Size-Dependent Effects of Nanoparticles on Enzymes in the Blood

Jul 15, 2014 - activation complex, (Kallikrein, Factor XII, and high molecular weight Kininogen) have previously been identified on NPs surfaces. We s...
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Letter pubs.acs.org/NanoLett

Size-Dependent Effects of Nanoparticles on Enzymes in the Blood Coagulation Cascade Elodie Sanfins,†,* Cecilia Augustsson,‡ Björn Dahlbac̈ k,‡ Sara Linse,† and Tommy Cedervall† †

Biochemistry and Structural Biology, Chemical Centre, Lund University, Lund, Sweden Division of Clinical Chemistry, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden



S Supporting Information *

ABSTRACT: Nanoparticles (NPs) are increasingly used in diagnostic and drug delivery. After entering the bloodstream, a protein corona will form around NPs. The size and curvature of NPs is one of the major characteristics affecting the composition of bound protein in the corona. Key initiators of the intrinsic pathway of blood coagulation, the contact activation complex, (Kallikrein, Factor XII, and high molecular weight Kininogen) have previously been identified on NPs surfaces. We show that the functional impact of carboxylmodified polystyrene NPs on these initiators of the intrinsic pathway is size dependent. NPs with high curvature affect the enzymatic activity differently from NPs with low curvature. The size dependency is evident in full blood plasma as well as in solutions of single coagulation factors. NPs induce significant alteration of the enzymatic activity in a size-dependent manner, and enzyme kinetics studies show a critical role for NPs surface area and curvature. KEYWORDS: Protein-nanoparticle, interaction, activity, kinetics, blood coagulation, nanosafety

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IX. FXI, 160 kDa glycoprotein, is activated by FXIIa. The major proteolytic activity of FXIa is toward FIX. This last reaction is the starting point of the common pathway of coagulation producing thrombin.7 (Figure 1) Nanoparticles (NPs) have chemical, physical, and biological properties that differ from those of larger particles of the same material. With decreasing particle size, the surface area increases in relation to its volume and mass, which potentially can change the surface reactivity.8 Because of their properties, nanomaterials (NMs) are increasingly used in medical applications such as diagnosis or drug delivery. However, once NPs have entered the bloodstream they can interact at the molecular level with proteins that form a protein corona. 9,10 NP−protein interactions can induce conformational changes and functional alterations in the proteins,11−18 which provide a molecular explanation for the observed biological impacts of NPs.19 Proteins from the coagulation cascade are often found in the corona around NPs.10,20,21 Moreover, different kinds of NPs can either hinder or promote blood coagulation.22−26 The effects of NPs on blood coagulation are related to size and curvature.22,27−29 Two hypotheses can be envisioned to explain the size and curvature effect: (i) the contact activation complex could not form correctly as the surface curvature on small NPs could sterically hinder correct complex formation. (ii) The function of proteins adsorbed on the NPs surface is modified because of conformational changes or steric interference with

lood coagulation is an amplification of sequential enzymatic steps that leads to the production of active thrombin, which cleaves fibrinogen to fibrin. Two pathways lead to the production of the fibrin clot: the intrinsic and extrinsic pathway (Figure 1). After a tissue trauma, the subendothelial tissue factor is expressed or exposed to the blood flow, which initiates the extrinsic pathway.1 In the absence of tissue damage, the blood still contains all the factors needed to start the coagulation cascade and a clot is formed through the intrinsic pathway.2 The initiating step of the intrinsic pathway is the surface-contact activation of the blood zymogen Factor XII (FXII), a 80 kDa glycoprotein, which is transformed to an active protease by either kallikrein (Kal) or by autoactivation.3 The amino-terminal region of the protein contains the major binding site for negatively charged surfaces, whereas the carboxy-terminal region contains the enzymatic active site. FXII activation is thought to occur upon contact or binding with material surfaces provided by glass, polyphosphates, collagen, or kaolin.4 The preference for anionic hydrophilic surface is also due to FXII’s cofactor, the high molecular weight kininogen (HMWK), which has a binding site for negatively charged surfaces. With a molecular weight of about 120 kDa, HMWK is a nonenzymatic cofactor central to contact activation reactions.5 A contact-activation complex, formed by prekallikrein (PK), factor XI (FXI), HMWK, and FXII, is thought to bring all the factors into reactive proximity and start the coagulation reaction cascade.6 PK is an 80 kDa protein that in plasma circulates in complex with HMWK. FXIIa cleaves PK into one 46 kDa heavy chain6 and one 36 or 33 kDa light chain. Kal activates FXII, plasminogen, and factor © 2014 American Chemical Society

