s2fieldsofstudy: Biology Psychology

text:
Differential Effects of apoE4 and Activation of ABCA1 on Brain and Plasma Lipoproteins

Apolipoprotein E4 (apoE4), the leading genetic risk factor for Alzheimer's disease (AD), is less lipidated compared to the most common and AD-benign allele, apoE3. We have recently shown that i.p. injections of the ATP-binding cassette A1 (ABCA1) agonist peptide CS-6253 to apoE mice reverse the hypolipidation of apoE4 and the associated brain pathology and behavioral deficits. While in the brain apoE is the main cholesterol transporter, in the periphery apoE and apoA-I both serve as the major cholesterol transporters. We presently investigated the extent to which apoE genotype and CS-6253 treatment to apoE3 and apoE4-targeted replacement mice affects the plasma levels and lipid particle distribution of apoE, and those of plasma and brain apoA-I and apoJ. This revealed that plasma levels of apoE4 were lower and eluted faster following FPLC than plasma apoE3. Treatment with CS-6253 increased the levels of plasma apoE4 and rendered the elution profile of apoE4 similar to that of apoE3. Similarly, the levels of plasma apoA-I were lower in the apoE4 mice compared to apoE3 mice, and this effect was partially reversed by CS-6253. Conversely, the levels of apoA-I in the brain which were higher in the apoE4 mice, were unaffected by CS-6253. The plasma levels of apoJ were higher in apoE4 mice than apoE3 mice and this effect was abolished by CS-6253. Similar but less pronounced effects were obtained in the brain. In conclusion, these results suggest that apoE4 affects the levels of apoA-I and apoJ and that the anti-apoE4 beneficial effects of CS-6253 may be related to both central and peripheral mechanisms.

Introduction
Apolipoprotein E (apoE) is the most abundant lipoprotein in the brain, where it is produced mostly by astrocytes [1]. ApoE is also synthesized in the periphery, typically by the liver and by macrophages [2], and is an important serum lipoprotein. ApoE exists as three major isoforms, termed apoE2, apoE3 and apoE4, of which apoE4 is the most prevalent genetic risk factor for Alzheimer's disease (AD) [3][4][5].
Studies performed in humans and corresponding mouse models which express apoE4, suggest that brain apoE4 is less lipidated compared to apoE3, the most common and AD-benign apoE allele [6][7][8][9]. These findings are supported by in vitro cell culture experiments which revealed that apoE4 is less effective than apoE3 in promoting cholesterol and phospholipid efflux [10][11][12]. ApoE lipidation is achieved by the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1, respectively), which stimulate apolipoprotein lipidation both in the brain and the periphery [13,14]. CSF samples extracted from AD patients have lower ex-vivo capacity for ABCA1-mediated cholesterol efflux compared to controls, an effect which is exacerbated in apoE4 carriers [15]. In the periphery, the main function of ABCA1 is to transport cholesterol and phospholipids from the plasma membrane of peripheral cells to lipid-free or lipid-poor apolipoprotein, such as apoE and apoA-I. This process which initiates the formation of high density lipoproteins (HDL) is called reverse cholesterol transport [16]. In the brain, ABAC1 stimulates the lipidation of apoE following its synthesis and secretion from astrocytes [17]. Interestingly, brain and peripheral apoE are produced and regulated independently and are, thus, parts of separate metabolic pools which do not mix [18,19].
ApoA-I is the major apolipoprotein of HDL in the periphery, where it plays an important role in reverse cholesterol transport. ApoA-I, though not synthesized in the brain, is present in the central nervous system (CNS), due to its ability to cross the blood brain barrier (BBB) from the periphery [19,20]. In view of the important role of apoA-I in lipid transport in the periphery, it is presumed that apoA-I plays a similar role in the brain [20][21][22]. Clusterin (apoJ) is another major apolipoprotein in the brain. It is less lipidated than apoE [23], and is believed to play a role in lipid transport and as a molecular chaperon associated with stress response [13,24].
We have recently shown, utilizing apoE-targeted replacement (TR) mice, that direct activation of the lipidating protein ABCA1 using intraparietal (i.p.) injections of the ABCA1-agonist peptide CS-6253 results in the accumulation of the peptide in the brain and in reversal of the hypolipidation of brain apoE4 which is observed in the non-treated mice [25]. Importantly, these effects were associated with reversal of the brain pathological effects of apoE4 and of the corresponding apoE4-driven cognitive impairments [25].
Since the protective anti-apoE4 effects of the ABCA1 agonist CS-6253 were observed following the peripheral application of this peptide and in view of the important role of ABCA1 in mediating the lipidation of apoE and apoA-I, we presently examined the extent to which the neuroprotective effects of the CS-6253 may be related to changes in the levels and extent of lipidation of peripheral apoE4 and/or to corresponding changes in the levels and lipidation of apoA-I and apoJ in the plasma and in the brain.

Mice
ApoE-TR mice, in which the endogenous mouse apoE was replaced by either human apoE3 or apoE4, were created by gene targeting as previously described [26]. The mice used were homozygous for the apoE3 (3/3) or apoE4 (4/4) alleles, and were purchased from Taconic, who back-crossed them to wild-type C57BL/6J mice (2BL/ 610; Harlan Laboratories) for 7 generations. The mice were further back-crossed in our lab for 3 additional generations with wildtype C57BL/6J mice (2BL/ 610; Harlan Laboratories). These mice are referred to herein as apoE3 and apoE4 mice, respectively. The apoE genotype of the mice was confirmed by PCR analysis, as described previously [27,28]. The mice were kept on standard chow-diet. All experiments were performed on age-matched male animals (4 months of age) and were approved by the Tel Aviv University Animal Care Committee. Every effort was made to reduce animal stress and to minimize animal usage. CS-6253 was kindly provided by Artery Therapeutics, Inc. and was administered according to a previously described in vivo protocol [29]. Accordingly, CS-6253 was injected intraperitoneally (i.p.) to 2.5-month-old male mice for 6 weeks (20mg/kg/48h, which translates to 0.5mg dissolved in 400μl of PBS per injection of a mouse weighing 25 grams). Corresponding control mice were injected with PBS (P5493, Sigma) in a similar manner. The mice were anesthetized with ketamine and xylazine, following termination of treatment, after which blood samples were collected and the mice were perfused transcardially with PBS. The brains were then removed from the PBS perfused mice and further processed for biochemical analysis, as outlined in the succeeding paragraphs.
Preparation of plasma and brain samples 1. Plasma samples. Freshly excised blood was drawn from the posterior vena cava of the anesthetized mice and collected into tubes containing 20μl of 10% EDTA to prevent blood clotting. The blood was centrifuged for 10 min at 3000 rpm at 4°C, after which the supernatant containing the plasma lipoproteins was collected and frozen at -70°C until used.
2. Hippocampal homogenates. Hippocampus was rapidly removed from one freshly excised brain hemisphere and rapidly frozen in liquid nitrogen after which it was stored frozen at −70°C until used. Frozen hippocampi were thawed, homogenized with a Teflon-glass homogenizer at 4°C with cold Tris-buffered saline (TBS) containing protease inhibitor mixture (P8340; Sigma), and phosphatase inhibitor mixture (P5726; Sigma). The homogenized hippocampi were then aliquoted and rapidly frozen in liquid nitrogen after which they were kept at -70°C until used.

