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Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials
Biomaterials 135 (2017) 1e9
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Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials Priscilla S. Briquez a, b, 1, Kristen M. Lorentz a, c, 1, Hans M. Larsson d, Peter Frey a, Jeffrey A. Hubbell a, b, * Institute of Bioengineering, Ecole Polytechnique F ed erale de Lausanne (EPFL), 1015 Lausanne, Switzerland Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637, United States c Anokion, Inc., Cambridge MA 02139, United States d Centre Hospitalier Universitaire Vaudois (CHUV), 1011 Lausanne, Switzerland a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 February 2017 Received in revised form 26 April 2017 Accepted 26 April 2017 Available online 29 April 2017
Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a humanderived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-b A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants. © 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Fibrin biomaterial Aprotinin Human Kunitz-type protease inhibitor Plasmin Protein engineering
1. Introduction Fibrin is extensively used in the clinic as a surgical sealant and continues to be explored in patients as a delivery vehicle for diverse therapeutics [1], including small molecule drugs [2], growth factors [3], and stem cells [4]. In these therapies, premature fibrinolysis not only leads to decreased treatment efficacy, but can additionally result in recurrent bleeding, leakage, or tissue dehiscence [5]. As such, many commercial fibrin sealants are stabilized with fibrinolysis inhibitors, such as the protein aprotinin or the small molecules tranexamic acid and ε-aminocaproic acid. However, as the latter two are simple lysine analogues, they are inherently less efficient than aprotinin in blocking the fibrinolysis cascade [6,7]. Moreover,
* Corresponding author. University of Chicago, ERC Room 369, 5640 S. Ellis Avenue, Chicago, IL 60637, United States. E-mail address: [email protected] (J.A. Hubbell). 1 These authors contributed equally.
there have been reports that they can negatively affect the physical and biological properties of fibrin [5]. Alternatively, aprotinin is a small, bovine-derived, Kunitz-type serine protease inhibitor that effectively inhibits the activity of several proteases, including plasmin, trypsin, chymotrypsin, and kallikrein [8]. Originally approved by the FDA in 1993, aprotinin quickly rose to popularity as a useful hemostatic agent, yet fell under debate later due to notable safety concerns spanning its systemic use and xenogeneic product origin [9,10]. Subsequent steps were taken to replace the bovine-derived aprotinin with a synthetically-produced, rather than animal-derived, bovine aprotinin in clinical fibrin glues, such as in Baxter's Tisseel/Tissucol®, to address those concerns. Despite synthetic derivation, the nonhuman nature of aprotinin continues to be associated with hypersensitivity reactions, although infrequent, ranging from skin rashes to lethal anaphylactic shock, particularly upon patient re-exposure. Indeed, about 2.5e4% of aprotinin-treated patients developed specific antibodies against the drug [11,12]. Thus, although
http://dx.doi.org/10.1016/j.biomaterials.2017.04.048 0142-9612/© 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
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aprotinin continues to be used clinically as a stabilizing factor in fibrin sealants, such as in Baxter's Tisseel/Tissucol® and Artiis® and Aventis Behring's Beriplast® P, there is a driving need for an effective human-derived protease inhibitor substitute that would allow exclusion of immunogenic, non-human protein from fibrin biomaterials. We sought to develop an effective human-derived broad-spectrum protease inhibitor, as a “humanized aprotinin”, for local use in fibrin formulations. Because aprotinin has not been found in the human proteome, we searched for human protein sequences homologous to aprotinin using BLASTp comparisons of aprotinin to homo sapiens proteins from the RefSeq database (National Center for Biotechnology Information, USA). We identified the human Kunitz-type serine Protease Inhibitor (KPI) domain in amyloid-b A4 precursor protein (APP), originally discovered by Ponte et al. [13], as being a potential candidate to substitute for aprotinin (Fig. 1). A structural comparison between KPI and aprotinin was first analyzed by Hynes et al. when reporting the X-ray structure of KPI [14]. KPI was shown to inhibit multiple proteases, notably plasmin, trypsin, chemotrypsin and factor XIa, but to lower extent than aprotinin [15e17], although the intrinsic inhibitory activities of the KPI domain can be augmented by point mutation in the sequence [17]. Here, we hypothesized that the KPI domain's apparent efficacy for local use in fibrin can be further improved by providing a mechanism for retaining it within the biomaterial. Our laboratory has previously developed protein engineering strategies for improved local protein drug delivery within fibrin. Specifically, fusion of aprotinin to a transglutaminase substrate domain derived from a2-plasmin inhibitor (a2PI1-8; NQEQVSPL) to allow its covalent crosslinking into fibrin matrices under the activity of Factor XIIIa, was found to substantially increase the stability of fibrin in vivo, by controlling its release kinetics [19].
Furthermore, fusion of protein growth factors to a high affinity extracellular matrix (ECM)-binding domain from placental growth factor-2 (PlGF-2123-144) resulted in their high e but non-covalent e retention into fibrin, leading to markedly enhanced local morphogenetic effects [20]. Here, we leveraged these two protein engineering strategies to develop an effective human-derived protease inhibitor for local use in fibrin sealants, comparing forms of human KPI domain comprising either an a2PI1-8 domain or a PlGF-2123-144 domain. We demonstrate that the engineered, fibrin-targeting KPIs were more effective than wild-type aprotinin in extending fibrin longevity, despite the intrinsically lower inhibitory activity of human KPI compared to bovine aprotinin. 2. Material and methods 2.1. Design, production and purification of recombinant KPI variants Complementary DNA (cDNA) encoding for the KPI domain of the human amyloid-b A4 precursor protein (APP-A4286-345) was obtained from the clone IRATp970C0386D (Source Bioscience, Berlin, Germany). Wild-type KPI and three fibrin-targeting KPI variants were designed with N-terminal, cleavable histidine tags for ease of purification (TABLE S1). Fibrin-targeting KPI variants were created by recombinant fusion of a transglutaminase substrate sequence of human a2-plasmin inhibitor (a2PI1-8) [19] or fusion of an extracellular matrix (ECM)-binding domain of human placental growth factor-2 (PlGF-2123-144) [20] to the C-terminus of the KPI domain, to facilitate covalent- or high affinity non-covalent interactions with fibrin, respectively. In the PlGF-2123-144A domain, the cysteine at position 142 was substituted with an alanine to prevent unnatural disulfide bridging between the fibrin targeting domain and protease
Fig. 1. Sequence and structure comparison of the protease inhibitors bovine-derived aprotinin and human-derived KPI. (A) Alignment of aprotinin and KPI sequences using CLUSTAL Omega (1.2.3) multiple sequence alignment program (European Bioinformatics Institute) demonstrates high degree of homology. Identical amino-acids (*) are highlighted in gray, and conserved substitutions (:) and semi-conserved substitutions (.) are denoted. (B) Ribbon-view structures of aprotinin (PDB database: 6PTI [18]), KPI (PDB database: 1AAP [14]), and their structural alignment (USCF Chimera software, University of California, USA) emphasize the structural similarity of the two protease inhibitors.