Received: May 20, 2014 Revised: July 8, 2014 Published: July 15, 2014 4736

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Figure 1. Coagulation cascade. Intrinsic pathway: Surface-mediated interactions in contact activation of plasma coagulation involve: PK, HMWK, FXII, FXI, and Kal. Suffixes “a” represent activated proteins. FXII “binds” to a negatively charged surface (represented by the gray box, e.g., glass, kaolin, or NPs). Binding induces a conformational change in FXII, ultimately leading to a transformation into an active-enzyme form FXIIa through a process known as autoactivation.3 In turn, FXIIa generated at the surface can cleave PK bound to the surface as a complex with HMWK. Ultimately, FXIIa activates FXI bound at the surface as a complex with HMWK to generate FXIa, leading to propagation of subsequent coagulation cascade reactions. Extrinsic pathway: the tissue factor (TF) produced during trauma induces the formation of FVIIa. These two pathways lead to the common pathway producing thrombin.

Figure 2. Thrombin generation assay. Carboxyl-modified polystyrene NPs activate the production of thrombin in plasma. Plasma was incubated in the presence or absence of different size of COOH-PS NPs and tested for thrombin generation using a modified thrombin generation assay (as described in Supporting Information). The first derivative, fluorescent units/min is shown. (a) Different size of COOH-PS NPs with the same surface area 12.5 m2/L. (b) Same NPs were used but with 1.25 m2/L surface area.

the active site. To distinguish between these two hypotheses, the impact of negatively charged carboxyl-modified polystyrene NPs (COOH-PS NPs) of different size, that is, different surface area and curvature, on complex formation and enzymatic activity was evaluated. Analysis of enzyme kinetics revealed a combination of a noncompetitive and an uncompetitive inhibition of Kal by carboxyl-modified polystyrene NPs (COOH-PS NPs) at four different sizes. The activity of FXIIa is maintained exclusively on the surface of 220 nm diameter COOH-PS NPs and furthermore the FXII zymogen is activated in the presence of 220 nm COOH-PS NPs. This demonstrates that the NPs curvature and surface area are essential parameters that modulate the activity of coagulation related enzymes and can alter the natural balance between antiand procoagulant mechanisms. First, we showed that in citrated stabilized plasma larger carboxyl-modified PS NPs (60 to 220 nm) are able to trigger

the production of thrombin while smaller particles (26 and 24 nm) are unable to do so (Figure 2 and Supporting Information Figure S3). As we used a modified thrombin generation assay (TGA) without tissue-factor, which targets the intrinsic coagulation pathway, the result indicates that the contact activation complex has a role in the generation of thrombin mediated by large NPs. The modified TGA shows variation between different pools of plasma. In Supporting Information Figure S3, the 60 nm NPs lead to less thrombin generation at a lower surface area (1.25 m2/L). However, the 26 and 24 nm NPs are still unable to trigger the generation of thrombin in any of the plasma pools. Next, we measured the activity of Kal and FXIIa bound to NPs. Briefly, aliquots of plasma were exposed to different concentration of various sizes of NPs. After incubation, the NPs were centrifuged and the activity of proteins adsorbed on the NPs surface was measured using a specific substrate of Kal and FXII, S-2302 (Figure 3a). The 220 4737

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Figure 3. Activity of the complex in plasma at a fixed weight per volume concentration. The specific activity of the contact activation complex was measured in the plasma in the presence of different size of NPs using the S-2302 substrate. The activity was measured on the pellet after centrifugation (a). The activity is represented as the percentage of control activity. Error bars represent standard deviation and the number of parallel samples is n = 3. (b) Each NPs pellets was also analyzed via Western blot for the presence of HMWK and FXII. Lane 1 to 3 correspond to 0.5, 1, and 2 mg/mL of NPs, respectively.