Immunoblots
1. SDS-electrophoresis. The hippocampal extracts and plasma samples were thawed and boiled for 10 min with 0.5% SDS and immunoblotted as previously described [30,31]. Gels were then transferred to a nitrocellulose membrane and stained with either goat anti-apoE Ab (1:10,000 for both hippocampal and plasma samples; Millipore), rabbit anti-apoA-I Ab (1:1000 for hippocampal homogenates and 1:5000 for plasma samples; Meridian Life Science, Inc.), and goat anti-apoJ Ab (1:5000 for hippocampal homogenates and 1:7000 for plasma samples; Santa Cruz). The immunoblot bands were all visualized using the ECL chemiluminescent substrate (Pierce), after which their intensity was visualized and quantified utilizing Image Lab software (Bio-Rad). GAPDH levels (mouse anti-GAPDH, 1:1000; Abcam) were used as gel-loading controls and the results are presented relative to the control apoE3 mice.
2. Blue native gels. This was performed with the hippocampal extracts, whereas the plasma samples were subjected to fast protein liquid chromatography (FPLC) as described below. Accordingly, the hippocampal homogenates were electrophoresed on 4-16% gels in the Native PAGE Novex Bis-Tris Gel System purchased from Novex and according to the manufacturer's instructions, as previously described [7,25]. Gels were next transferred to PVDF membranes and stained with either goat anti-apoE Ab (1:10,000; Millipore), rabbit anti-apoA-I Ab (1:1000; Meridian Life Science, Inc.), or goat anti-apoJ Ab (1:1000; Santa Cruz).
The immunoblot bands were all visualized using the ECL chemiluminescent substrate (Pierce).

Fast protein liquid chromatography (FPLC) and cholesterol and triglycerides measurements
Total plasma cholesterol and triglycerides were measured using commercial kits (Chol, Roche/ Hitachi, Roche Diagnostics; Infinity, Thermo Electron). For FPLC, four samples from each mouse group were pooled together to generate 200μl of plasma extract, which was loaded and subjected to high resolution size exclusion/fast protein liquid chromatography (FPLC) using a Superose 6 column (Amersham Pharmacia Biotech AB, Piscataway, NJ), as previously described [32]. This process was performed twice and the results presented are the pool of the two columns performed for each group, as was determined by immunoblotting of the different FPLC fractions utilizing the appropriate anti-apolipoprotein Ab.

Statistical analysis
The experimental design consisted of two genotypes (apoE3 and apoE4) and two treatments (control and CS-6253) and the results were analyzed using 2-way ANOVA testing with STA-TISTICA software (version 8.0; StatSoft). Only after 2-way ANOVA retrieved significant results, further post hoc Fisher analysis was performed to test for individual effects, and these findings are depicted in the figures. The results presented correspond to the mean ± SEM, and are normalized relative to control apoE3 mice. Each of the four groups contained n = 8-10 mice for apoJ, apoE and cholesterol analysis, and n = 5 mice for apoA-I analysis. For FPLC analysis, the fractions from each column were run in a western blot, and the area under the emerging curve was calculated and is depicted beneath the curve.

Results
1. The effect of CS-6253 and the apoE genotype on plasma lipoproteins 1.1. ApoE. The effects of apoE genotype and CS-6253 on the plasma levels of apoE and on the levels and type of lipid particles with which apoE is associated were assessed by SDS gel electrophoresis and FPLC. As shown in Fig 1, the total levels of apoE in the plasma were lower in the control apoE4 mice compared to the corresponding apoE3 mice (1.00 ± 0.03 versus 0.59 ± 0.06 for control apoE3 and control apoE4 mice, respectively). This effect was virtually abolished by the CS-6253 treatment, which elevated the levels of apoE in the treated-apoE4 mice while having no effect on the corresponding plasma level of the CS-6253-treated apoE3 mice (0.87 ± 0.05 versus 0.80 ± 0.03 for CS-6253-treated apoE3 and CS-6253-treated apoE4, respectively). Two-way ANOVA of these results revealed a significant effect of genotype x treatment (p = 0.02). Further post hoc analysis revealed that the levels of apoE were significantly lower in the control apoE4 mouse group compared to the control apoE3 mice (p<0.0001), and that the CS-6253 treatment significantly increased the levels of apoE in the apoE4-treated mice (p = 0.003). The extent to which this CS-6253-driven increase in plasma apoE4 is associated with changes in the size distribution of the apoE4 particles was assessed by FPLC, followed by apoE immunoblot analysis of the FPLC fractions. This revealed, in accordance with previous observations, that in the mouse plasma apoE elutes mostly as HDL particles [26,33], and that the elution profile of plasma apoE was affected by both apoE genotype and CS-6253 ( Fig 1B). Analysis of the total levels of apoE eluted from the column (i.e., the sum of apoE from all fractions) revealed a pattern similar to that obtained from the SDS analysis of total apoE levels in the plasma (compare Fig 1A and 1B). A marked difference also emerged in the elution profiles of apoE3 and apoE4, whereas apoE of the control apoE4 mice eluted from the column about 4 fractions earlier than the corresponding apoE3 mice, the CS-6253 treatment shifted the elution profile of apoE4 towards that of the apoE3 mice, which was not affected by this treatment. As the observed effect of CS-6253 was to shift the apoE4 peak towards the apoE3 peak, namely, towards fractions 28-35, the results were quantitated by focusing on these fractions (Fig 1B, right bottom panel). This revealed that the distinct decrease in apoE4 particles in this size range was markedly reversed by CS-6253. Taken together, these findings suggest that the total levels of plasma apoE are lower in the apoE4 mice compared to those of the apoE3 mice, and that CS-6253, which had no effect on plasma apoE3, had a pronounced effect on the plasma apoE4 particles, namely it shifted the apoE4 towards particles which elute later from the column. The mechanism underlying the finding that in the plasma CS-6253 seems to alter the plasma apoE4 particle ( Fig 1B) will be addressed in the discussion.

ApoA-I.
ApoA-I is a central HDL lipoprotein, and is a major substrate of ABCA1-induced lipidation in the periphery [34]. Therefore, we next examined the effects of ABCA1 activation and of apoE genotype on the levels and particle size distribution of plasma apoA-I. As can be seen in Fig 2A, the levels of apoA-I were lower in the control apoE4 mice compared to control apoE3 mice, and CS-6253 treatment increased the levels of apoA-I of the apoE4 mice to that of the apoE3 mice, without having an effect on the corresponding levels in the apoE3 mice (1.00 ± 0.04 versus 0.64 ± 0.03 for control apoE3 and control apoE4 mice, respectively, and 1.00 ± 0.05 versus 0.82 ± 0.03 for CS-6253-treated apoE3 and CS-6253-treated apoE4 mice, respectively). Two-way ANOVA of these results revealed a significant effect of genotype x treatment (p = 0.037), and further post hoc analysis revealed that the levels of apoA-I in the apoE4 control mice were significantly lower compared to those of the corresponding apoE3 mice (p<0.0001), and were significantly increased following treatment (p = 0.007). FPLC analysis revealed that the apoA-I fractions of the four mouse groups had a similar size distribution peaking at around fraction 33 (Fig 2B), which is later than the elution of the apoE particles (compare Figs 1B and 2B). Furthermore, and in accordance with the total measurements of apoA-I (Fig 2A), the levels of the FPLC-fractionated apoA-I were lower in the control apoE4 mice than those of the corresponding apoE3 mice, and this effect was partially reversed by CS-6253 treatment (Fig 2B).
1.3. ApoJ. We next examined the effects of apoE genotype and CS-6253 on plasma apoJ. As shown in Fig 3, the total levels of apoJ were significantly higher in the apoE4 control mice compared to the corresponding apoE3 mice (1.00 ± 0.05 versus 2.01 ± 0.16 for control apoE3 and control apoE4 mice, respectively). Importantly, this effect was completely abolished by CS-6253 treatment, which had no effect on the apoE3 mice (1.04 ± 0.04 versus 0.90 ± 0.05 for CS- 6253-treated apoE3 and CS-6253-treated apoE4 mice, respectively). Two-way ANOVA revealed a significant genotype x treatment effect (p<0.0001). Further post hoc analysis revealed that the levels of apoJ were significantly higher in the control apoE4 mouse group compared to the control apoE3 mice (p<0.0001), and that the CS-6253 treatment significantly reduced the levels of apoJ in the apoE4-treated mice (p<0.0001). FPLC analysis revealed a similar pattern for all groups with a single peak climaxing at fraction 34. Quantitation of these FPLC results revealed, in accordance with the total apoJ measurements (Fig 3A), that they were higher in the control apoE4 mice relative to the corresponding apoE3 mice, and that this effect was abolished by CS-6253 (Fig 3B). The fact that the apoJ particles eluted later than the apoA-I and apoE particles (i.e., the apoJ peak was observed at fraction 34, while the apoE peak was observed at around fraction 28) is in accordance with previous findings that apoJ is associated with smaller lipoprotein particles compared to apoE and apoA-I [23,35].