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inhibitor domain. All KPI variants were inserted into the expression vector pXLG (PECF Core Facility-EPFL, Lausanne, Switzerland) under an IgGk signal sequence, for expression in suspension-adapted HEK293E cells over 7 days, as described previously [20,21]. In one experiment that requires a high amount of inhibitors, KPI-PlGF-2123144A was substituted by KPI-PlGF-2123-152 for technical reasons of protein availability; both KPI-PlGF-2123-144A and KPI-PlGF-2123-152 behaved very similarly (Fig. S1), and no effect of substitution of the C at position 142 with A was apparent. KPI-PlGF-2123-152 was produced following the same procedure than KPI-PlGF-2123-144A. KPI variants were purified from the HEK culture supernatants by histidine-affinity chromatography (HisTrap HP, GE Healthcare, Little € Chalfond, UK) using an Akta FPLC protein purification system (GE Healthcare). For wild-type KPI and KPI-a2PI1-8, removal of the histidine tag was achieved by digestion with bovine thrombin (GE Healthcare) followed by size exclusion chromatography (HiLoad 16/ 60 Superdex 75, GE Healthcare), and storage in TBS pH 7.4 at 80 C. For KPI-PlGF-2123-144A and KPI-PlGF-2123-152, histidine tags were not removed, as unspecific cleavage of the proteins seemed to occur. Protein concentration was determined by absorbance at 280 nm using a Nanodrop spectrophotometer (Thermo Fisher Scientific) and corrected with the extinction coefficient given by the ProtParam bioinformatics tool (Expasy, Swiss Institute of Bioinformatics, Switzerland). Protein purity was assessed under reducing conditions by SDS-PAGE analysis. All protein batches were validated for low endotoxin content (<5 EU/mg of protein) by lipopolysaccharides detection using HEK-Blue mTLR4 cells (Invivogen, San Diego CA, USA). 2.2. Assessment of bioactivity of recombinant KPI variants via plasmin inhibition A plasmin inhibition assay was used to assess the bioactivity of KPI variants, based on the degradation of a fluorogenic plasmin substrate, namely N-succinyl-Ala-Phe-Lys-7-amido-4-methylcoumarin acetate salt (Sigma Aldrich, St. Louis MO, USA). To determine the Km between plasmin and its substrate, increasing concentrations of the substrate ranging from 8 nM to 3 mM were exposed to 2.5 nM of human plasmin (Roche, Basel, Switzerland) in PBS pH 7.4 at room temperature. Plasmin substrate degradation was monitored by fluorescence measurements (ex/em ¼ 345/438 nm) every 100 s over 10 min using €nnedorf, Switzerland). Initial vea plate reader (Safire II, Tecan, Ma locities of the reactions were determined by linear interpolation and were further converted from relative fluorescence unit (RFU) per time to a product concentration per time using a standard curve of full substrate degradation (linear conversion: DProduct [mM] ¼ 0.01 DFluorescence [RFU]). The Km was determined as the substrate concentration at the half-maximal velocity, interpolated from a curve fit of the substrate concentrations plotted against the initial velocities (Prism v5.0a, GraphPad, La Jolia CA, USA). The inhibitory constant (Ki) of the KPI variants and plasmin were determined by incubating increasing concentrations (0.3 nMe30 mM) of KPI variants with 2.5 nM of human plasmin in the presence of 100 mM of the fluorogenic plasmin substrate. Substrate degradation was monitored by fluorescence measurements, as detailed above. The half maximal inhibitory concentration IC50 was determined by interpolation using a sigmoidal curve fitting of the logarithm of KPI variant concentrations plotted against relative initial velocities. Kis were calculated according to the formula: Ki ¼ IC50 ÷ (1 þ [S] K1 m ) with [S] the substrate concentration. The Ki of bovine aprotinin (Roche) and plasmin was determined similarly. 2.3. Preparation of fluorescent fibrinogen Lyophilized fibrinogen (FIB3 e Plasminogen-, von Willebrand
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Factor- and Fibronectin-depleted, purity >95%; Enzyme Research Laboratories, South Bend IN, USA) was resuspended in sterile endotoxin-free water, dialyzed against HEPES buffer (150 mM NaCl, 20 mM HEPES, pH 7.4), and stored at 80 C. Fluorescent fibrinogen was prepared by reacting 100 mg of fibrinogen with 0.1 mg of Alexa Fluor 680 NHS ester (Thermo Fisher Scientific) in 0.1 M sodium bicarbonate buffer pH 8.3 for 2 h at room temperature. Labeled fibrinogen was purified from unconjugated dye by gel filtration using PD-10 desalting columns (Sephadex G-25 M, GE Healthcare). 2.4. In vitro inhibition of fibrinolysis Fibrin matrices (75 mL) were made of 10 mg/mL of fibrinogen (25% w/w fluorescent fibrinogen), 2 U/mL of thrombin (Sigma Aldrich), 4 U/mL of FXIIIa, 5 mM CaCl2 and variable concentrations of KPI variant or aprotinin (Roche). After mixing reagents, fibrin matrices were incubated in a humid chamber for 1 h at 37 C, 5% CO2 to ensure complete polymerization. To model in vivo plasmin conditions, fluorescently-labeled fibrin matrices were transferred into 1 mL of TBS pH 7.4, containing physiologically relevant levels of plasmin (2.5 nM) [22], and incubated at 37 C, 5% CO2 until matrices completely degraded. The plasmin-containing supernatant was completely collected and replenished daily until full degradation of the gel. Fluorescence of remaining gel was quantified using an IVIS Spectrum in vivo imaging system (PerkinElmer, Waltham MA, USA) and analyzed with the associated Living Image software (Caliper, PerkinElmer). The same procedures were used to prepare fibrin matrices with various concentrations of fibrinogen (4, 8 or 10 mg/ mL), inhibitors (1e25 mM) and gel volumes (18.75, 37.5 or 75 mL) when optimizing parameters for extended gel longevity in vitro. 2.5. In vivo inhibition of fibrinolysis in a subcutaneous implantation mouse model All animal experimentation was approved by the ethical committee and veterinary authority of the Canton de Vaud, Switzerland. Fibrin gels (100 mL) were prepared as described above, using 12 mg/mL fibrinogen (10% w/w fluorescent fibrin) and 40 mM KPI variants or aprotinin. Fibrin gels were implanted subcutaneously on the back of female BALB/c mice, 8e10 weeks of age. As analgesia, mice were administered with Buprenorphine at 0.05 mg/ kg before the surgery and again every 12 h approximately for 2 days. During surgical procedures, animals were anesthetized with induction of 4% isoflurane followed by maintenance at 2% isoflurane. They were placed on a heating pad, with eye-protective artificial tears. The dorsal skin was shaved and disinfected with Betadine, followed by 70% ethanol. Two lateral skin incisions of about 8 mm were performed on each side of the spine axis, and small subcutaneous pockets were created by blunt tissue dissection. Fibrin matrices were inserted into the pockets, and incisions were closed by interrupted Prolene 4-0 sutures (Ethicon, Sommerville NJ, USA). Fibrin gel degradation was imaged by an IVIS Spectrum in vivo imaging system every 6 days and analyzed with the associated software (PerkinElmer, Caliper). Gel fluorescence was quantified in radiant efficiency units, in a 1-cm diameter region of interest and plotted against time to compare the efficacies of the inhibitors. The degradation speed at day 18 was calculated as the slope of gel degradation between 12 and 24 days using semi-log line interpolation. 2.6. Immunohistochemistry of explanted fibrin matrices Fifteen days after in vivo gel implantation, some mice were euthanized for immunohistochemistry analyses of partially degraded fibrin matrices. Degraded gels with surrounding skin
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were explanted and embedded into cryomatrices (Tissue-Tek O.C.T., Sakura Finetek, Alphen aan den Rijn, Netherlands) and frozen on dry-ice. Tissues were sliced at 8 mm thickness and immunohistochemistry was performed to detect fibrin matrices and KPI inhibitor variants. Briefly, tissues were fixed 2 min in ice-cold acetone, washed in PBS pH 7.4, and blocked with 10% normal horse serum for 1 h at room temperature. Then, tissues were washed and incubated overnight at 4 C in the presence of anti-human fibrinogen antibody (Pierce HYB 051-07-02, Thermo Fisher Scientific) and anti-human KPI antibody (AHP665, AbD Serotec) diluted in PBS with 1% normal serum. The following day, tissues were washed in PBS, and further incubated with AlexaFluor-647-conjugated antimouse antibody and Alexa-Fluor-488-conjugated anti-rabbit antibody (Thermo Fisher Scientific) for 1 h at room temperature, after which they were washed again in PBS. Tissues were mounted for imaging confocal microscopy (LSM700 Inverted, Zeiss). To prevent staining variability between the different conditions and allow for visual comparison, all immunohistochemical procedures were performed in parallel using common pre-mixed reagents, and images were taken using fixed parameters. 2.7. Statistical data analysis Statistical analyses were performed using Prism v5.0a (GraphPad). Statistical significance was given for p-values < 0.05. Multiple comparisons were performed either by ANOVA using Bonferroni's post-test or by Kruskal-Wallis statistics with Dunns post-test for the in vivo experimentation, in which the normality of the data was rejected by Kolmogorov-Smirnov test. Statistical tests are detailed in figure legends. 3. Results 3.1. Generation of KPI variants We engineered fibrin-targeting KPI variants by fusing a transglutaminase substrate domain of human a2-plasmin inhibitor (a2PI1-8) [19] or an ECM-binding domain of placental growth factor 2 (PlGF-2123-144A) [20] to the C-terminus of wild-type KPI, to enable covalent- or high affinity non-covalent interactions with fibrin, respectively (Fig. 2A). KPI variants were purified by affinity and size exclusion chromatography, to reach a high purity level, as revealed by SDS-PAGE (Fig. 2B). On 15% polyacrylamide gels, KPI variants migrated at sizes ranging between 12 and 15 kDa, slightly higher than their theoretical molecular weights of 6.9 kDa for wild-type KPI, 8.0 kDa for KPI-a2PI1-8 and 11.2 kDa for KPI-PlGF-2123-144A (Fig. 2B). 3.2. Wild-type and engineered KPI variants inhibit plasmin We determined the bioactivity of KPI variants using a plasmin inhibition assay. First, we measured the Km between the fluorogenic plasmin substrate N-succinyl-Ala-Phe-Lys-7-amido-4methylcoumarin acetate salt and plasmin (Fig. 2C). More precisely, we tested 12 concentrations of the fluorogenic substrate, ranging from 8 nM to 3 mM, and monitored the formation of cleaved fluorescent product when incubated with physiological level of plasmin (2.5 nM) [22]. The apparent Km was calculated by a Michaelis-Menten kinetic fit, and was found to be 269.6 mM. This allowed us to further determined the Ki of wild-type and engineered KPI variants (Fig. 2D, E). We tested 11 concentrations of KPI variants for their ability to inhibit 2.5 nM of plasmin, and calculated the IC50 values and subsequent Ki constants for each variant. We found Kis of 29.4 nM, 46.0 nM, and 31.7 nM for KPI, KPI-a2PI1-8 and KPI-PlGF-2123-144A respectively (Fig. 2D, E), thus demonstrating that