Figure 4. Activation of purified FXII zymogen. (a) FXII zymogen was incubated at 0.4 μM with increasing concentration of 220, 93, 60, and 26 nm nanoparticles for 30 min at 37 °C and then an activity assay was then performed on each samples. The reaction velocity is plotted against the NPs surface area for the four sort of NPs. Error bars represent standard deviation and the number of parallel samples is n = 3. Each sample was centrifuged 30 min at 13 000g. The supernatant and the NPs pellet were separated and the pellet was washed with PBS buffer. The proteins from the NPs pellet and supernatant were analyzed by Western blot with a specific antibody raised against FXII. Lane 0 is the FXII without NPs; lane 1 to 6 are the proteins in the pellet and supernatant of 220 nm (b,c), 93 nm (d,e) 60 nm (f,g), and 26 nm (h,i) NPs.

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zymogen or FXIIa were found in the supernatant of the samples containing 60 and 93 nm NPs, showing that at these concentrations, FXII is strongly bound to the NPs. In the presence of the 26 nm NPs, no activation of the FXII zymogen was observed (Figure 4a). The smallest NPs (26 nm) are known to be difficult to spin down. Nevertheless, the protein adsorbed on the NPs shown in Figure 4h,i attest that these particles in the presence of protein are able to sediment. Moreover, the Western blot analysis confirmed that the major form of the protein is the zymogen. From now on, we measure the impact of NPs on FXIIa to evaluate in detail the impact of NPs on triggering the sequential cascade of coagulation by the active enzyme rather than their impact on the enzyme activation per se. To study the impact of NPs curvature on the blood coagulation activation complex only, purified Kal and FXIIa were exposed to increasing size of NPs (from 26 to 220 nm) at a constant concentration (0.1 mg/mL) thus corresponding to decreasing surface area. As shown in Figure 5a, there is an inverse correlation between the NPs surface area and the protein activity for Kal and FXIIa. More precisely, the 26 and 60 nm NPs have a stronger effect on the activity compared to