Plasma cholesterol and triglycerides.
Measurements of the total plasma cholesterol levels revealed a small decrease in the apoE4 mice compared to apoE3 mice and that CS-6253 had no effect on the plasma cholesterol levels of both mouse groups (64.2 mg/dL ± 3.5 versus 57.0 mg/dL ± 3.9 for control apoE3 and control apoE4, respectively, and 70.9 mg/dL ± 4.0 versus 59.9 mg/dL ± 2.7 for CS-6253-treated apoE3 and CS-6253-treated apoE4 mice, respectively; see Fig 4). FPLC revealed that the cholesterol of the four mouse groups eluted as a single HDLpeak which co-eluted closely with apoA-I (peak at fractions 31-32 in Figs 2B and 4B), and whose levels, in accordance with the total cholesterol measurements, were somewhat lower in the apoE4 mice groups compared to the corresponding apoE3 mice (Fig 4B). Measurements of the total levels of triglycerides in the plasma revealed that like the cholesterol levels, they were slightly lower in the apoE4 control mice relative the control apoE3 mice (35.26 mg/dL ± 3.90 and 27.79 mg/dL ± 1.18 for the control apoE3 and control apoE4 mice, respectively), and that the triglycerides levels of both moue groups were not affected by CS-6253 (34.22 mg/dL ± 4.51 and 28.31 mg/dL ± 4.33 for the CS-6253-treated apoE3 and CS-6253-treated apoE4 mice, respectively).

The effect of CS-6253 and apoE genotype on brain lipoproteins
We have previously shown that in the brain apoE4 is hypolipidated relative to apoE3 and that this effect is counteracted by peripheral application of CS-6253, which crosses the BBB and activates ABCA1 [25]. We presently examined the extent to which these effects are associated with changes in either the levels or extent of lipidation of brain apoA-I and apoJ. We first examined the levels of apoA-I in the hippocampus of apoE3-and apoE4-TR mice ( Fig 5A). As can be seen, the total levels of apoA-I were higher in the apoE4 control mice compared to the control apoE3 mice, and were unaffected by the CS-6253 treatment (1.00 ± 0.06 versus 1.26 ± 0.06 for control apoE3 and control apoE4 mice, respectively, and 0.96 ± 0.09 versus 1.24 ± 0.08 for CS-6253-treated apoE3 and CS-6253-treated apoE4 mice, respectively). Twoway ANOVA revealed a significant effect for genotype (p = 0.003). Further post hoc analysis revealed that the levels of the control apoE4 mice were significantly higher than those of the corresponding apoE3 mice, and that the treated-apoE4 mice showed similarly higher levels of apoA-I compared to the treated apoE3 mice (p = 0.03 and p = 0.02, respectively). Native gel electrophoresis revealed that the apoA-I particles migrated as two bands, of apparent molecular weight of about 25kDa and 45-50 kDa, and that there were no differences in the size distribution of the apoA-I bands between the different groups. However, the intensities of the bands in Results of the total cholesterol levels (mean ± SEM; n = 8-10 per group) are depicted as mg/dL. White bars correspond to apoE3 mice, whereas black bars correspond to apoE4 mice. Fig 4B depicts the cholesterol eluted from the FPLC column of the different mice groups (□ and ■ correspond to control apoE3 and apoE4 mice, respectively, whereas △ and ▲ correspond to CS-6253-treated apoE3 and apoE4 mice, respectively). the control and CS-6253-treated apoE4 mouse groups were higher than those of the corresponding apoE3 mice (Fig 5B), which is in accordance with the results obtained by measurements of the total apoA-I levels in the hippocampus (Fig 5A).
Lastly, we assessed the effects of apoE-genotype and CS-6253 on the levels and extent of lipidation of brain apoJ. This revealed that the levels of apoJ in the control mice were slightly higher in the apoE4 mice compared to the corresponding apoE3 mice, and were decreased by CS-6253 in the apoE4 mice following treatment (1.00 ± 0.04 versus 1.13 ± 0.07 for control apoE3 and apoE4 mice, respectively, and 1.09 ± 0.04 versus 0.92 ± 0.04 for CS-6253-treated apoE3 and apoE4 mice, respectively; see Fig 6A). Two-way ANOVA revealed a significant effect for group x treatment (p = 0.006). Post hoc analysis of the results revealed that the levels of apoJ in the CS-6253-treated apoE4 mice was significantly lower than those of the control apoE4 mice (p = 0.005). Subsequent native apoJ immunoblotting revealed that the size distribution of the brain apoJ particles was similar in all mouse groups, and was unaffected by either genotype or treatment (Fig 6B). Importantly, the presently observed size distribution of the apoJ particles (~100 to 700 kDa, Fig 6B) was smaller than that of the corresponding brain apoE particles [~200 to 1000 kDa; [25]]. This is accordance with previous observations that in the brain apoE resides in in larger lipoprotein particles than apoJ [23]. The intensities of the brain apoJ staining in the native gel of the CS-6253-treated apoE4 mice was lower than those of the other groups as was observed in the SDS gel (Compare Fig 6A and 6B).