all KPI variants inhibit plasmin to similar extent. As a comparison, the more powerful inhibitor, aprotinin, was estimated to have a Ki of about one order of magnitude less [15] (Fig. 2D, E). 3.3. Engineered KPI variants slow down plasmin-mediated fibrinolysis in vitro We then evaluated the ability of engineered KPI variants to protect against fibrinolysis in vitro, and compared them to the clinically relevant inhibitor, aprotinin. Fluorescent fibrin matrices containing 15 mM of inhibitors were incubated with plasmin, and their degradation was monitored daily by fluorescence decay measurements (Fig. 3A, B). We observed that fibrin gels containing wild-type KPI completely degraded after 5 days, similarly to fibrin without inhibitor, showing that wild-type KPI did not provide protection against plasmin-mediated fibrinolysis at the tested concentration. In contrast, the longevity of fibrin matrices containing aprotinin was extended to 8 days. KPI-PlGF-2123-144A and KPI-a2PI1-8 further outperformed the aprotinin control by significantly prolonging the longevity of fibrin gels to 11 and 15 days, respectively (Fig. 3B). We then sought to further optimize relative concentrations of fibrinogen and inhibitors in vitro before assessing efficacy of KPI variants in vivo. We screened 3 fibrin concentrations, 5 inhibitor concentrations, and 3 gel volumes to determine the influence of these parameters on degradation rates of fibrin matrices, by using methods analogous to those previously described (Fig. 4A, B; Fig. S2). In this experiment, we replaced KPI-PlGF-2123-144A by KPIPlGF-2123-152 because of technical reasons, after having confirmed that both variants displayed the same binding properties to fibrin and inhibitory activity on plasmin (Fig. S1). The slowest matrix degradation rate was observed for matrices made of 12 mg/mL fibrinogen and 25 mM of KPI-a2PI1-8. Increasing inhibitor concentration resulted in an increase in gel longevity, as expected, for all inhibitors except wild-type KPI, which did not show detectable levels of efficacy at any of the tested concentrations (Fig. 4A, B). Although KPI-a2PI1-8 demonstrated the best protection against in vitro fibrinolysis at the highest tested concentration of 25 mM, higher concentrations may be further beneficial, since a plateau in efficacy of protection had not been noted even at the highest inhibitor concentration. In addition to increases in inhibitor concentrations, it was demonstrated that increases in fibrin concentrations and gel volumes contributed to longer matrix longevity, as expected (Fig. 4C; Fig. S2). 3.4. KPI-a2PI1-8 extends the longevity of fibrin matrices in vivo To study the anti-fibrinolytic properties of engineered KPIs in vivo, fluorescent fibrin matrices containing 40 mM of the different inhibitors were implanted subcutaneously in the back of mice. Similar degradation rates were observed for all groups during the first 12 days, after which only fibrin matrices containing KPI-a2PI1-8 showed a significant increase in longevity (Fig. 5A). This was further correlated with a simultaneous decrease in fibrin degradation rate (Fig. 5B). Consequently, gels supplemented with KPI-a2PI1-8 lasted for more than 36 days whereas those without inhibitor were not detectable after 24 days, representing a gel longevity extension of about 50% (Fig. 5A, C). Surprisingly, in this experimental model, neither aprotinin nor KPI-PlGF-2123-144A resulted in protection against fibrinolysis as compared to the ‘no inhibitor’ group. Likewise, fibrin containing wild-type KPI degraded similarly to fibrin without inhibitor. Lastly, we evaluated the retention of engineered KPI variants into fibrin matrix in vivo by immunohistochemistry performed on explanted gel samples. KPI-a2PI1-8 and, to lower extent, KPI-PlGF-2123-144A, were each detectable at 15 days post-
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Fig. 2. Design and characterization of wild-type and engineered KPI variants. (A) KPI was fused to C-terminal fibrin-targeting domains a2PI1-8 or PlGF-2123-144A. (B) KPI variant size and purity were assessed by SDS-PAGE. KPI, KPI-a2PI1-8 and KPI-PlGF-2123-144A migrated at sizes ranging from 12 to 15 kDa. (C) Determination of Michaelis constant (Km) of plasmin and N-succinyl-Ala-Phe-Lys-7-amido-4-methylcoumarin acetate salt plasmin substrate (n ¼ 6, mean ± SEM). (D) Bioactivity of KPI variants characterized via a plasmin inhibition assay (n ¼ 4, mean ± SEM). (E) Inhibition constant Ki were calculated from IC50 values obtained by data interpolation using a sigmoidal curve fitting (n ¼ 4, mean ± SEM). All KPI variants have similar inhibitory activities on plasmin, with Kis ranging from 29 to 46 nM. Aprotinin Ki was determined experimentally and corresponded to published data [15].
Fig. 3. Engineered KPI-a2PI1-8 and KPI-PlGF-2123-144A slow the rate of fibrinolysis in vitro. Fluorescent fibrin gels containing 15 mM of KPI variants or aprotinin were incubated in presence of 2.5 nM of plasmin. (A) Representative images of fluorescent fibrin gel degradation over time. (B) Gel degradation was monitored by quantification of fluorescence decay. Both KPI-a2PI1-8 and KPI-PlGF-2123-144A inhibit plasmin and prolong the longevity of fibrin matrices compared to matrices containing aprotinin or no inhibitor. KPI-a2PI1-8 offered the best protection against fibrinolysis (n ¼ 4 gels, mean ± SEM, ANOVA with Bonferroni post-hoc test).
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Fig. 4. Optimization of fibrinogen and inhibitor concentrations to enhance fibrin longevity in vitro. Three concentrations of fibrin and 5 concentrations of inhibitors were tested to compare the degradation time of fluorescently-labeled fibrin matrices in the presence of 2.5 nM plasmin. Gel degradation was quantified daily by fluorescence measurement and the time of complete degradation was defined by the absence of fluorescence. (A) Fibrin matrix longevity during plasmin exposure, as a function of fibrin and inhibitor concentrations. Tables indicate days at which gels were fully degraded. (B) Comparison of the inhibitors, at various concentrations, on the degradation time of 12 mg/mL fibrin matrices. (C) Influence of fibrin concentration on the degradation time of matrices supplemented with KPI-a2PI1-8.
implantation, thus reflecting the functional and strong efficacies of a2PI1-8 and PlGF-2123-144A domains in sequestering KPI within the fibrin matrices (Fig. 5D). 4. Discussion The goal of this study was to develop a human-derived protease inhibitor variant that would confer extended longevity to fibrin matrix in vitro and in vivo. This inhibitor candidate may provide potential as a clinically relevant substitute to the currently used xenogeneic inhibitor, aprotinin, and the less-potent small molecule inhibitors currently utilized within fibrin glues. We selected the Kunitz Protease Inhibitor (KPI) domain of the amyloid beta A4 precursor protein as the core to our variant protein, due to its small, nonglycosylated structure and reported homology to the functionally potent, bovine-derived inhibitor, aprotinin [16] (Fig. 1A and B). Our design comprised a fusion of the inhibitor core to a fibrintargeting domain to confer sufficient matrix protection from fibrinolysis by way of local, extended sequestration of the KPI domain within the fibrin matrix. Hence, we compared two protein engineering strategy; the variant KPI-a2PI1-8 is covalently immobilized to fibrin during normal polymerization through the enzymatic activity of the coagulation transglutaminase Factor XIIIa, whereas KPI-PlGF-2123-144A binds with a high but non-covalent affinity to fibrin. Standard methods were used to fuse the fibrin-targeting sequences a2PI1-8 [19] and PlGF-2123-144 [20] to the C-terminus of the KPI domain (Fig. 2A), and all KPI variants were successfully
expressed in HEK293 cells and highly purified (Fig. 2B). By determining the IC50 and Ki of each KPI variants toward a key fibrinolytic protease, plasmin [23], we found that engineered KPI variants displayed similar bioactivity to wild-type KPI (Fig. 2C, D, E) [17], so demonstrating that the molecular design of the engineered KPIs did not impair their inhibitory properties against plasmin. In an in vitro model of inhibition of plasmin-mediated fibrinolysis, we then showed that engineered KPI-a2PI1-8 and KPI-PlGF-2123-144A variants outperformed wild-type KPI and, more interestingly, aprotinin, despite its 10-fold higher intrinsic inhibitory power (Fig. 3A, B) [15,17]. Because intrinsic inhibitory bioactivity of KPI variants toward plasmin were highly similar, such differences in inhibitory efficacies can be attributed to the levels of inhibitor sequestration into fibrin, thus supporting that covalent immobilization of KPI mediated by a2PI1-8 improves retention compared to the noncovalent affinity conferred by PlGF-2123-144A. Even though this result was rather expected, as covalent binding is the higher possible level of protein immobilization, incorporation of high doses of KPI-a2PI1-8 could have had increased inhibitor concentration at the risk of interference with the degree of fibrin polymerization. Indeed, a2PI1-8 is permissive to forming isoglutamyl lysine isopeptide bonds at a2PI-fibrin as well as fibrin-fibrin crosslinking sites. This limitation was the rational for the design of KPI-PlGF-2123-144A variant, which facilitates high affinity binding of the inhibitor to fibrin while avoiding potential interference of fibrin polymerization. In our study however, we did not observe significant impaired fibrin polymerization and subsequent reduced fibrin gel longevity, for all tested concentrations of KPI-a2PI1-8
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Fig. 5. KPI-a2PI1-8 extends fibrin matrix longevity in vivo when implanted subcutaneously. Fluorescent fibrin matrices containing 40 mM of KPI variants or aprotinin were implanted subcutaneously in mice. (A) In vivo gel degradation was quantified through fluorescence measurements every 6 days. KPI-a2PI1-8 significantly increased fibrin longevity compared to the other inhibitors (n 7 for KPI-a2PI1-8 and ‘no inhibitor’ groups, n 4 in remaining groups, mean ± SEM, Kruskal-Wallis with Dunns post-hoc test). (B) Apparent gel degradation rate at day 18 as a function of the different inhibitors (n 7 for KPI-a2PI1-8 and ‘no inhibitor’ groups, n 4 in other groups, mean ± SEM, Kruskal-Wallis with Dunns post-hoc test). (C) Representative images of fluorescence decay measurements of subcutaneous fibrin implants loaded with the different inhibitors over time. (Blue circles: region-of-interests of 1 cm in diameter). (D) Immunohistochemistry of subcutaneously implanted fibrin matrices containing the different KPI inhibitor variants at 15 days post-implantation (blue: DAPI/ cell nuclei; red: fibrinogen; green: human KPI). Both KPI-a2PI1-8 and KPI-PlGF-2123-144A remain sequestered into fibrin matrices compared to wild-type KPI. Scale bar ¼ 100 mm.