nm COOH-PS NPs have strong positive effect on the activity of the complex. In comparison, smaller NPs have a weak effect on the complex activity. The presence of Kal, FXII, and HMWK was evaluated by recovering bound proteins on the NPs in the pellet by adding 4× SDS sample buffer and analyzing the proteins by Western blot. HMWK and FXII are both found on the 60, 93, and 220 nm NPs but not on 26 nm NPs (Figure 3b). Surprisingly, Kal is not present on any NPs used in this study when mixed with plasma. During the centrifugation assay, proteins that are weakly bound to the NPs will not be detected by Western blot analysis of NPs pellet. In the TGA, no centrifugation step is used, therefore weak interactions with the contact activation complex may have an impact the generation of thrombin. These experiments demonstrate that the NPs-dependent activation of the contact activation complex in plasma requires NPs with low curvature. A possible explanation could lie in the need for sufficiently large and plane surfaces where the FXII, Kal, and HMWK can simultaneously interact with the NPs negative surface. In plasma, the initiation of the intrinsic coagulation cascade by FXII and HMWK is known to rely on the interaction of these proteins with negatively charged surfaces. We extrapolated the hydrodynamic diameters of FXI, HMWK, FXII, and Kal from their molecular weight and obtained values of 9.5, 8.6, 7.5, and 7.5 nm, respectively. Considering that three of these proteins need to interact with negatively charged surfaces to be activated, one can conceive that only NPs with a large enough hydrodynamic diameter (e.g., 220 nm) can provide sufficient surface area and low curvature to accommodate/adsorb this large protein-complex while conserving the protein conformation (no distortion of the protein structure) and the near proximity of the proteins in the complex and their function/ activity. To confirm the activation of the contact activation complex pathway, we measured the activation of the purified FXII zymogen into FXIIa by NPs of different size. The zymogen FXII is incubated with increasing concentrations of NPs at 37 °C for 30 min. After incubation, the activity is measured using a chromogenic substrate specific for activated FXIIa. This allows us to measure the activation of FXII by NPs. Figure 4a shows an increase in FXII activation in the presence of 220 nm NPs with a maximum around 7.5 m2/L. Smaller NPs, 60 and 93 nm, needed less surface area to activate the FXII zymogen. This experiment confirms that the NPs with a diameter over 60 nm are able to activate FXII, the first zymogen of the intrinsic coagulation cascade. We could not measure FXIIa activity in the presence of 26 nm COOH-NPs, indicating that such small NPs could not activate FXIIa. The centrifugation assays showed the presence of FXII or FXIIa either in the pellet (Figure 4b) or in the supernatant (Figure 4c) of 220 COOH PS NPs. The FXII zymogen (80 kDa Band) is clearly present in the supernatant of samples where no activity could be measured. More interestingly, the 52 kDa band representing the FXIIa is present in the pellet indicating that the active form of the factor is adsorbed on the NPs surface. At the NPs concentration where the FXII is activated, we can clearly see that the FXII zymogen band disappears from the supernatant while the FXIIa band appears on the NPs. For the 93 nm (Figure 4d,e) and the 60 nm (Figure 4f,g), there is a mix between 80 and 52 kDa bands in the pellet. At the concentrations where the FXIIa activity is optimal (3 and 4 m2/ L respectively for the 60 and 93 nm NPs), the major protein is the 52 kDa band suggesting that only FXIIa is present at these concentrations with high enzymatic activity. Little or no FXII

Figure 5. Fixed weight per volume concentration and fixed surface area. (a) Kal and FXIIa were incubated with 0.1 mg/mL of different size NPs (from 26 to 220 nm) for 30 min at 37 °C then the remaining activity of the proteins were measured using the method described in Supporting Information. Dark bars represent FXIIa remaining activity; light gray bars represent Kal activity. The curvature of each NP is plotted (triangles). As a comparison, the surface area of 0.1 mg/mL of each NP size is noted next to each NPs diameter. Error bars represent standard deviation and the number of parallel samples is n = 6. (b) Kal and FXIIa were incubated with different size NPs (from 26 to 220 nm) at a fixed surface area of 6 m2/L (the NPs concentration in mg/mL is indicated near each diameter on the x-axis) for 30 min at 37 °C then the remaining activity of the proteins were measured using the method described in Supporting Information. Dark bars represent FXIIa remaining activity; light gray bars represent Kal activity. Error bars represent standard deviation and the number of parallel samples is n = 6. 4739

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Figure 6. Kinetics of FXIIa activity in the presence of NPs. Purified FXIIa (0.4 μM) was incubated with 26 nm (a), 60 nm (b), 93 nm (c), and 220 nm (d) COOH-PS NPs for 30 min at 37 °C. Aliquots of each sample were then assayed with increasing concentration of substrate (from 125 μM to 1 mM) and initial rate was plotted against substrate concentration. Data for each NPs concentration were fitted, as for Kal, with the Michaelis− Menten equation (solid lines, eq 1). Western Blot analysis (insets): Purified FXII (0.4 μM) was incubated with increasing concentration of NPs (2, 1, 0.5 mg/mL) for 30 min at 37 °C and then each sample was centrifuged for 30 min at 37 °C and the remaining proteins in the supernatants were analyzed by Western blot (see Material and Methods in Supporting Information). Error bars represent standard deviation and the number of parallel samples is n = 6.