Discussion
Brain apoE4 is hypolipidated relative to apoE3 [7,25] and we have recently shown that treatment with the ABCA1 agonist CS-6253 reverses the hypolipidation of apoE4 and concomitantly counteracts the apoE4-driven brain pathology and cognitive impairments [25]. We presently investigated the extent to which these effects are related to plasma apoE4 and to the effects of apoE genotype and CS-6253 on brain and plasma apoJ and apoA-I levels and lipidation. The results thus obtained and the mechanisms underlying them are addressed below.
The apoE levels in the plasma of the control apoE4 mice were found to be lower than the corresponding levels in apoE3 mice (Fig 1A). Furthermore, there is a marked difference in the size of the apoE particles between the genotypes and their association with HDL-cholesterol, as depicted by the FPLC analysis (Figs 1B and 4B). These effects, namely the lower levels of apoE in apoE4 carriers and the difference in HDL-association, are in accordance with previous findings obtained from human studies in plasma and CSF [12,[36][37][38] and with sera and brain from different mouse models expressing human apoE [39][40][41][42].
In contrast to apoA-I, whose FPLC profile almost completely overlaps that of the HDL-cholesterol, the plasma of apoE co-elutes with both low-cholesterol containing particles (fractions 21-28) and high-cholesterol containing particles (fractions [28][29][30][31][32][33][34][35]. This is in accordance with findings showing that the majority of HDL is associated with apoA-I, whereas only about 10% of HDL is associated with apoE [43,44]. It is important to note that the control apoE4 profile is closely associated with the low-cholesterol containing particles, whereas the control apoE3 has a larger overlap with the HDL-cholesterol FPLC profile (compare Figs 1B and 4B which show a greater overlap between cholesterol and apoE3 following FPLC than between apoE4 and cholesterol). This suggests that the plasma apoE4 particles contain less cholesterol than the apoE3 particles. This is in accordance with the observation that apoE4 down regulates ABCA1 activity and cholesterol efflux by macrophages [45].
The major effect of CS-6253 treatment on the apoE4-containing particles is a shift from the low-cholesterol particles to the high-cholesterol containing particles, a shift that increases the overlap between the apoE4 FPLC profile and that of the HDL-cholesterol, and renders it similar to that of the apoE3 FPLC profile. This finding corresponds to the observation that in the brain, CS-6253 accelerates ABCA1-driven cholesterol efflux [46], thus shifting apoE4 particles to higher molecular weight species [25], which suggests increased lipidation of the apoE4 particles. Importantly, the plasma apoE3 particles are not affected by CS-6253.
With regards to the observation that apoE levels in the control apoE4 mice were lower compared to those in the corresponding apoE3 mice, it has been suggested that this is related to the decreased stability of apoE4 resulting in the susceptibility of apoE4 to proteolysis [12]. The enhanced vulnerability of apoE4 for degradation might relate to its lipidation state, causing it to be less stable [44]. Thus, treatment with CS-6253 which altered the lipid content of the apoE4 particle may contribute to its improved stability and increased levels.
Taken together, these findings suggest that like in the brain [25], the total levels of plasma apoE are lower in the apoE4 mice compared to those of the apoE3 mice and are less lipidated, and that CS-6253, which had no effect on either brain or plasma apoE3, has a pronounced effect on brain and plasma apoE4-lipid particles. In the plasma, this results in a shift of the apoE4 particles towards particles which co-elute with HDL-cholesterol, whereas in the brain the CS-6253 treatment increased the levels of apoE4 particles with decreased mobility [25].
It should be noted that while in the brain, apoE plays a major role in the distribution of lipids to cells, both during development and following injury [47,48], in the periphery-it is mainly involved in reverse cholesterol transport to the liver [49,50]. The distinct roles of apoE in the different pathways may reflect specific effects of apoE genotype and CS-6253 treatment on the apoE particles size and composition. Additional experiments, including measurements of the ratio of apoE to cholesterol and phospholipids and proteome analysis of these particles are needed to further the understanding of the effects of apoE genotype on the structure and size of the plasma lipoprotein particles.
ApoE4 is associated with cerebrovascular pathology and is a risk factor for vascular dementia [51,52]. Preliminary histochemical findings suggest that, unlike the brain parenchyma [53], the cerebrovascular system of young 4-months old apoE4 mice is not markedly affected by apoE4 (in preparation), suggesting that the overall pathological effects of apoE4 in these mice and the associated protective effects of CS-6253 are largely brain parenchyma-driven. This assertion is also supported by the present observation that CS-6253 does not affect the levels of either cholesterol or triglycerides in the plasma (Fig 4).
ApoE4 affects brain and plasma apoA-I levels in opposite directions. We presently report that the levels of plasma apoA-I are lower in the control apoE4 mice compared to the apoE3 mice, corresponding to the apoE levels, which were also lower in the control apoE4 mice. Similar results, depicting lower levels of apoA-I, were obtained from plasma of human apoE4 carriers [38]. Taken together, this suggests a possible regulatory link between apoA-I and apoE. The inter-relationship between apoA-I and apoE has been previously shown in the periphery, namely that apoA-I can induce the secretion of apoE, possibly through modulation of the apoE recycling process [54][55][56]. Alternatively, it has been shown that apoE3 supports the shift of apoA-I to HDL more so than apoE4, thus presumably stabilizing apoA-I [41,57,58]. Since apoE4 is associated with decreased levels of ABCA1, which are induced by CS-6253 treatment [25], it is possible that the apoE-apoA-I positive correlation under these condition is mediated via ABCA1 induction; however, it cannot be ruled out that this effect is due to direct cross-talk between the apolipoproteins. It remains to be determined whether in the plasma, apoA-I or apoE are the main target for ABCA1-induced lipidation, and which triggers the down-stream effects.
In contrast to the findings in the plasma, in the brain, apoA-I levels were elevated by apoE4 and were not affected by CS-6253 (Fig 5). The size of the brain apoA-I particles was smaller than those of the corresponding brain apoE and apoJ particles and is composed mainly of monomers and dimers (Fig 5B). This is in agreement with the finding that in the brain, apoE and apoA-I reside in different particles [59] and that apoA-I is less lipidated than apoE [14,59]. The reason that apoA-I is less lipidated than apoE could be because apoA-I, unlike apoE, is synthesized peripherally and accumulates in the brain by crossing the BBB at the choroid plexus and that delipidated apoA-I accumulates readily in the brain [60].
ApoA-I plays a major role in peripheral cholesterol transport, and it is likely that it also plays an analogous role in the CNS [61][62][63][64]. However, data is sparse when addressing the interaction between apoA-I and apoE in the brain. The fact that the leading apolipoprotein in the periphery is apoA-I, while in the CNS it is apoE, may explain the differences in the levels of apoA-I in the plasma and in the brain of apoE4 mice. Accordingly, it is possible that the observed apoE4-driven increase in brain apoA-I is driven by a compensatory mechanism via which the increase in brain apoA-I levels counteracts lipid transport related deficits due to apoE4 and its decreased levels. Since the total brain levels of apoE4 are not affected by CS-6253 and remain lower than those of the corresponding apoE3 mice [25], such a mechanism is not expected to be affected by CS-6253, as indeed is the case. It is presumed that a different mechanism is at play in the plasma.
The observation that the levels of apoA-I in the plasma and brain are affected differently by apoE4 is in accordance with the finding that there is no correlation between the levels of apoA-I in the sera and CSF of individual subjects [65]. It still remains to be determined which plasma-related mechanism underlie the apoE4-driven decrease in plasma apoA-I and the reversal of this effect by CS-6253.
The levels of apoJ in the plasma of naïve apoE4 mice are significantly higher than those of the corresponding apoE3 mice, and this effect is reversed by treatment with CS-6253, which has no effect on the apoE3 mice (Fig 3A). A similar but less pronounced effect was observed in the brain (Fig 6A). The size and extent of lipidation of brain apoJ particles are smaller than those of apoE [i.e., brain apoJ particles range between~200-700 kDa (Fig 5B), whereas the brain apoE particles ranged between~200-1000 kDa; [25]], which is in accordance with previous findings [23,35]. A similar effect was observed in the plasma, where the apoE particles eluted from the FPLC column at an earlier fraction than the apoJ particles, as previously described [compare Figs 1B and 3B [35]].
ApoJ serves both as a lipid transport protein and as a molecular chaperon [66][67][68]. The promoter region of the apoJ gene contains response elements for transcription factors involved in the cellular stress response and increased expression of apoJ is observed in several pathological conditions including AD, head trauma and epilepsy [for review, see [65]]. It is thus possible that the observed apoE4-driven increase in apoJ levels corresponds to a protective response to apoE4. This interpretation is supported by the observation that the anti-apoE4 protective effects of CS-6253 [25] are associated with complete abolition of the effects of apoE4 on apoJ (Figs 4 and 6). The cellular and molecular mechanisms underlying the elevation of apoJ by apoE4 are not known. However, the finding that the lipidation of apoJ is not affected by the CS-6253 treatment suggests that apoJ is less sensitive to ABCA1 modulation, as was previously suggested [13,14]. This may indicate an indirect mechanism possibly mediated by apoE4-driven stress [69,70].
In conclusion, this study shows that the levels of plasma apoE of apoE4 mice are substantially lower than those of the corresponding apoE3 mice and that this effect is associated with structural changes in the corresponding plasma apoE particles. These effects are accompanied by a compensatory increase in the levels of brain apoA-I and a significant increase in the levels of apoJ in the plasma. Treatment with the ABCA1 agonist CS-6253 which reverses the brain pathological effects of apoE4 and the associated cognitive impairments [25], reverses the effects of apoE4 on the lipidation and aggregation of plasma apoE4 without affecting the total corresponding apoE3 levels. This treatment abolishes the effects of apoE4 on plasma apoA-I and apoJ but not on the levels of brain apoA-I suggesting that the plasma apoJ levels are apoE4 stress responders whereas the levels of brain apoA-I are a compensatory response to the decreased levels of brain apoE4, which are unchanged by CS-6253 treatment.


extfieldsofstudy: Medicine Chemistry

text:
Structural and haemostatic features of pharmaceutical heparins from different animal sources: challenges to define thresholds separating distinct drugs

Heparins extracted from different animal sources have been conventionally considered effective anticoagulant and antithrombotic agents despite of their pharmacological dissimilarities. We performed herein a systematic analysis on the physicochemical properties, disaccharide composition, in vitro anticoagulant potency and in vivo antithrombotic and bleeding effects of several batches of pharmaceutical grade heparins obtained from porcine intestine, bovine intestine and bovine lung. Each of these three heparin types unambiguously presented differences in their chemical structures, physicochemical properties and/or haemostatic effects. We also prepared derivatives of these heparins with similar molecular weight differing exclusively in their disaccharide composition. The derivatives from porcine intestinal and bovine lung heparins were structurally more similar with each other and hence presented close anticoagulant activities whereas the derivative from bovine intestinal heparin had a higher proportion of 6-desulfated α-glucosamine units and about half anticoagulant activity. Our findings reasonably indicate that pharmaceutical preparations of heparin from different animal sources constitute distinct drugs, thus requiring specific regulatory rules and therapeutic evaluations.