(Fig. 4A, B), suggesting that we did not reach its limiting dose. As a proof-of-concept toward a future potential clinical application of KPI inhibitors in fibrin sealants, we used an in vivo model
of subcutaneous implantation of fibrin matrix, with optimized inhibitors and fibrin concentrations (Fig. 4A, B, C). In this model, KPIa2PI1-8 significantly extended the longevity of fibrin by about 50%
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by slowing fibrin degradation, whereas none of the other inhibitors showed efficacy, including the clinically-relevant aprotinin (Fig. 5A, B, C). The lack of efficacy of wild-type KPI and aprotinin (which is a wild-type homologous to KPI domain) is likely related to diffusive loss, since wild-type KPI was not detectable in fibrin implants at 15 days post-implantation (Fig. 5D) [19]. In contrast, KPI-a2PI1-8 and KPI-PlGF-2123-144A had strong abilities to bind and be retained into fibrin biomaterials for extended time in vivo, as demonstrated by immunohistochemistry analyses at 15 days post-implantation (Fig. 5D). Consequently, the lack of in vivo efficacy of KPI-PlGF2123-144A against fibrinolysis is unexpected; it may be explained by an insufficient level of sequestered inhibitor within the gel, or potentially by a lack of functional stability of the KPI-PlGF-2123-144A variant. Alternatively, while fusion of PlGF-2123-144A to KPI did not impair its specific inhibitory activity toward plasmin, it is possible that it could affect the KPI-related inhibition of other proteases involved in the fibrinolysis cascade. Indeed, KPI is known to be a broad-spectrum protease inhibitor, as is aprotinin, which is assumed to increase its efficacy in fibrin protection in comparison to more specific inhibitors (e.g. tranexamic or ε-aminocaproic acids) [6,7]. Therefore, it would be interesting to further assess whether the engineered KPI variants retained their broadspectrum protease inhibition characteristic, as a follow-up to this study that mainly focused on plasmin-mediated fibrinolysis. Finally, let us note that the concentration of KPI-a2PI1-8 used in vivo to demonstrate its potential for use in fibrin sealant is in the range of the aprotinin concentration currently used in the clinically approved Tisseel® sealant (Baxter Healthcare Corporation) [23], which contains about 30e50 mM of aprotinin (1125e1875 KIU/mL) [19]. On the other hand, the concentration of fibrinogen in Tisseel® (35e55 mg/mL) is about 3e5 times higher than in our fibrin formulation (12 mg/mL). However, we showed in a previous study that such increase in fibrinogen concentration did not directly convert into an extended fibrin material longevity, and that fibrinolytic inhibitors assessed at lower fibrin concentrations can remain relevant for use in commercially available fibrin sealant products [19]. 5. Conclusion We engineered a human-derived broad-spectrum protease inhibitor to propose an alternative to the clinical use of bovine aprotinin in fibrin-based tissue sealants and tissue engineering matrices. We particularly focused our work on the sequestration of a homologous human-derived KPI into fibrin, by fusing KPI to either a transglutaminase substrate domain a2PI1-8 that allows the covalent incorporation of the inhibitor into fibrin matrix, or to a high affinity fibrin-binding domain, PlGF-2123-144A, that permits its noncovalent retention. We found that both engineered variants improved the longevity of fibrin in vitro compared to wild-type KPI and aprotinin. In vivo, the engineered KPI-a2PI1-8 variant significantly increased the longevity of fibrin material by about 50%, thus outperforming the clinically used aprotinin. While the intrinsic inhibitory activity of KPI-a2PI1-8 could be further improved by point mutation [17], we believe that its human origin and simple structure yet highly functional design, contribute to its potential for manufacturability as a clinically appealing substitute to aprotinin as a fibrinolysis inhibitor in fibrin sealants and matrices. Disclosure The transglutaminase-mediated immobilization technology €ssidescribed herein is the subject of patents filed by the Eidgeno sche Technische Hochschule Zürich (ETHZ, Zürich, Switzerland), upon which J.A.H. is named as an inventor. The super-affinity based
retention technology described herein is the subject of a patent de rale de Lausanne application filed by the Ecole Polytechnique Fe (EPFL, Lausanne, Switzerland), upon which J.A.H and P.S.B are named as inventors. Acknowledgements and funding sources The authors would like to acknowledge Dr. Eleonora Simeoni for assistance on animal experimentations, Dr. Elif Vardar for fruitful discussions, Jean-Philippe Gaudry for help on protein purification, €lle J. Be zy for technical assistance. Xavier Quaglia-Thermes and Mae de rale de This research was funded by the Ecole Polytechnique Fe Lausanne, Lausanne, Switzerland and the National Institutes of Health (DP3DK108215). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2017.04.048. References [1] D.M. Albala, Fibrin sealants in clinical practice, Cardiovasc. Surg. (2003), http:// dx.doi.org/10.1016/S0967-2109(03)00065-6. [2] J.Z. Fu, J. Li, Z.L. Yu, Effect of implanting fibrin sealant with ropivacaine on pain after laparoscopic cholecystectomy, World J. Gastroenterol. WJG (2009), http://dx.doi.org/10.3748/wjg.15.5851. [3] P.L. Danielsen, M.S. Agren, L.N. Jorgensen, Platelet-rich fibrin versus albumin in surgical wound repair: a randomized trial with paired design, Ann. Surg. 251 (2010) 825e831, http://dx.doi.org/10.1097/SLA.0b013e3181d3548c. [4] D. Garcia-Olmo, D. Herreros, I. Pascual, J.A. Pascual, E. Del-Valle, J. Zorrilla, et al., Expanded adipose-derived stem cells for the treatment of complex perianal fistula: a phase II clinical trial, Dis. Colon Rectum 52 (2009) 79e86, http:// dx.doi.org/10.1007/DCR.0b013e3181973487. [5] W. Furst, A. Banerjee, H. Redl, Comparison of structure, strength and cytocompatibility of a fibrin matrix supplemented either with tranexamic acid or aprotinin, J. Biomed. Mater. Res. 82B (2007) 109e114, http://dx.doi.org/ 10.1002/jbm.b.30711. [6] E.S. Schouten, A.C. van de Pol, A.N.J. Schouten, N.M. Turner, N.J.G. Jansen, C.W. Bollen, The effect of aprotinin, tranexamic acid, and aminocaproic acid on blood loss and use of blood products in major pediatric surgery: a metaanalysis, Pediatr. Crit. Care Med. 10 (2009) 182e190, http://dx.doi.org/ 10.1097/PCC.0b013e3181956d61. [7] M. Sperzel, J. Huetter, Evaluation of aprotinin and tranexamic acid in different in vitro and in vivo models of fibrinolysis, coagulation and thrombus formation, J. Thromb. Haemost. 5 (2007) 2113e2118, http://dx.doi.org/10.1111/ j.1538-7836.2007.02717.x. [8] H.-M. Kang, M.H. Kalnoski, M. Frederick, W.L. Chandler, The kinetics of plasmin inhibition by aprotinin in vivo, Thromb. Res. 115 (2005) 327e340, http://dx.doi.org/10.1016/j.thromres.2004.09.015. [9] S. Westaby, Aprotinin: twenty-five years of claim and counterclaim, J. Thorac. Cardiovasc. Surg. 135 (2008) 487e491, http://dx.doi.org/10.1016/ j.jtcvs.2008.01.002. [10] A.M. Mahdy, Perioperative systemic haemostatic agents, Br. J. Anaesth. 93 (2004) 842e858, http://dx.doi.org/10.1093/bja/aeh227. €th, M. Zühlsdorf, H. Dalichau, P.G. Kirchhoff, H. Kuppe, et al., [11] W. Dietrich, P. Spa Anaphylactic reactions to aprotinin reexposure in cardiac surgery: relation to antiaprotinin immunoglobulin G and E antibodies, Anesthesiology 95 (2001), 64e71e discussion 5Ae6A. [12] A.M. Scheule, W. Beierlein, S. Arnold, F.S. Eckstein, J.M. Albes, G. Ziemer, The significance of preformed aprotinin-specific antibodies in cardiosurgical patients, Anesth. Analgesia 90 (2000) 262e266, http://dx.doi.org/10.1213/ 00000539-200002000-00005. [13] P. Ponte, P. Gonzalez-DeWhitt, J. Schilling, J. Miller, D. Hsu, B. Greenberg, et al., A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors, Nature 331 (1988) 525e527, http://dx.doi.org/10.1038/331525a0. [14] T.R. Hynes, M. Randal, L.A. Kennedy, C. Eigenbrot, X-ray crystal structure of the protease inhibitor domain of Alzheimer's amyloid. beta.-protein precursor, Biochemistry 29 (1990) 10018e10022, http://dx.doi.org/10.1021/ bi00495a002. [15] H. Fritz, G. Wunderer, Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs, Arzneimittelforschung 33 (1983) 479e494. [16] H. Zollner, Handbook of Enzyme Inhibitors, VCH Verlagesgesellschaft, Weinheim, 1993. [17] W.E. Van Nostrand, A.H. Schmaier, R.S. Siegel, S.L. Wagner, W.C. Raschke, Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitztype protease inhibitor domain of the amyloid beta-protein precursor, J. Biol.