12.5 m2/L, carboxyl-modified polystyrene 60 nm NPs act as an activator of the coagulation cascade (as seen in the modified thrombin generation assay, Figure 2) at 6 m2/L these NPs act as an inhibitor of the contact activation system depending on the plasma pools (Figure 2 and Supporting Information Figure S3). In comparison, we observe a larger effect observed for Kal, which corroborates the previous results implying that FXIIa is more prone to be stabilized on the surface of larger NPs compared to Kal (Figure 5b). Our results support previous observation by Sperling et al., who studied the ability of carboxyl-modified surfaces to activate the contact activation system. They found the strongest activation rate of FXII for 100% −COOH coated glass or silicon surfaces.30 The mechanisms behind the curvature-dependent effects on the enzymatic activity can be further understood by performing kinetics studies. FXIIa purified from human plasma was incubated with increasing concentrations of NPs with diameters ranging from 26 to 220 nm (Supporting Information Table S1). The initial rate (Vi) of the enzymatic reaction at different concentrations of substrate was measured in the presence of 0.4 μM FXIIa (estimated concentration of FXIIa in human plasma31) and different concentration of PS-COOH NPs. (Figure 6) Data were fitted on the Michaelis−Menten eq 1 using the GraphPad Prism Software

the 93 and 220 nm NPs. The 93 or 220 nm NPs have almost no effect on the activity of the FXIIa whereas there is almost a 50% decrease of Kal activity in the presence of 93 nm NPs. Figure 5a shows for each NP the mean curvature (which is the inverse of the radius obtained by DLS measurements, Supporting Information Table S3). These data show that the activity of the FXIIa increases as the curvature of NPs decreases. For NPs with a diameter of 60 nm or smaller, the curvature of the NPs surface is too high to act as a surfacecontact activator, whereas above 60 nm the NPs surface has a positive effect on FXIIa activity. It is noteworthy that for a defined concentration, the surface area and the curvature of a NP increase as the diameter of the NPs decreases. Thus, the present experiment suggests that the gain of activity observed for FXIIa in the presence of 220 nm NPs is rather due to a lower surface curvature than to the increase of surface area. To further characterize the effect of surface area and curvature of NPs, we measured the activity of Kal and FXIIa in a fixed NP surface area (6 m2/L) for different NP sizes. We observe an increase of FXIIa activity on the NPs with low curvature (93 and 220 nm, Figure 5b). The effect of the 26 nm NPs at surface area of 6 m2/L on Kal and FXIIa activity is very low (0.028 mg/mL) indicating that there is small or no effect on protease activity. On the contrary, the 60 nm NPs act as a good inhibitor of FXIIa and Kal activity; when exposing the enzymes to 6 m2/L NPs surface area, the activity of FXII and Kal decreases to 50 and 75%, respectively. At a surface area of

V0 = 4740

Vmax . [S] K m + [S]

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Figure 7. Kinetics of Kal activity in the presence of NPs. Purified Kal (0.6 μM) was incubated with 26 nm (a), 60 nm (b), 93 nm (c), and 220 nm (d) COOH-PS NPs for 30 min at 37 °C. Aliquots of each sample were then assayed with increasing concentration of substrate (from 125 μM to 1 mM) and initial rate was plotted against substrate concentration. Data were then fitted with the Michaelis−Menten equation for each NPs concentration (solid lines, eq 1). Western Blot analysis (insets): purified Kal (0.6 μM) was incubated with increasing concentration of NPs (2, 1, 0.5 mg/mL) for 30 min at 37 °C then each sample was centrifuged for 30 min at 37 °C and the remaining proteins in the supernatants were analyzed by Western blot (see Supporting Information). Error bars represent standard deviation and the number of parallel samples is n = 6.