Heparin is widely employed in medicine as anticoagulant and antithrombotic agent. The clinical use of heparin has started about 65 years ago, which places it within the oldest biologic drugs still in use 1 ; furthermore, it is the second leading biologics currently in use, just behind insulin 2 . Heparin is a unique sulfated glycosaminoglycan (GAG), with one of the highest anionic charges of natural biomolecules, and can be considered as a major representative of a carbohydrate-based drug 3 . Its use is essential for procedures of extracorporeal circulation during cardiovascular surgeries and hemodialysis 4 . The mechanism of action of heparin on the coagulation system is well characterized at molecular level and acts by carbohydrate-protein interaction resulting in proteases inhibition mediated by serpins 1 .
World consumption of heparin has been increasing significantly mainly due to more precise diagnosis of thromboembolic diseases and the increasing importance given to their prevention. However, the heparin production is restricted by its dependence of animal tissues as basic raw material. Different tissues from different species of mammals were already employed over the years for manufacturing heparin with pharmaceutical purposes; originally from dog liver, then from bovine lung and lately from porcine intestine 5 . In some Latin American and Islamic countries bovine intestine is also a source of heparin 6 . Currently, Chinese industries produce approximately 60% of the heparin consumed in the world using porcine intestine as raw material 7 .
The current production of heparin mostly based on a single animal source and concentrated in a single country raises a "supply insecurity", which lead the FDA -USA to evaluate the reintroduction of lung or intestine bovine heparins 8 . Possibly, such argument will be extended to other countries, demanding new information and regulatory rules on the chemical structures and haemostatic features of heparins obtained from sources other than porcine intestine.
Heparin is a linear polysaccharide mostly constituted for 4-linked units of α -L-iduronic acid 2-sulfate (α -IdA-2S) and α -D-glucosamine N,6-disulfated (α -GlcN-N,6diS). Other minority units found in the molecule are non-sulfated α -L-iduronic acid, α -D-glucosamine N-acetylated or N, 3, 6-trisulfated, and β -D-glucuronic acid residues (not epimerized to α -L-iduronic acid). The proportion of these units in the heparin polymer directly relates to the tissue used for its extraction, notably: the proportion of N-acetylated α -D-glucosamine residues is lower in bovine lung heparin 9 and the amount of 6-sulfated α -D-glucosamine units is significantly lower in bovine than in porcine intestinal heparin 10,11 . In addition to these structural differences, heparins obtained from different sources also differ in their average molecular weight 9 .
The structural variations among heparins obtained from different sources induce significant changes on their anticoagulant effects, including the dosage required for the prevention of thrombosis and leading to a bleeding adverse effect. For example, it is necessary to use higher quantity (in weight basis) of bovine intestinal than porcine intestinal heparin to achieve the same anticoagulant and antithrombotic effects. These differences have direct implications in the clinical use of heparin, such as the higher dosages of protamine necessary to neutralize bovine than porcine intestinal heparins 10,11 .
The objective of the present study was to answer two essential questions involving the production and therapeutic use of heparin: (1) Are heparins obtained from different sources distinct drugs? (2) Which are the analytical thresholds separating these distinct heparins? To answer these questions, we undertook a systematic analysis on physicochemical properties, disaccharide composition, in vitro anticoagulant activity and in vivo antithrombotic and bleeding effects of pharmaceutical heparins obtained from porcine intestine (HP-I), bovine intestine (HB-I) and bovine lung (HB-L). We also prepared and analyzed derivatives of these heparins with similar molecular weight but differing in their disaccharide units. Our findings allowed us to clarify many aspects of these questions concerning the pharmacological features of heparins produced from different animal sources.