P.S. Briquez et al. / Biomaterials 135 (2017) 1e9 Chem. 270 (1995) 22827e22830. [18] A. Wlodawer, J. Nachman, G.L. Gilliland, W. Gallagher, C. Woodward, Structure of form III crystals of bovine pancreatic trypsin inhibitor, J. Mol. Biol. 198 (1987) 469e480. [19] K.M. Lorentz, S. Kontos, P. Frey, J.A. Hubbell, Engineered aprotinin for improved stability of fibrin biomaterials, Biomaterials (2010) 1e9, http:// dx.doi.org/10.1016/j.biomaterials.2010.08.109. [20] M.M. Martino, P.S. Briquez, E. Guc, F. Tortelli, W.W. Kilarski, S. Metzger, et al., Growth factors engineered for super-affinity to the extracellular matrix enhance tissue healing, Science 343 (2014) 885e888, http://dx.doi.org/
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10.1126/science.1247663. [21] N. Muller, P. Girard, D.L. Hacker, M. Jordan, F.M. Wurm, Orbital shaker technology for the cultivation of mammalian cells in suspension, Biotechnol. Bioeng. 89 (2005) 400e406, http://dx.doi.org/10.1002/bit.20358. [22] W.L. Chandler, M.C. Alessi, M.F. Aillaud, P. Vague, I. Juhan-Vague, Formation, inhibition and clearance of plasmin in vivo, Haemostasis 30 (2000) 204e218, http://dx.doi.org/10.1159/000054136. [23] H. Baxter, TISSEEL, 2015, pp. 1e17. www.baxterhealthcare.com.audownloads healthcareprofessionalscmipitisseelpi.pdf.
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Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials Priscilla S. Briquez a, b, 1, Kristen M. Lorentz a, c, 1, Hans M. Larsson d, Peter Frey a, Jeffrey A. Hubbell a, b, * Institute of Bioengineering, Ecole Polytechnique F ed erale de Lausanne (EPFL), 1015 Lausanne, Switzerland Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637, United States c Anokion, Inc., Cambridge MA 02139, United States d Centre Hospitalier Universitaire Vaudois (CHUV), 1011 Lausanne, Switzerland a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 February 2017 Received in revised form 26 April 2017 Accepted 26 April 2017 Available online 29 April 2017
Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a humanderived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-b A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants. © 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Fibrin biomaterial Aprotinin Human Kunitz-type protease inhibitor Plasmin Protein engineering
1. Introduction Fibrin is extensively used in the clinic as a surgical sealant and continues to be explored in patients as a delivery vehicle for diverse therapeutics [1], including small molecule drugs [2], growth factors [3], and stem cells [4]. In these therapies, premature fibrinolysis not only leads to decreased treatment efficacy, but can additionally result in recurrent bleeding, leakage, or tissue dehiscence [5]. As such, many commercial fibrin sealants are stabilized with fibrinolysis inhibitors, such as the protein aprotinin or the small molecules tranexamic acid and ε-aminocaproic acid. However, as the latter two are simple lysine analogues, they are inherently less efficient than aprotinin in blocking the fibrinolysis cascade [6,7]. Moreover,
* Corresponding author. University of Chicago, ERC Room 369, 5640 S. Ellis Avenue, Chicago, IL 60637, United States. E-mail address: [email protected] (J.A. Hubbell). 1 These authors contributed equally.
there have been reports that they can negatively affect the physical and biological properties of fibrin [5]. Alternatively, aprotinin is a small, bovine-derived, Kunitz-type serine protease inhibitor that effectively inhibits the activity of several proteases, including plasmin, trypsin, chymotrypsin, and kallikrein [8]. Originally approved by the FDA in 1993, aprotinin quickly rose to popularity as a useful hemostatic agent, yet fell under debate later due to notable safety concerns spanning its systemic use and xenogeneic product origin [9,10]. Subsequent steps were taken to replace the bovine-derived aprotinin with a synthetically-produced, rather than animal-derived, bovine aprotinin in clinical fibrin glues, such as in Baxter's Tisseel/Tissucol®, to address those concerns. Despite synthetic derivation, the nonhuman nature of aprotinin continues to be associated with hypersensitivity reactions, although infrequent, ranging from skin rashes to lethal anaphylactic shock, particularly upon patient re-exposure. Indeed, about 2.5e4% of aprotinin-treated patients developed specific antibodies against the drug [11,12]. Thus, although
http://dx.doi.org/10.1016/j.biomaterials.2017.04.048 0142-9612/© 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
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aprotinin continues to be used clinically as a stabilizing factor in fibrin sealants, such as in Baxter's Tisseel/Tissucol® and Artiis® and Aventis Behring's Beriplast® P, there is a driving need for an effective human-derived protease inhibitor substitute that would allow exclusion of immunogenic, non-human protein from fibrin biomaterials. We sought to develop an effective human-derived broad-spectrum protease inhibitor, as a “humanized aprotinin”, for local use in fibrin formulations. Because aprotinin has not been found in the human proteome, we searched for human protein sequences homologous to aprotinin using BLASTp comparisons of aprotinin to homo sapiens proteins from the RefSeq database (National Center for Biotechnology Information, USA). We identified the human Kunitz-type serine Protease Inhibitor (KPI) domain in amyloid-b A4 precursor protein (APP), originally discovered by Ponte et al. [13], as being a potential candidate to substitute for aprotinin (Fig. 1). A structural comparison between KPI and aprotinin was first analyzed by Hynes et al. when reporting the X-ray structure of KPI [14]. KPI was shown to inhibit multiple proteases, notably plasmin, trypsin, chemotrypsin and factor XIa, but to lower extent than aprotinin [15e17], although the intrinsic inhibitory activities of the KPI domain can be augmented by point mutation in the sequence [17]. Here, we hypothesized that the KPI domain's apparent efficacy for local use in fibrin can be further improved by providing a mechanism for retaining it within the biomaterial. Our laboratory has previously developed protein engineering strategies for improved local protein drug delivery within fibrin. Specifically, fusion of aprotinin to a transglutaminase substrate domain derived from a2-plasmin inhibitor (a2PI1-8; NQEQVSPL) to allow its covalent crosslinking into fibrin matrices under the activity of Factor XIIIa, was found to substantially increase the stability of fibrin in vivo, by controlling its release kinetics [19].
Furthermore, fusion of protein growth factors to a high affinity extracellular matrix (ECM)-binding domain from placental growth factor-2 (PlGF-2123-144) resulted in their high e but non-covalent e retention into fibrin, leading to markedly enhanced local morphogenetic effects [20]. Here, we leveraged these two protein engineering strategies to develop an effective human-derived protease inhibitor for local use in fibrin sealants, comparing forms of human KPI domain comprising either an a2PI1-8 domain or a PlGF-2123-144 domain. We demonstrate that the engineered, fibrin-targeting KPIs were more effective than wild-type aprotinin in extending fibrin longevity, despite the intrinsically lower inhibitory activity of human KPI compared to bovine aprotinin. 2. Material and methods 2.1. Design, production and purification of recombinant KPI variants Complementary DNA (cDNA) encoding for the KPI domain of the human amyloid-b A4 precursor protein (APP-A4286-345) was obtained from the clone IRATp970C0386D (Source Bioscience, Berlin, Germany). Wild-type KPI and three fibrin-targeting KPI variants were designed with N-terminal, cleavable histidine tags for ease of purification (TABLE S1). Fibrin-targeting KPI variants were created by recombinant fusion of a transglutaminase substrate sequence of human a2-plasmin inhibitor (a2PI1-8) [19] or fusion of an extracellular matrix (ECM)-binding domain of human placental growth factor-2 (PlGF-2123-144) [20] to the C-terminus of the KPI domain, to facilitate covalent- or high affinity non-covalent interactions with fibrin, respectively. In the PlGF-2123-144A domain, the cysteine at position 142 was substituted with an alanine to prevent unnatural disulfide bridging between the fibrin targeting domain and protease
Fig. 1. Sequence and structure comparison of the protease inhibitors bovine-derived aprotinin and human-derived KPI. (A) Alignment of aprotinin and KPI sequences using CLUSTAL Omega (1.2.3) multiple sequence alignment program (European Bioinformatics Institute) demonstrates high degree of homology. Identical amino-acids (*) are highlighted in gray, and conserved substitutions (:) and semi-conserved substitutions (.) are denoted. (B) Ribbon-view structures of aprotinin (PDB database: 6PTI [18]), KPI (PDB database: 1AAP [14]), and their structural alignment (USCF Chimera software, University of California, USA) emphasize the structural similarity of the two protease inhibitors.
