in plasma as high molecular weight MMP/TIMP complexes (preferentially, but not only, as MMP-2/TIMP-2 and MMP-9/TIMP-1 complexes) [7]. The discrepancy in MMP and TIMP concentrations between serum and plasma was for the first time reported about fifteen years ago [39] but, only starting from 1996 [14], the influence of blood sampling process on MMP and TIMP concentrations and zymographic profiles started to be carefully evaluated [as reviewed in 13]. While MMP-2 (activity and concentration) shows no significant difference between plasma and serum, levels and zymographic profile of MMP-1, MMP-8, MMP-9 forms and TIMP-1 are strongly affected by several anticoagulants [13,31]. It is well known that platelets and leukocytes contain several TIMP and MMP that may be extracellularly released upon activation or during aggregation [40–42]. The use of anticoagulants (e.g., citrate) limits the mobilization of gelatinase-rich granules of human neutrophils [42,43]; the use of high molecular weight heparin in blood collection devices has led to contrasting results [13]. Belonging to the highly-sulphated glycosaminoglycan family, high molecular weight heparins are widely used as anticoagulants [33], even though new unexpected functions of heparin are emerging and add to its known physiological role [44]. As suggested by our data, we can distinguish two main possible effects of heparin: a) “indirect effect” (pre-analytical phase, depending on anticoagulant effects on white blood cells), that accounts for the correlation between lower values of MMP-9 and TIMP-1 in LH plasma and their diminished release from leukocytes/platelets; in agreement, serum samples contained higher levels of both MMP-9 and TIMP-1 than in LH plasma. Moreover, the unchanged MMP-2 concentrations agree with its missing release from the blood cells. b) the “direct effect” (effects of heparin on enzyme/protein), that may account for the modified zymographic pattern (APMA activation) and concentrations of gelatinases and inhibitors obtained increasing the amounts of heparin in test tubes (detachment of TIMP protein from circulating MMP/TIMP complexes). For the first time we suggest that high molecular weight heparin differentially affects gelatinase measurements: it does not modify the blood concentrations of MMP-2 measured by ELISA and the MMP-2 zymographic profile (Figs. 1A and 5A, respectively). Contrary to what was observed for MMP-2, collection of MMP-9 in lithium heparin may have two possible effects: 1) the presence of heparin in test tube (preanalytical phase) limits the release of MMP-9 forms from platelets and leukocytes (indirect effect). In fact, LH plasma contained lower concentrations of MMP-9 respect to serum (Fig. 1C): when the time between sample collection and centrifugation was increased, MMP-9

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levels were the highest in serum whereas lithium heparin limited their release in plasma (Fig. 1D). 2) the treatment/addition of heparin after blood sample separation (post-analytical phase) increases the amount and activity of circulating MMP-9 (direct effect). In fact, the heparin treatment of previously separated plasma and serum samples increases both the gelatinolytic activity of the pro-MMP-9 forms (Fig. 5A) and the MMP-9 concentration (Fig. 4A), in a time- and dose-dependent manner. This direct effect of heparin is not linked to the release from leukocytes but probably due to the detachment of MMP-9 from circulating complexes. In agreement to the ability of heparin to interact with MMP protein, we also found that heparin in conjunction to APMA is able to activate the zymogen forms of MMP-2. In fact, the heparin treatment increases the gelatinolytic activity of the pro-MMP-9 form (Fig. 5A) but does not cause the detachment of the pro-domain of MMPs (Fig. 5B lane 2). Interestingly, while APMA alone is known to activate MMP9 zymogen but not pro-MMP-2, the combined heparin/APMA treatment results in the activation of pro-MMP-2 zymogen, as shown by the molecular weight shift of MMP-2 gelatinolytic bands (Fig. 5B lane 4). On this basis, we hypothesize that heparin may increase MMP-9 activity and may alter the displacement of the MMP-2 pro-domain. Starting from the evidence that MMP-2 circulating in blood is mainly complexed with TIMP-2 [5,6,13], we sought to determine the effect of high molecular weight heparin on TIMP-2 release from its complex circulating in blood. According to previous data [14–18,21,26,27] showing different concentration of TIMPs in plasma collected by several anticoagulants, here we found that TIMP-2 concentrations circulating in blood are strongly affected by heparin. In fact, LH plasma showed significantly higher levels of TIMP-2 respect to serum with and without clot accelerator (Fig. 3C). Moreover, by increasing the time between sample collection and centrifugation, TIMP-2 in LH plasma showed an increased concentration significantly higher respect to serum (Fig. 3D). In particular, the presence of heparin in plastic blood collection devices (30 IU/mL) shows a significant increase of TIMP-2 recovery in plasma with a direct time-dependent correlation (Y = 575x − 550, r2 = 0.989, p b 0.0001) (Fig. 3D). Moreover, we also revealed that increasing amounts of heparin lead to a significant enhancement of TIMP-2 release (both in free and complexed forms) from the circulating MMP/TIMP complex (Fig. 4B), showing a significant dose-dependent relationship (Y = 108x − 34, r2 = 0.957, p b 0.0001). In conclusion, on the basis of our ELISA quantitative and zymographic qualitative MMP and TIMP analyses and in agreement with previous evidences of the involvement of heparin in TIMP release [7,13–18,21,27,30], we demonstrate for the first time that high molecular weight heparin (without any differences among its different salts) does not affect the gelatinolytic activity and concentration of MMP-2 (as shown in Figs. 1A and 2). Moreover, we found that heparin may strongly limit the release of MMP-9 and TIMP-1 from leukocytes and platelets (as shown in Figs. 1C, 2 and 3A). On the other hand, in heparinized plasma we found the highest concentrations of TIMP-2 (as shown in Fig. 3C), released from white blood cells in a time- and dose-dependent fashion (as shown in Figs. 3D