Results
Physicochemical Properties. Pharmaceutical heparins obtained from different animal sources were initially analyzed via anion exchange chromatography. HB-I (Fig. 1, blue lines) elutes as a broader peak and with lower concentration of NaCl than HP-I (Fig. 1, red lines) or HB-L ( Fig. 1, green line). One hundred and seventeen batches of HB-I and 25 of HP-I were analyzed, showing reproducible elution profiles as those illustrated in Fig. 1. In contrast, only one batch of HB-L was analyzed; this is because its production was ended in the 1980's and thus commercially unavailable. HB-I and HP-I batches showed no contaminations with dermatan sulfate or oversulfated chondroitin sulfate while a discrete contamination with dermatan sulfate (< 5%) was observed in the HB-L batch (Fig. 1). We also quantitatively evaluated the capacity of retention of these heparins on the anion exchange column at different NaCl concentrations. With NaCl concentrations up to 1.9 M, 56.2% of HB-I (6 batches), 27.7% of HP-I (5 batches) and 35.5% of HB-L (1 batch) were eluted from the column (Supplementary Table S1). This clearly demonstrates that the different heparins have distinct ionic properties, especially those extracted from bovine intestine.
The molecular weight of these heparins was investigated via gel permeation chromatography, using a set of columns previously calibrated with the heparin sodium molecular-weight calibrant from USP and the 2nd low-molecular-weight heparin molecular-weight standard from NIBSC ( Fig. 2A). HP-I and HB-I batches (Fig. 2B, red lines and Fig. 2C, blue lines, respectively) showed very low variability within and between themselves unlike HB-L, which had a noticeably reduced molecular weight distribution (Fig. 2C, green line). The M w , M p , M n and PD of these heparins are list in Table 1. HP-I and HB-I are more alike one each other, nevertheless differing significantly (P < 0.05) in their M p , M n and PD values ( Table 1). All the six batches of HP-I comply with the requirements for molecular weight range defined by the  Table S2). Only one within the six batches of HB-I analyzed did not comply with USP recommendations regarding the ratio M 8,000 → 16,000 Da/M 16,000 → 24,000 Da 12 .
Disaccharide composition. NMR analysis of the three heparins revealed some coincident signals on 1D 1 H NMR spectra in the ranges of 4.8-5.6 and 1.9-2.2 ppm (Fig. 3A). Despite these analogies, HB-I showed significant structural differences in comparison with the other two heparin types (see vertical arrows in Fig. 3A); mainly with regard to its higher proportion of 6-desulfated α -glucosamine units, clearly identified by the anomeric signal at 5.31 ppm 10,11 . The lack of sulfate in the position 6 of α -glucosamine unit also results in a modest downfield shift of H1 and H5 of the neighbor 2-sulfated α -iduronic acid (Fig. 3A). In addition, HB-I contains a higher proportion of β -glucuronic acid linked to N-sulfated α -glucosamine (H1 at 5.58 ppm) and lower proportion of α -glucosamine N,3,6-trisulfated (H1 at 5.52 ppm) and non-sulfated α -iduronic acid (H1 at 5.01 ppm) (Fig. 3A). In contrast, HB-L and HP-I showed only subtle variations in their structures; the only noticeable difference was the low intensity of the N-acetyl signal at 2.06 ppm in HB-L, indicating lower proportion of N-acetylated α -glucosamine, as previously reported 9 .
1D 1 H NMR analysis performed with a large number of batches of HP-I and HB-I yielded 1D 1 H spectra with high similarities within the batches and a marked difference between each heparin type. To further assess the regularity and reproducibility of the chemical composition of these heparins we weighted and fitted as concatenate plots the spectra of 20 batches of each HB-I and HP-I (Fig. 4). As expected, the average plots of HB-I and HP-I batches were fairly similar within and different between themselves (Fig. 4). Although these plots lack resolution and HB-I and HB-L (blue and green lines in panel C, respectively) were determined using a set of TSK gel G4000 SW/G3000 SW columns. Retention times and relative molecular weight of the calibrants (low-molecular-weight heparin -NIBSC and heparin sodium -USP) were used to derive the calibration curve (fit to a third order polynomial) depicted in the panel A. Dashed black lines on panels B,C show the elution of the heparin sodium molecular-weight calibrant from USP. The eluents were monitored via differential refractive index.    to discriminate the double-peaks of H1 and H5 signals of the 2-sulfated α -iduronic acid units, they can be considered a useful analytical tool to compare multiple batches of heparin. Other relevant information derived from 1D 1 H spectra was the level of purity of these heparins; we were able to detect mixtures of HB-I and HP-I up to 4% on weight basis (Supplementary Figure S1). We further analyzed the different heparins via 2D NMR techniques. 1 H -13 C HSQC spectra (Fig. 3B,D) were used to identify and quantify the constituent units of the heparins, including their minority components, as previously described 13 . These spectra are particularly useful to identify residues of β -D-glucuronic acid, which are superimposed by the HOD signal in 1D 1 H spectra. Three distinct regions on the HSQC spectra were clearly ascribed to the anomeric signals of α -glucosamine, α -iduronic acid and β -glucuronic acid + units of the linkage region ( Fig. 3B,D, dotted squares). The 1 H and 13 C chemical shifts of the anomeric units from each heparin type are available in the Supplementary Table S3. The constituent units of these heparins were qualitatively similar but differ in their proportions (Table 2), especially the units from HB-I, which showed: greater heterogeneity of α -glucosamine units, mainly because its 6-desulfation (signal 5, Table 2); higher proportion of N-sulfated α -glucosamine linked to β -glucuronic instead α -iduronic acid (signal 4); and lower content of β -glucuronic acid linked to N-glucosamine N,3,6-trisulfated (signal 9), a component of the binding region to antithrombin 14 . We also observed that HB-L has more uniform composition than HP-I and HB-I, especially due to the sulfation pattern of α -glucosamine units and lower content of β -glucuronic acid ( Table 2).
The connection among the different α -glucosamine units and 2-sulfated α -iduronic acid of each heparin type was further investigated via phase-sensitive 1 H-1 H TOCSY (Supplementary Figure S2), which discern signals of intra-residue spin systems from those of inter-residue ROES [15][16][17] . This approach allows determining the  Figure S2). Briefly, the most intense anti-phase signal shows the linkage in the subsequent sugar residue. The connection of the anomeric signal of 6-desulfated α -glucosamine (C1) and the two slightly down-shifted H1 and H5 signals of 2-sulfated α -iduronic acid (namely I1-C and I5-C, respectively) was previously speculated correlating their intensities 10 . Now we unambiguously demonstrated that C1 signal of 6-desulfated α -glucosamine and A1 signal of N,6-disulfated α -glucosamine connect with signals I1-C/I5-C and I1-A/I5-A, respectively, of 2-sulfated α -iduronic acid (Supplementary Figure S2). The set of 1D and 2D NMR analysis presented above clearly demonstrate that the disaccharide composition of HB-I markedly differs from those of HB-L and HP-I.

Anticoagulant and antithrombotic activities and bleeding tendency. The in vitro anticoagulant
potencies of the different heparins were determined based on clotting assays (APTT) (Fig. 5A) and on their anti-FIIa (Fig. 5B) and anti-FXa (Fig. 5C) activities. When analyzed separately, each heparin type presented similar (P > 0.05) specific activity (IU mg −1 ) on the three anticoagulant assays (Supplementary Table S4), except for the HB-L APTT, which was significantly lower than its anti-FIIa and anti-FXa activities (Supplementary Table S4). The comparison among the heparins through these assays revealed that HP-I has significantly higher anticoagulant potency (P < 0.05) than HB-I and HB-L, which also shows slightly but significant differences (P < 0.05) between them on anti-FIIa and anti-FXa assays (Fig. 5). This result indicates that despite of the structural resemblance between HP-I and HB-L, their in vitro anticoagulant activities are quite different.
To assess the in vivo antithrombotic activities of the three heparins we employed a classical venous model of experimental thrombosis after intravascular administration of the drug of interest 18 . These assays were performed using a single representative batch of each heparin. Higher doses of HB-I and HB-L than HP-I administered on weight basis (mg kg −1 body weight) were necessary to achieve a satisfactory antithrombotic effect (Fig. 5D, inset). However, the three heparin types generate similar dose vs. response curves with doses based on their anticoagulant activities (IU kg −1 body weight) (Fig. 5D). As expected, these in vivo assessments have reasonably reproduced the results obtained in the in vitro anticoagulant assays.
The bleeding tendencies of each heparin type were evaluated in rats 19 , before and after their neutralizations with protamine (Fig. 5E). Doses with similar anticoagulant potency (~200 IU kg −1 body weight) of one representative batch of each heparin type and saline (control) were intravascular administered and afterward the bleeding was measured. The hemorrhagic tendencies of the three heparins were similar among themselves (P > 0.05) and significant higher (P < 0.05) than the control (Fig. 5E). Animals treated with protamine (2 mg kg −1 body weight) showed no significant differences (P > 0.05) in comparison with the control. Therefore, the set of results presented Preparation and analysis of heparins with similar molecular weight. HP-I and HB-L share similarities on their disaccharide composition but differ on their anticoagulant potencies. The most notable difference between these two heparins is their molecular weight (Fig. 2 20 . To confirm if the anticoagulant potency of HB-L is impaired by its molecular weight we prepared derivatives from the three heparin types with similar molecular weight range using a semi-preparative gel permeation column (Fig. 6A). One batch of each heparin type was pooled into five distinct fractions (F1 to F5), of which the fraction F3 yielded derivatives with similar molecular weight range and closest M w , M p , M n and PD values (Fig. 6A, Table 1). The molecular weight profiles of the F3 derivatives were afterward checked using a set of analytical gel permeation columns (Fig. 6B). F3 derivatives from HP-I and HB-L elute from the column as narrow and coincident peaks while HB-I presented a shoulder shifted towards the V t (Fig. 6B), which reflects in a slightly lower M n value (Table 1). Nevertheless, the three F3 derivatives have showed remarkable molecular weight homogeneity, with F3 derivatives from HP-I and HB-I showing lower and HB-L higher molecular weight than their unfractionated parental heparins ( Table 1).
The disaccharide composition of F3 derivatives were investigated via 1D 1 H (Fig. 6C-E) and 2D 1 H -13 C HSQC (Supplementary Figure S3, Table 2) NMR spectra. All the three derivatives were structurally similar to their parental-unfractionated heparins (Fig. 6C-E), except for minor differences in the region of β -glucuronic acid + linkage region, especially in the spectrum of the derivative from HB-L (Supplementary Figure S3). Therefore, we successfully have produced preparations of the heparins with similar molecular weights differing exclusively in their disaccharide compositions in a way similar to their parental-unfractionated molecules.
Anticoagulant activities of F3 derivatives. Once a reasonable amount of the F3 derivatives were gathered, we evaluated their in vitro anticoagulant activities via APTT, anti-FIIa and anti-FXa assays and then compared with their parental-unfractionated heparin batches (Fig. 6F-H). F3 derivative from HP-I and HB-I showed no significant differences (P > 0.05) in comparison with their parental-unfractionated batches (Fig. 6F-H). In contrast, the F3 derivative from HB-L presented a substantial increase in its anticoagulant effect, becoming significantly more potent (P < 0.05) than its parental-unfractionated batch on all the three assays (Fig. 6F-H).    Comparing the F3 derivatives among themselves we clearly observed those from HP-I and HB-L with similar anticoagulant effect (~170 IU mg −1 ), especially on their anti-FIIa and anti-FXa activities (Fig. 6F-H); however, the derivative from HB-I is noticeably less potent on all assays (< 100 I.U. mg −1 ), possibly due to its structural characteristics such as the marked 6-desulfation of the α -glucosamine units. Through these results we can now establish a strict correlation between the disaccharide composition and anticoagulant activity of heparin excluding the influence of the molecular weight.