strong negative effect on the Ceff of the FXIIa but the effect is independent of the NPs concentration. In contrast, the Ceff is not affected by the presence of 60 and 93 nm NPs (Supporting Information Table S1). Moreover, the catalytic constant does not decrease in the presence of 220 nm NPs and further is significantly increased for the lower concentration of NPs. Centrifugation assays followed by Western blot analysis were performed on the purified FXIIa exposed to increasing concentrations of NPs (0.5, 1, 2 mg/mL). In the inset of Figure 6, we can see that there is a dose-dependent increase of FXIIa in the supernatant as the concentration of NPs decreases. Furthermore, FXIIa appears to be completely adsorbed on the 220 nm NPs as almost no signal is visible in the supernatants. These centrifugation assays explicitly show that the proteins are cosedimented with the NPs as proteins disappear from the supernatant and confirm that they are adsorbed on the NPs surface. Overall the results indicate that the NPs inhibition mechanism of 26, 60, and 93 nm NPs follow an uncompetitive inhibition model confirmed by the decrease of both Km and kcat with increasing concentration of NPs. This model implies that the inhibitor only binds to the enzyme−substrate complex. However, the decrease of Vmax can also be the result of a noncompetitive model of inhibition where the inhibitor can bind to the free enzyme and the enzyme−substrate complex. Thus, FXIIa inhibition by COOH-PS NPs could be a combined effect of an uncompetitive and a noncompetitive mechanism. The inhibitor interacts with the free enzyme, preventing the

where V0 is the initial rate of the reaction, Vmax is the maximum initial velocity of the reaction, [S] is the concentration of substrate, and Km is the Michaelis−Menten constant. Km is related to the inverse of the enzyme affinity toward its substrate and as the Michaelis−Menten constant decreases the apparent affinity of the enzyme for its substrate increases. For the NPs with a diameter of 26, 60, and 93 nm, there is a clear decrease of Vmax and Km as the concentration of NPs increases (Supporting Information Table S1). More strikingly, the 220 nm NPs have a positive effect on FXIIa in terms of initial rate (V0). To further characterize the impact of the NPs on the catalytic activity of FXIIa, we calculated the catalytic efficiency (Ceff., eq 2) of the enzyme in the presence of the different NPs concentrations/sizes

Ceff =

kcat Km

(2)

where kcat is the catalytic constant (eq 3) and [Et] is the total concentration of enzyme during the reaction kcat =

Vmax [Et]

(3)

The Ceff is a useful tool for characterizing the effect of NPs on enzymes as it considers both the impact on the enzymes capacity to bind its ligands (Km) and its ability to process the chemical reaction (kcat). In presence of NPs, 26 nm NPs have a 4741

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Figure 8. The specific activity of the contact activation complex. (a) The residual activity of FXIIa in the presence of 220 and 26 nm NPs was measured in buffer containing only HMWK (0.6 μM) or both Kal (0.6 μM) and HMWK (0.6 μM). (b) The residual activity of Kal in the presence of 220 and 26 nm NPs was measured in buffer containing only HMWK (0.6 μM) or both FXIIa (0.4 μM) and HMWK (0.6 μM). Error bars represent standard deviation and the number of parallel samples is n = 3.

zymogen (Figure 4) observed specifically for the largest 220 nm NPs. Similarly to FXIIa kinetic studies, analyses have been performed on Kal. Kal, 0.6 μM, was incubated with increasing concentrations of different size of NPs ranging from 26 to 220 nm (Figure 7). As shown in Figure 7, the Vmax clearly decreases as the concentration of NPs increases. Likewise for all sizes of NPs, the Km constant decreases with the increase of the NPs concentration. Furthermore, the loss of activity is dosedependent as the concentration of NPs increases. There is approximately 50% loss of Ceff in the presence of 26 and 220 nm COOH-PS NPs, but this enzymatic parameter is not affected by the NPs concentration (Supporting Information Table S2). This result implies that the loss of enzymatic activity (Vmax decreasing) of the Kal is somewhat compensated by an apparent gain of substrate affinity (Km decreasing). Surprisingly, although the affinity and catalytic constant change, the catalytic efficiency remains unchanged in the presence of 60 and 93 nm in comparison with the control. This result suggests that the enzyme inactivation may be due to a steric effect of the NPs preventing the substrate either to reach or to escape the active site of the enzyme, leading in both cases to blocked catalytic reactions. Centrifugation assays followed by Western blot analysis were performed on purified Kal exposed to increasing concentrations of NPs (0.5, 1, 2 mg/mL). In the inset of Figure 7, we can see that there is a dose-dependent increase of Kal in the