P.S. Briquez et al. / Biomaterials 135 (2017) 1e9
inhibitor domain. All KPI variants were inserted into the expression vector pXLG (PECF Core Facility-EPFL, Lausanne, Switzerland) under an IgGk signal sequence, for expression in suspension-adapted HEK293E cells over 7 days, as described previously [20,21]. In one experiment that requires a high amount of inhibitors, KPI-PlGF-2123144A was substituted by KPI-PlGF-2123-152 for technical reasons of protein availability; both KPI-PlGF-2123-144A and KPI-PlGF-2123-152 behaved very similarly (Fig. S1), and no effect of substitution of the C at position 142 with A was apparent. KPI-PlGF-2123-152 was produced following the same procedure than KPI-PlGF-2123-144A. KPI variants were purified from the HEK culture supernatants by histidine-affinity chromatography (HisTrap HP, GE Healthcare, Little € Chalfond, UK) using an Akta FPLC protein purification system (GE Healthcare). For wild-type KPI and KPI-a2PI1-8, removal of the histidine tag was achieved by digestion with bovine thrombin (GE Healthcare) followed by size exclusion chromatography (HiLoad 16/ 60 Superdex 75, GE Healthcare), and storage in TBS pH 7.4 at 80 C. For KPI-PlGF-2123-144A and KPI-PlGF-2123-152, histidine tags were not removed, as unspecific cleavage of the proteins seemed to occur. Protein concentration was determined by absorbance at 280 nm using a Nanodrop spectrophotometer (Thermo Fisher Scientific) and corrected with the extinction coefficient given by the ProtParam bioinformatics tool (Expasy, Swiss Institute of Bioinformatics, Switzerland). Protein purity was assessed under reducing conditions by SDS-PAGE analysis. All protein batches were validated for low endotoxin content (<5 EU/mg of protein) by lipopolysaccharides detection using HEK-Blue mTLR4 cells (Invivogen, San Diego CA, USA). 2.2. Assessment of bioactivity of recombinant KPI variants via plasmin inhibition A plasmin inhibition assay was used to assess the bioactivity of KPI variants, based on the degradation of a fluorogenic plasmin substrate, namely N-succinyl-Ala-Phe-Lys-7-amido-4-methylcoumarin acetate salt (Sigma Aldrich, St. Louis MO, USA). To determine the Km between plasmin and its substrate, increasing concentrations of the substrate ranging from 8 nM to 3 mM were exposed to 2.5 nM of human plasmin (Roche, Basel, Switzerland) in PBS pH 7.4 at room temperature. Plasmin substrate degradation was monitored by fluorescence measurements (ex/em ¼ 345/438 nm) every 100 s over 10 min using €nnedorf, Switzerland). Initial vea plate reader (Safire II, Tecan, Ma locities of the reactions were determined by linear interpolation and were further converted from relative fluorescence unit (RFU) per time to a product concentration per time using a standard curve of full substrate degradation (linear conversion: DProduct [mM] ¼ 0.01 DFluorescence [RFU]). The Km was determined as the substrate concentration at the half-maximal velocity, interpolated from a curve fit of the substrate concentrations plotted against the initial velocities (Prism v5.0a, GraphPad, La Jolia CA, USA). The inhibitory constant (Ki) of the KPI variants and plasmin were determined by incubating increasing concentrations (0.3 nMe30 mM) of KPI variants with 2.5 nM of human plasmin in the presence of 100 mM of the fluorogenic plasmin substrate. Substrate degradation was monitored by fluorescence measurements, as detailed above. The half maximal inhibitory concentration IC50 was determined by interpolation using a sigmoidal curve fitting of the logarithm of KPI variant concentrations plotted against relative initial velocities. Kis were calculated according to the formula: Ki ¼ IC50 ÷ (1 þ [S] K1 m ) with [S] the substrate concentration. The Ki of bovine aprotinin (Roche) and plasmin was determined similarly. 2.3. Preparation of fluorescent fibrinogen Lyophilized fibrinogen (FIB3 e Plasminogen-, von Willebrand
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Factor- and Fibronectin-depleted, purity >95%; Enzyme Research Laboratories, South Bend IN, USA) was resuspended in sterile endotoxin-free water, dialyzed against HEPES buffer (150 mM NaCl, 20 mM HEPES, pH 7.4), and stored at 80 C. Fluorescent fibrinogen was prepared by reacting 100 mg of fibrinogen with 0.1 mg of Alexa Fluor 680 NHS ester (Thermo Fisher Scientific) in 0.1 M sodium bicarbonate buffer pH 8.3 for 2 h at room temperature. Labeled fibrinogen was purified from unconjugated dye by gel filtration using PD-10 desalting columns (Sephadex G-25 M, GE Healthcare). 2.4. In vitro inhibition of fibrinolysis Fibrin matrices (75 mL) were made of 10 mg/mL of fibrinogen (25% w/w fluorescent fibrinogen), 2 U/mL of thrombin (Sigma Aldrich), 4 U/mL of FXIIIa, 5 mM CaCl2 and variable concentrations of KPI variant or aprotinin (Roche). After mixing reagents, fibrin matrices were incubated in a humid chamber for 1 h at 37 C, 5% CO2 to ensure complete polymerization. To model in vivo plasmin conditions, fluorescently-labeled fibrin matrices were transferred into 1 mL of TBS pH 7.4, containing physiologically relevant levels of plasmin (2.5 nM) [22], and incubated at 37 C, 5% CO2 until matrices completely degraded. The plasmin-containing supernatant was completely collected and replenished daily until full degradation of the gel. Fluorescence of remaining gel was quantified using an IVIS Spectrum in vivo imaging system (PerkinElmer, Waltham MA, USA) and analyzed with the associated Living Image software (Caliper, PerkinElmer). The same procedures were used to prepare fibrin matrices with various concentrations of fibrinogen (4, 8 or 10 mg/ mL), inhibitors (1e25 mM) and gel volumes (18.75, 37.5 or 75 mL) when optimizing parameters for extended gel longevity in vitro. 2.5. In vivo inhibition of fibrinolysis in a subcutaneous implantation mouse model All animal experimentation was approved by the ethical committee and veterinary authority of the Canton de Vaud, Switzerland. Fibrin gels (100 mL) were prepared as described above, using 12 mg/mL fibrinogen (10% w/w fluorescent fibrin) and 40 mM KPI variants or aprotinin. Fibrin gels were implanted subcutaneously on the back of female BALB/c mice, 8e10 weeks of age. As analgesia, mice were administered with Buprenorphine at 0.05 mg/ kg before the surgery and again every 12 h approximately for 2 days. During surgical procedures, animals were anesthetized with induction of 4% isoflurane followed by maintenance at 2% isoflurane. They were placed on a heating pad, with eye-protective artificial tears. The dorsal skin was shaved and disinfected with Betadine, followed by 70% ethanol. Two lateral skin incisions of about 8 mm were performed on each side of the spine axis, and small subcutaneous pockets were created by blunt tissue dissection. Fibrin matrices were inserted into the pockets, and incisions were closed by interrupted Prolene 4-0 sutures (Ethicon, Sommerville NJ, USA). Fibrin gel degradation was imaged by an IVIS Spectrum in vivo imaging system every 6 days and analyzed with the associated software (PerkinElmer, Caliper). Gel fluorescence was quantified in radiant efficiency units, in a 1-cm diameter region of interest and plotted against time to compare the efficacies of the inhibitors. The degradation speed at day 18 was calculated as the slope of gel degradation between 12 and 24 days using semi-log line interpolation. 2.6. Immunohistochemistry of explanted fibrin matrices Fifteen days after in vivo gel implantation, some mice were euthanized for immunohistochemistry analyses of partially degraded fibrin matrices. Degraded gels with surrounding skin