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and 4B). Finally, we demonstrate that heparin is able to favour the detachment of the prodomain of MMP-2 from zymogen form, in conjunction to activating agents such as APMA (as shown in Fig. 5B). The possible mechanisms explaining our results obtained by heparin may be due to its direct and indirect effects on MMP/TIMP complex circulating in blood. Heparin may detach each component of the complex, increasing TIMP-2 levels in heparinized plasma. It is noteworthy that BioTrack™ TIMP-2 ELISA assay reveals only free TIMP-2 and that complexed with the active form of MMP-2, but not TIMP-2 complexed with the precursor/zymogen of MMP-2. This may explain the higher levels of TIMP-2 in plasma collected by LH tubes (Fig. 3C) and the TIMP-2 time- and dose-dependent increasing levels by heparin (Figs. 3D and 4B). The absence of any variation of MMP-2 concentrations by heparin (Figs. 1A and B) may be related to the characteristic of BioTrack™ MMP-2 ELISA assay, recognizing free proMMP-2 and proMMP-2 complexed with TIMP-2. The effects of heparin on the MMP-2/TIMP-2 complex may cause the release of proMMP-2, favouring the displacement, but not the detachment of the MMP-2 prodomain, finally allowing its removal by various activating agents (like SDS, APMA, etc.) [7]. Our hypothesis of an heparin-induced prodomain displacement may explain also the increased activity of gelatinase B (MMP-9) without causing the decrease of its molecular weight (Fig. 5A), in agreement with the well-known capability of some compounds (such as sodium dodecylsulphate) to stimulate the gelatinolytic activity of MMP without altering molecular mass [45,46]. Finally, our results show that an indirect and direct effect of heparin on the measurement of circulating MMPs and TIMPs should be distinguished. The indirect effect of heparin (preanalytical phase) results in lower concentrations of MMP-9 and TIMP-1 in plasma, preventing the release of these analytes from the blood cells in consequence of coagulation and fibrinolysis [13]. In addition, the direct effect of heparin is due to its involvement in the MMP/TIMP complex degradation and MMP prodomain displacement and affects their analyses by ELISA and zymography. Thus, to avoid pitfalls or misinterpretations for both TIMP and MMP quantitative analyses and zymography profiles the use of heparin as anticoagulant for plasma collection can only be recommended under strong standardized conditions and taking into consideration both direct and indirect effects of high molecular weight heparin on these analytes. Acknowledgment We thank Ms. Eleonor Cencherle for the assistance in the English correction. References [1] McCawley LJ, Matrisian LM. Matrix metalloproteinases: they're not just for matrix anymore! Curr Opin Cell Biol 2001;13:534–40. [2] Malemud CJ. Matrix metalloproteinases (MMPs) in health and disease: an overview. Front Biosci 2006;11:1696–701. [3] Mannello F, Luchetti F, Falcieri E, Papa S. Multiple roles of matrix metalloproteinases during apoptosis. Apoptosis 2005;10:19–24.

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