substrate to enter the catalytic site and decreasing the turnover of the enzymatic reaction (steric hindrance). Moreover, the NPs interact with the enzyme−substrate complex increasing the apparent affinity of the enzyme for its substrate. In contrast, large NPs (220 nm) have a positive effect on the FXIIa activity, as shown by a higher turnover rate (kcat) and the positive impact on the Ceff in the presence of 220 nm NPs. These results probably mean that proteins adsorbed on the surface of the NPs are functional and even more active than free protein. Furthermore, the impact of the curvature of the NPs is correlated with the gain of FXIIa activity. The FXII is known to bind and be activated upon contact to negatively charged surfaces.4 FXII adsorbed on negatively charged polystyrene NPs could be stabilized and its enzymatic activity enhanced. Chen and collaborators32 have hypothesized that negatively charged surface could induce ordered FXII adsorption. Using sum frequency generation vibrational spectroscopy, they confirmed specific conformations/orientations that facilitates FXII activation and makes it more susceptible to proteolytic cleavage. Furthermore, Griffin had already in 1978 estimated that surface-bound FXII is around 500 times more susceptible than soluble FXII to proteolytic activation by Kal in the presence of HMWK.33 The stabilization of FXIIa and the activation of the FXII zymogen on NPs surface are in good agreement with the low curvature of the NPs that is likely to enable a functional conformation of the enzyme, supporting the particular behavior of FXIIa and the activation of the FXII 4742

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supernatant as the concentration of NPs decreases. The centrifugation assays explicitly show that the protein is adsorbed on the NPs surface for all sizes. These results are in agreement with recent mass-spectrometry studies that showed the presence of Kal adsorbed on the surface of polystyrene NPs after incubation in human plasma.34 Similarly to FXIIa inhibition by 26, 60, and 93 nm COOHPS NPs, the mechanism of inhibition of Kal by all sizes of NPs used in this study is a mix between uncompetitive and noncompetitive inhibition mechanism indicated by the fact that both Km and kcat decrease in the presence of increasing concentrations of NPs. Moreover, NPs not only bind to the enzyme−substrate complex but also to the free enzyme. Finally, we measured the direct impact of polystyrene NPs on the complete contact activation complex by coincubation of Kal, FXIIa, and their cofactor HMWK at their estimated plasma concentration31 with different concentrations of NPs and measured the specific activity of each enzyme (Figure 8). The HMWK, as a cofactor, has a protective effect in this experiment. In the presence of HMWK, the inhibition of Kal and FXIIa by the NPs is lost but more surprisingly the positive effect of 220 nm NPs on FXIIa activity disappears as well. Thus, in this protein-purified model of the contact activation complex the HMWK appears to counteract the effect of the NPs by either coating the NP surface and/or interacting with the FXIIa and Kal. In conclusion, we have studied the interaction of the contact activation complex involved in the initiation of the intrinsic pathway of coagulation with carboxyl-modified polystyrene NPs. We have characterized a strong interaction between the proteins and the NPs as well as a subsequent functional alteration. Because of the important impact of the surface curvature of the NPs we observe both positive and negative effects on the proteins activity when exposed to negatively charged surfaces. Our results demonstrate that NPs can modify the natural balance of coagulation and act as anti- or procoagulant surfaces depending on their surface curvature and surface area. As activation, stabilization, and inhibition of enzyme can be achieved, these results provide a better understanding of the bioactivity of NPs in physiological environment and the role played by NPs−proteins interactions in their toxicity mechanism.



ASSOCIATED CONTENT

Detailed tables of kinetic constants, DLS measurements, TEM images, and description of the material and methods. This material is available free of charge via the Internet at http:// pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: elodie.sanfi[email protected]. Notes

The authors declare no competing financial interest.



REFERENCES

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ACKNOWLEDGMENTS

This work was supported by grants from the Lawski Foundation (ES) and the Nanometer Structure Consortium at Lund University (TC) and by the Swedish Research Foundation (#70413 to BD). 4743

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