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were explanted and embedded into cryomatrices (Tissue-Tek O.C.T., Sakura Finetek, Alphen aan den Rijn, Netherlands) and frozen on dry-ice. Tissues were sliced at 8 mm thickness and immunohistochemistry was performed to detect fibrin matrices and KPI inhibitor variants. Briefly, tissues were fixed 2 min in ice-cold acetone, washed in PBS pH 7.4, and blocked with 10% normal horse serum for 1 h at room temperature. Then, tissues were washed and incubated overnight at 4 C in the presence of anti-human fibrinogen antibody (Pierce HYB 051-07-02, Thermo Fisher Scientific) and anti-human KPI antibody (AHP665, AbD Serotec) diluted in PBS with 1% normal serum. The following day, tissues were washed in PBS, and further incubated with AlexaFluor-647-conjugated antimouse antibody and Alexa-Fluor-488-conjugated anti-rabbit antibody (Thermo Fisher Scientific) for 1 h at room temperature, after which they were washed again in PBS. Tissues were mounted for imaging confocal microscopy (LSM700 Inverted, Zeiss). To prevent staining variability between the different conditions and allow for visual comparison, all immunohistochemical procedures were performed in parallel using common pre-mixed reagents, and images were taken using fixed parameters. 2.7. Statistical data analysis Statistical analyses were performed using Prism v5.0a (GraphPad). Statistical significance was given for p-values < 0.05. Multiple comparisons were performed either by ANOVA using Bonferroni's post-test or by Kruskal-Wallis statistics with Dunns post-test for the in vivo experimentation, in which the normality of the data was rejected by Kolmogorov-Smirnov test. Statistical tests are detailed in figure legends. 3. Results 3.1. Generation of KPI variants We engineered fibrin-targeting KPI variants by fusing a transglutaminase substrate domain of human a2-plasmin inhibitor (a2PI1-8) [19] or an ECM-binding domain of placental growth factor 2 (PlGF-2123-144A) [20] to the C-terminus of wild-type KPI, to enable covalent- or high affinity non-covalent interactions with fibrin, respectively (Fig. 2A). KPI variants were purified by affinity and size exclusion chromatography, to reach a high purity level, as revealed by SDS-PAGE (Fig. 2B). On 15% polyacrylamide gels, KPI variants migrated at sizes ranging between 12 and 15 kDa, slightly higher than their theoretical molecular weights of 6.9 kDa for wild-type KPI, 8.0 kDa for KPI-a2PI1-8 and 11.2 kDa for KPI-PlGF-2123-144A (Fig. 2B). 3.2. Wild-type and engineered KPI variants inhibit plasmin We determined the bioactivity of KPI variants using a plasmin inhibition assay. First, we measured the Km between the fluorogenic plasmin substrate N-succinyl-Ala-Phe-Lys-7-amido-4methylcoumarin acetate salt and plasmin (Fig. 2C). More precisely, we tested 12 concentrations of the fluorogenic substrate, ranging from 8 nM to 3 mM, and monitored the formation of cleaved fluorescent product when incubated with physiological level of plasmin (2.5 nM) [22]. The apparent Km was calculated by a Michaelis-Menten kinetic fit, and was found to be 269.6 mM. This allowed us to further determined the Ki of wild-type and engineered KPI variants (Fig. 2D, E). We tested 11 concentrations of KPI variants for their ability to inhibit 2.5 nM of plasmin, and calculated the IC50 values and subsequent Ki constants for each variant. We found Kis of 29.4 nM, 46.0 nM, and 31.7 nM for KPI, KPI-a2PI1-8 and KPI-PlGF-2123-144A respectively (Fig. 2D, E), thus demonstrating that
all KPI variants inhibit plasmin to similar extent. As a comparison, the more powerful inhibitor, aprotinin, was estimated to have a Ki of about one order of magnitude less [15] (Fig. 2D, E). 3.3. Engineered KPI variants slow down plasmin-mediated fibrinolysis in vitro We then evaluated the ability of engineered KPI variants to protect against fibrinolysis in vitro, and compared them to the clinically relevant inhibitor, aprotinin. Fluorescent fibrin matrices containing 15 mM of inhibitors were incubated with plasmin, and their degradation was monitored daily by fluorescence decay measurements (Fig. 3A, B). We observed that fibrin gels containing wild-type KPI completely degraded after 5 days, similarly to fibrin without inhibitor, showing that wild-type KPI did not provide protection against plasmin-mediated fibrinolysis at the tested concentration. In contrast, the longevity of fibrin matrices containing aprotinin was extended to 8 days. KPI-PlGF-2123-144A and KPI-a2PI1-8 further outperformed the aprotinin control by significantly prolonging the longevity of fibrin gels to 11 and 15 days, respectively (Fig. 3B). We then sought to further optimize relative concentrations of fibrinogen and inhibitors in vitro before assessing efficacy of KPI variants in vivo. We screened 3 fibrin concentrations, 5 inhibitor concentrations, and 3 gel volumes to determine the influence of these parameters on degradation rates of fibrin matrices, by using methods analogous to those previously described (Fig. 4A, B; Fig. S2). In this experiment, we replaced KPI-PlGF-2123-144A by KPIPlGF-2123-152 because of technical reasons, after having confirmed that both variants displayed the same binding properties to fibrin and inhibitory activity on plasmin (Fig. S1). The slowest matrix degradation rate was observed for matrices made of 12 mg/mL fibrinogen and 25 mM of KPI-a2PI1-8. Increasing inhibitor concentration resulted in an increase in gel longevity, as expected, for all inhibitors except wild-type KPI, which did not show detectable levels of efficacy at any of the tested concentrations (Fig. 4A, B). Although KPI-a2PI1-8 demonstrated the best protection against in vitro fibrinolysis at the highest tested concentration of 25 mM, higher concentrations may be further beneficial, since a plateau in efficacy of protection had not been noted even at the highest inhibitor concentration. In addition to increases in inhibitor concentrations, it was demonstrated that increases in fibrin concentrations and gel volumes contributed to longer matrix longevity, as expected (Fig. 4C; Fig. S2). 3.4. KPI-a2PI1-8 extends the longevity of fibrin matrices in vivo To study the anti-fibrinolytic properties of engineered KPIs in vivo, fluorescent fibrin matrices containing 40 mM of the different inhibitors were implanted subcutaneously in the back of mice. Similar degradation rates were observed for all groups during the first 12 days, after which only fibrin matrices containing KPI-a2PI1-8 showed a significant increase in longevity (Fig. 5A). This was further correlated with a simultaneous decrease in fibrin degradation rate (Fig. 5B). Consequently, gels supplemented with KPI-a2PI1-8 lasted for more than 36 days whereas those without inhibitor were not detectable after 24 days, representing a gel longevity extension of about 50% (Fig. 5A, C). Surprisingly, in this experimental model, neither aprotinin nor KPI-PlGF-2123-144A resulted in protection against fibrinolysis as compared to the ‘no inhibitor’ group. Likewise, fibrin containing wild-type KPI degraded similarly to fibrin without inhibitor. Lastly, we evaluated the retention of engineered KPI variants into fibrin matrix in vivo by immunohistochemistry performed on explanted gel samples. KPI-a2PI1-8 and, to lower extent, KPI-PlGF-2123-144A, were each detectable at 15 days post-
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Fig. 2. Design and characterization of wild-type and engineered KPI variants. (A) KPI was fused to C-terminal fibrin-targeting domains a2PI1-8 or PlGF-2123-144A. (B) KPI variant size and purity were assessed by SDS-PAGE. KPI, KPI-a2PI1-8 and KPI-PlGF-2123-144A migrated at sizes ranging from 12 to 15 kDa. (C) Determination of Michaelis constant (Km) of plasmin and N-succinyl-Ala-Phe-Lys-7-amido-4-methylcoumarin acetate salt plasmin substrate (n ¼ 6, mean ± SEM). (D) Bioactivity of KPI variants characterized via a plasmin inhibition assay (n ¼ 4, mean ± SEM). (E) Inhibition constant Ki were calculated from IC50 values obtained by data interpolation using a sigmoidal curve fitting (n ¼ 4, mean ± SEM). All KPI variants have similar inhibitory activities on plasmin, with Kis ranging from 29 to 46 nM. Aprotinin Ki was determined experimentally and corresponded to published data [15].
Fig. 3. Engineered KPI-a2PI1-8 and KPI-PlGF-2123-144A slow the rate of fibrinolysis in vitro. Fluorescent fibrin gels containing 15 mM of KPI variants or aprotinin were incubated in presence of 2.5 nM of plasmin. (A) Representative images of fluorescent fibrin gel degradation over time. (B) Gel degradation was monitored by quantification of fluorescence decay. Both KPI-a2PI1-8 and KPI-PlGF-2123-144A inhibit plasmin and prolong the longevity of fibrin matrices compared to matrices containing aprotinin or no inhibitor. KPI-a2PI1-8 offered the best protection against fibrinolysis (n ¼ 4 gels, mean ± SEM, ANOVA with Bonferroni post-hoc test).
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Fig. 4. Optimization of fibrinogen and inhibitor concentrations to enhance fibrin longevity in vitro. Three concentrations of fibrin and 5 concentrations of inhibitors were tested to compare the degradation time of fluorescently-labeled fibrin matrices in the presence of 2.5 nM plasmin. Gel degradation was quantified daily by fluorescence measurement and the time of complete degradation was defined by the absence of fluorescence. (A) Fibrin matrix longevity during plasmin exposure, as a function of fibrin and inhibitor concentrations. Tables indicate days at which gels were fully degraded. (B) Comparison of the inhibitors, at various concentrations, on the degradation time of 12 mg/mL fibrin matrices. (C) Influence of fibrin concentration on the degradation time of matrices supplemented with KPI-a2PI1-8.