Discussion
Currently heparin is defined by most pharmacopeias as a single biological drug, regardless the animal source used for its production 1 . Heparin was initially mass-produced from bovine lung and lately from porcine intestine 5 . Some Latin American and Islamic countries also employ heparin from bovine intestine as a therapeutic alternative 6 . However, the transition from HB-L to HP-I and the concomitant introduction of HB-I were not accompanied by new regulations or clinical evaluations by the health authorities.
Here we performed analytical assessments (361 batches) of pharmaceutical heparins obtained from these different animal sources (viz. porcine and bovine intestinal mucosa and bovine lung), which showed different physicochemical features such as their ionic properties and the lower molecular weight of HB-L. Besides, these heparins also presented differences in their disaccharide compositions; HP-I and HB-L present more alike structures, with similar sulfation patterns in their α -glucosamine units and 2-sulfated α -iduronic acid proportions; and bleeding tendency (E) assays. Data from the in vitro assays (panels A-C) were expressed as median (CI 95%) and mean (□ ) and compared via One-way ANOVA with Bonferroni post hoc test. The anticoagulant activities (IU mg −1 , as dry weight) were determined based on standard curves obtained with the 6 th International Heparin Standard (NBISC). In vivo antithrombotic activities (normalized results) were expressed as IU kg −1 body weight (a single sigmoidal curve for the three heparin types) in panel D and as mg kg −1 body weight in panel D inset (mean ± SD, n ≥ 5). Bleeding tendencies (panel E) were expressed as ratios of blood loss of animals treated with the heparins (200 IU kg −1 body weight) and saline (control, in black). In part of the assays heparins were neutralized with 2 mg kg −1 body weight of protamine before the bleeding measurement. Full and empty squares, circles and triangles represent different experimentalists.
Scientific RepoRts | 6:35619 | DOI: 10.1038/srep35619 however, HB-L has considerably lower proportion of β -glucuronic acid. In contrast, HB-I shows a remarkably distinct structure, with higher 6-desulfated α -glucosamine and lower β -glucuronic acid linked to N-glucosamine N,3,6-trisulfated proportions. Despite of the structural resemblances between HP-I and HB-L, these heparins presented dissimilar anticoagulant activities, with HB-L potency close to HB-I and markedly lower than HP-I. Differences among heparins obtained from different animal sources were recently reported elsewhere [21][22][23] . The results obtained via NMR spectroscopy presented in these articles also suggest that heparins from porcine and bovine intestine have dissimilar structures. However, none of these works thoroughly correlate the molecular weight and disaccharide composition of those different heparins with their anticoagulant and antithrombotic potencies, as reported here. Besides, these works assessed a limited number of heparin samples, differently of our work, which assessed several different pharmaceutical grade heparins commercially available for therapeutic use.
We also investigated the impact of the molecular weight on the anticoagulant activity of these different heparins. The fractions F3 derived from the three heparin types differ exclusively in their disaccharide units. F3 derivatives from HP-I and HB-L showed disaccharide compositions and anticoagulant activities more alike to each other. In contrast, HB-I, showed major differences in its structure, especially regarding the high proportion of 6-desulfated α -glucosamine residues, and remarkably lower (~50%) anticoagulant activity. These results clearly demonstrate the pivotal role of the molecular weight on the anticoagulant activity of heparin. A previous study showed it is possible to separate via ion-exchange chromatography HB-I polymers enriched in 6-sulfated α -glucosamine units, with improved anticoagulant activity 11 . Taking into consideration that heparin is a heterogeneous and polydisperse polymer, purification processes aiming for a specific molecular weight range or ionic property can result in preparations with improved anticoagulant potencies.
In the last decades the knowledge on the pharmaceutical and biochemical features of heparin increased notably, mainly as result of the advances in the analytical tools to investigate its complex structure (i.e. NMR) and biological effects 10,11,24 . These advances have allowed more precise and comprehensive quality control analysis of pharmaceutical preparations of heparin. An example of the necessity of these thorough quality assessments was the case of heparin contamination with oversulfated chondroitin sulfate, an event with great repercussion within the health community and associated with several deaths 25 . Another important advance has been the production of HP-I with enhanced specific activity (IU mg −1 ), which can be then administrated as lower doses (in weight basis) 5 and hence requiring lower doses of protamine for its neutralization.
Clinical use of unfractionated heparin requires an extra caution because the dose necessary to produce the desired anticoagulant or antithrombotic effect is very close to those inducing adverse effects, especially bleeding. Furthermore, heparin therapy is usually employed in serious clinical situations, such as cardiovascular surgeries or prevention and treatment oh thromboembolism episodes. As demonstrated here and elsewhere 10,11 . HB-I requires about two-fold higher doses (in weight basis) than HP-I to achieve the same anticoagulant and antithrombotic effect. In consequence, HB-I will demand higher doses of protamine than HPI to be effectively neutralized, thus avoiding bleeding incidents. This was observed on in vitro assays 10,11 .
Few studies comparing the efficacy and/or safety of heparins obtained from different sources were already performed e.g. 26,27 . The most notable adverse effect reported was the increased incidence of thrombocytopenia associated with HB-L in comparison with HP-I 28 . The concomitant clinical use of HP-I and HB-I in Brazil precipitated several bleeding incidents during cardiovascular surgeries, drawing attention of the regulatory authorities 29,30 . The only clinical study comparing HP-I and HB-I was performed with patients undergoing hemodialysis 31 ; in this study both heparins were similarly efficient and safe, but showed different anticoagulant activities due to an inadequate determination of the therapeutic formulation.
Our results thoroughly indicate that heparins from different animal sources consist in effective but distinct drugs, this is because: (1) HB-I presents distinct chemical structure and anticoagulant efficacy from HP-I; (2) HB-L and HP-I present preponderant structure more alike each other, however, their anticoagulant efficacy and physicochemical properties (mainly the molecular weight) significantly differ; (3) HB-L and HB-I have fairly similar anticoagulant effect, but greatly differ in their chemical structures.
More than one century has passed since the discovery of heparin, during this time, the understanding on its nature and uses has grown dramatically 32 . Even so, heparin stills continuously challenging us with new questions concerning its structure, mechanism of action, manufacturing, quality controls, etc. Nowadays, when similarity of biological drugs is a central theme in pharmacology, the heparins from different animal sources approached here show to us new challenges, perhaps as a paradigm to define thresholds separating distinct biological drugs. Anion-exchange chromatography. Heparins were analyzed via anion-exchange chromatography using an IonPac AS11-HC column (Dionex; Sunnyvale, USA) equilibrated with 10 mM Tris-HCl with 0.5 M NaCl, pH 7.4, linked to a HPLC system (Shimadzu; Kioto, Japan) as previously described 33 . Heparins (200 μ g of each), standard heparin (200 μ g), dermatan sulfate (50 μ g) and oversulfated chondroitin sulfate (30 μ g) were applied to the column, washed with equilibration buffer (10 mL) and then eluted using a linear gradient of 0.4 → 2.5 M NaCl (40 mL), at a flow rate of 0.5 mL.min −1 . The eluent was continuously monitored via UV (A 215nm ). The background produced by the NaCl gradient was subtracted from the chromatograms obtained applying the glycosaminoglycans (GAGs).

Heparins and standard glycosminoglycans samples.
Determination of the molecular weight. Molecular weight distribution of heparins was determined via gel permeation chromatography using a set of TSK gel G4000 SW × 1 and G3000 SW × 1 columns, both 7.5 mm × 300 mm i.d. (Tosoh; Tokyo, Japan), linked to a HPLC system (Shimadzu) and equilibrated with 0.1 M ammonium acetate. The combination of G4000 and G3000 columns spread the molecular weight exclusion limits yielding well-marked separations. The heparins and standard GAGs (200 μ g of each) were eluted with equilibration buffer at 40 °C, flow rate of 0.3 mL min −1 and monitored by differential refractive index. The columns were calibrated using a low-molecular-weight heparin molecular-weight standard (Lot No. 05/112) from NIBSC and a heparin sodium molecular-weight calibrant from USP (Lot No. FOL4830). Average molecular weight (M w ), peak molecular weight (Mp), number average molecular weight (M n ) and polydispersity degree (PD) were calculated as previously described 5 . Preparation of F3 derivatives. F3 derivatives of each heparin type were prepared via gel permeation chromatography using a Peptide HR 10//30 column (GE healthcare, Chicago, United States) linked to a HPLC system (Shimadzu, Tokyo, Japan) and equilibrated with 0.5 M ammonium acetate (pH 5.0). Heparins (20 mf of each type) were eluted with the equilibration buffer at a flow rate of 0.5 mL.min −1 and monitored by differential refractive Scientific RepoRts | 6:35619 | DOI: 10.1038/srep35619 index. Fractions of 0.5 mL were collected, pooled as indicated in Fig. 6A and lyophilized. Sodium salt of the F3 derivatives was obtained following Dowex 50 Wx8 (H + ) exchange.
Analysis of heparins by NMR. 1 H one-dimensional (1D) and 1 H and 13 C two-dimensional (2D) nuclear magnetic resonance (NMR) spectra of heparins batches were recorded using a Bruker DRX 800 MHz apparatus (Bruker; Billerica, USA) with a triple resonance probe, as detailed previously 13,15,16 . About 20 mg of each batch were dissolved in 0.5 mL 99.9% deuterium oxide (Cambridge Isotope Laboratories; Tewksbury, USA). All spectra were recorded at 35 °C with HOD suppression by presaturation. 1D 1 H NMR spectrum of 361 batches were recorded with 64 scans and inter-scan delay of 1 second. 1D spectra of 20 batches of each HB-I and HP-I were weighted using AMIX software (Bruker) to generate concatenate fits. 2D phase sensitive 1 H -1 H TOCSY (total correlated spectroscopy) and 1 H -13 C HSQC (heteronuclear single quantum coherence) spectra were recorded using states-time proportion phase incrementation (TPPI) for quadrature detection in the indirect dimension. Phase-sensitive 1 H-1 H TOCSY spectra run as previously described 15,16 , with 4046 × 400 points with a spin-lock field of 10 kHz and a mix time of 80 ms. 1 H-13 C HSQC spectra run with 1024 × 256 points and globally optimized alternating phase rectangular pulses (GARP) for decoupling 13 . Chemical shifts are displayed relative to external trimethylsilylpropionic acid at 0 ppm for 1 H and relative to methanol for 13 C. Activated partial thromboplastin time assay (APTT). Human plasma (100 μ L) and various concentrations of the heparins were incubated for 2 min at 37 °C with 100 μ L of APTT reagent (kaolin bovine phospholipid reagent from Biolab-Merieux AS; Rio de Janeiro, Brazil). After incubation 100 μ L of 25 mM CaCl 2 was added to the mixture and the clotting time recorded in an Amelung KC4A coagulometer (Heinrich Amelung GmbH; Lemgo, Germany). The results where expressed as the ratio of clotting time in the presence (T) and absence (T 0 ) of different volumes (2-10 μ L) of the heparins samples (50 μ g mL −1 ) or the 6 th International Heparin Standard (10 IU mL −1 ) and then fit to second order polynomial curves 31 . Anticoagulant potencies of the heparins samples as IU mg −1 (as dry weight) were derived from the parameters obtained in the 6 th International Heparin Standard fit.
In vitro anti-FXa and anti-FIIa activities. The heparins were subject of amidolytic activity assessments of thrombin (FIIa) or factor Xa (FXa) by measuring the hydrolysis of chromogenic substrates as previously described 10,11 . Incubations were performed in 96-well plates with 10 nM antithrombin, 2.0 nM FXa or thrombin (all from Hematologic Technologies; Essex Junction, USA) and 0 to 2 μ g mL −1 heparins (dry weight) in TS/PEG buffer (0.02 M Tris/HCl, 0.15 M NaCl and 1.0 mg mL −1 polyethylene glycol 8,000, pH 7.4). FXa or FIIa were added lastly to trigger the reaction. After incubation for 60 s at 37 °C, residual FXa or FIIa activities were determined by the addition of 100 μ M of chromogenic substrates S-2765 or S-2238, respectively (both from Chromogenix; Molndal, Sweden). Absorbance (405 nm) was recorded for 300 s in a Thermomax Microplate Reader (American Devices; Sunnyvile, California). Anti-FXa and anti-FIIa activities were obtained by parallel line assays against the 6 th International Heparin Standard (NIBSC) using SoftMax Pro5.4.1 software (American Devices; Sunnyvile, California) and validated methods recommended by the pharmacopeias. When the results obtained with HB-I were adjusted to IU mL −1 in the assays, they generate coincident curves with the 6 th International Heparin Standard (NIBSC).

Assessment of venous antithrombotic activity.
Antithrombotic activity was measured in Wistar rats using rabbit brain thromboplastin (Biolab-Merieux AS, Rio de Janeiro, Brazil) as thrombogenic stimulus 18 . Animal experiments were carried out in accordance with the institutional guidelines and regulations. All experimental protocols were approved by the licensing committee of the Federal University of Rio de Janeiro (Protocol No. UFRJ-CCS-015). Rats (both sexes, ~200 g body weight, five animals per dose) were anesthetized with an intramuscular injection of 100 mg kg −1 body weight of ketamine (Cristalia; São Paulo, Brazil) and 16 mg kg −1 body weight of xylazine (Bayer AS; Leverkusen, Germany). Different doses of the heparins were infused into the right carotid artery and were letting to circulate for 5 min. The inferior vena cava was isolated and then thromboplastin (5 mg kg −1 body weight) was slowly injected intravenously and after 1 min 0.7 cm of isolated vena cava was clamped off using distal and proximal sutures. After 20 min stasis the thrombus formed inside the occluded segment was carefully pulled out, washed with phosphate-buffered saline, dried for 1 h at 60 °C and weighted. The results were expressed as a ratio of thrombus weight in the presence and absence of the heparins.
Bleeding effect. Wistar rats (both sexes, ~200 g body weight) were anesthetized as described above. A cannula was inserted into the right carotid artery for administration of the heparins at a dose of ~200 IU kg −1 body weight. After heparins circulate for 5 min the tail tip was cut (3 mm of diameter) and carefully immersed in 30 mL distilled water at room temperature. Blood loss was quantified 60 min later by measuring the hemoglobin dissolved in the water via spectrophotometry (absorbance 540 nm) and then deduced from a standard curve based on known volumes of blood. The results were expressed as ratios of blood loss of heparin treated and saline treated animals 19 . In some assays the heparins were neutralized after circulate for 5 minutes with 2 mg kg −1 body weight of protamine sulfate (Valeant, São Paulo, Brazil) and then the bleeding assay was carried out as described above. One mg of protamine neutralizes 100 IU of heparin 34 . Statistical analysis. Statistical analyses were performed using Sigma Stat software (Systat; San Diego, USA) and OriginPro 8 software (OriginLab; Northtampton, USA).