implantation, thus reflecting the functional and strong efficacies of a2PI1-8 and PlGF-2123-144A domains in sequestering KPI within the fibrin matrices (Fig. 5D). 4. Discussion The goal of this study was to develop a human-derived protease inhibitor variant that would confer extended longevity to fibrin matrix in vitro and in vivo. This inhibitor candidate may provide potential as a clinically relevant substitute to the currently used xenogeneic inhibitor, aprotinin, and the less-potent small molecule inhibitors currently utilized within fibrin glues. We selected the Kunitz Protease Inhibitor (KPI) domain of the amyloid beta A4 precursor protein as the core to our variant protein, due to its small, nonglycosylated structure and reported homology to the functionally potent, bovine-derived inhibitor, aprotinin [16] (Fig. 1A and B). Our design comprised a fusion of the inhibitor core to a fibrintargeting domain to confer sufficient matrix protection from fibrinolysis by way of local, extended sequestration of the KPI domain within the fibrin matrix. Hence, we compared two protein engineering strategy; the variant KPI-a2PI1-8 is covalently immobilized to fibrin during normal polymerization through the enzymatic activity of the coagulation transglutaminase Factor XIIIa, whereas KPI-PlGF-2123-144A binds with a high but non-covalent affinity to fibrin. Standard methods were used to fuse the fibrin-targeting sequences a2PI1-8 [19] and PlGF-2123-144 [20] to the C-terminus of the KPI domain (Fig. 2A), and all KPI variants were successfully
expressed in HEK293 cells and highly purified (Fig. 2B). By determining the IC50 and Ki of each KPI variants toward a key fibrinolytic protease, plasmin [23], we found that engineered KPI variants displayed similar bioactivity to wild-type KPI (Fig. 2C, D, E) [17], so demonstrating that the molecular design of the engineered KPIs did not impair their inhibitory properties against plasmin. In an in vitro model of inhibition of plasmin-mediated fibrinolysis, we then showed that engineered KPI-a2PI1-8 and KPI-PlGF-2123-144A variants outperformed wild-type KPI and, more interestingly, aprotinin, despite its 10-fold higher intrinsic inhibitory power (Fig. 3A, B) [15,17]. Because intrinsic inhibitory bioactivity of KPI variants toward plasmin were highly similar, such differences in inhibitory efficacies can be attributed to the levels of inhibitor sequestration into fibrin, thus supporting that covalent immobilization of KPI mediated by a2PI1-8 improves retention compared to the noncovalent affinity conferred by PlGF-2123-144A. Even though this result was rather expected, as covalent binding is the higher possible level of protein immobilization, incorporation of high doses of KPI-a2PI1-8 could have had increased inhibitor concentration at the risk of interference with the degree of fibrin polymerization. Indeed, a2PI1-8 is permissive to forming isoglutamyl lysine isopeptide bonds at a2PI-fibrin as well as fibrin-fibrin crosslinking sites. This limitation was the rational for the design of KPI-PlGF-2123-144A variant, which facilitates high affinity binding of the inhibitor to fibrin while avoiding potential interference of fibrin polymerization. In our study however, we did not observe significant impaired fibrin polymerization and subsequent reduced fibrin gel longevity, for all tested concentrations of KPI-a2PI1-8
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Fig. 5. KPI-a2PI1-8 extends fibrin matrix longevity in vivo when implanted subcutaneously. Fluorescent fibrin matrices containing 40 mM of KPI variants or aprotinin were implanted subcutaneously in mice. (A) In vivo gel degradation was quantified through fluorescence measurements every 6 days. KPI-a2PI1-8 significantly increased fibrin longevity compared to the other inhibitors (n 7 for KPI-a2PI1-8 and ‘no inhibitor’ groups, n 4 in remaining groups, mean ± SEM, Kruskal-Wallis with Dunns post-hoc test). (B) Apparent gel degradation rate at day 18 as a function of the different inhibitors (n 7 for KPI-a2PI1-8 and ‘no inhibitor’ groups, n 4 in other groups, mean ± SEM, Kruskal-Wallis with Dunns post-hoc test). (C) Representative images of fluorescence decay measurements of subcutaneous fibrin implants loaded with the different inhibitors over time. (Blue circles: region-of-interests of 1 cm in diameter). (D) Immunohistochemistry of subcutaneously implanted fibrin matrices containing the different KPI inhibitor variants at 15 days post-implantation (blue: DAPI/ cell nuclei; red: fibrinogen; green: human KPI). Both KPI-a2PI1-8 and KPI-PlGF-2123-144A remain sequestered into fibrin matrices compared to wild-type KPI. Scale bar ¼ 100 mm.
(Fig. 4A, B), suggesting that we did not reach its limiting dose. As a proof-of-concept toward a future potential clinical application of KPI inhibitors in fibrin sealants, we used an in vivo model
of subcutaneous implantation of fibrin matrix, with optimized inhibitors and fibrin concentrations (Fig. 4A, B, C). In this model, KPIa2PI1-8 significantly extended the longevity of fibrin by about 50%
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by slowing fibrin degradation, whereas none of the other inhibitors showed efficacy, including the clinically-relevant aprotinin (Fig. 5A, B, C). The lack of efficacy of wild-type KPI and aprotinin (which is a wild-type homologous to KPI domain) is likely related to diffusive loss, since wild-type KPI was not detectable in fibrin implants at 15 days post-implantation (Fig. 5D) [19]. In contrast, KPI-a2PI1-8 and KPI-PlGF-2123-144A had strong abilities to bind and be retained into fibrin biomaterials for extended time in vivo, as demonstrated by immunohistochemistry analyses at 15 days post-implantation (Fig. 5D). Consequently, the lack of in vivo efficacy of KPI-PlGF2123-144A against fibrinolysis is unexpected; it may be explained by an insufficient level of sequestered inhibitor within the gel, or potentially by a lack of functional stability of the KPI-PlGF-2123-144A variant. Alternatively, while fusion of PlGF-2123-144A to KPI did not impair its specific inhibitory activity toward plasmin, it is possible that it could affect the KPI-related inhibition of other proteases involved in the fibrinolysis cascade. Indeed, KPI is known to be a broad-spectrum protease inhibitor, as is aprotinin, which is assumed to increase its efficacy in fibrin protection in comparison to more specific inhibitors (e.g. tranexamic or ε-aminocaproic acids) [6,7]. Therefore, it would be interesting to further assess whether the engineered KPI variants retained their broadspectrum protease inhibition characteristic, as a follow-up to this study that mainly focused on plasmin-mediated fibrinolysis. Finally, let us note that the concentration of KPI-a2PI1-8 used in vivo to demonstrate its potential for use in fibrin sealant is in the range of the aprotinin concentration currently used in the clinically approved Tisseel® sealant (Baxter Healthcare Corporation) [23], which contains about 30e50 mM of aprotinin (1125e1875 KIU/mL) [19]. On the other hand, the concentration of fibrinogen in Tisseel® (35e55 mg/mL) is about 3e5 times higher than in our fibrin formulation (12 mg/mL). However, we showed in a previous study that such increase in fibrinogen concentration did not directly convert into an extended fibrin material longevity, and that fibrinolytic inhibitors assessed at lower fibrin concentrations can remain relevant for use in commercially available fibrin sealant products [19]. 5. Conclusion We engineered a human-derived broad-spectrum protease inhibitor to propose an alternative to the clinical use of bovine aprotinin in fibrin-based tissue sealants and tissue engineering matrices. We particularly focused our work on the sequestration of a homologous human-derived KPI into fibrin, by fusing KPI to either a transglutaminase substrate domain a2PI1-8 that allows the covalent incorporation of the inhibitor into fibrin matrix, or to a high affinity fibrin-binding domain, PlGF-2123-144A, that permits its noncovalent retention. We found that both engineered variants improved the longevity of fibrin in vitro compared to wild-type KPI and aprotinin. In vivo, the engineered KPI-a2PI1-8 variant significantly increased the longevity of fibrin material by about 50%, thus outperforming the clinically used aprotinin. While the intrinsic inhibitory activity of KPI-a2PI1-8 could be further improved by point mutation [17], we believe that its human origin and simple structure yet highly functional design, contribute to its potential for manufacturability as a clinically appealing substitute to aprotinin as a fibrinolysis inhibitor in fibrin sealants and matrices. Disclosure The transglutaminase-mediated immobilization technology €ssidescribed herein is the subject of patents filed by the Eidgeno sche Technische Hochschule Zürich (ETHZ, Zürich, Switzerland), upon which J.A.H. is named as an inventor. The super-affinity based
retention technology described herein is the subject of a patent de rale de Lausanne application filed by the Ecole Polytechnique Fe (EPFL, Lausanne, Switzerland), upon which J.A.H and P.S.B are named as inventors. Acknowledgements and funding sources The authors would like to acknowledge Dr. Eleonora Simeoni for assistance on animal experimentations, Dr. Elif Vardar for fruitful discussions, Jean-Philippe Gaudry for help on protein purification, €lle J. Be zy for technical assistance. Xavier Quaglia-Thermes and Mae de rale de This research was funded by the Ecole Polytechnique Fe Lausanne, Lausanne, Switzerland and the National Institutes of Health (DP3DK108215). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2017.04.048. References [1] D.M. Albala, Fibrin sealants in clinical practice, Cardiovasc. Surg. (2003), http:// dx.doi.org/10.1016/S0967-2109(03)00065-6. [2] J.Z. Fu, J. Li, Z.L. Yu, Effect of implanting fibrin sealant with ropivacaine on pain after laparoscopic cholecystectomy, World J. Gastroenterol. WJG (2009), http://dx.doi.org/10.3748/wjg.15.5851. [3] P.L. Danielsen, M.S. Agren, L.N. Jorgensen, Platelet-rich fibrin versus albumin in surgical wound repair: a randomized trial with paired design, Ann. Surg. 251 (2010) 825e831, http://dx.doi.org/10.1097/SLA.0b013e3181d3548c. [4] D. Garcia-Olmo, D. Herreros, I. Pascual, J.A. Pascual, E. Del-Valle, J. Zorrilla, et al., Expanded adipose-derived stem cells for the treatment of complex perianal fistula: a phase II clinical trial, Dis. Colon Rectum 52 (2009) 79e86, http:// dx.doi.org/10.1007/DCR.0b013e3181973487. [5] W. Furst, A. Banerjee, H. Redl, Comparison of structure, strength and cytocompatibility of a fibrin matrix supplemented either with tranexamic acid or aprotinin, J. Biomed. Mater. Res. 82B (2007) 109e114, http://dx.doi.org/ 10.1002/jbm.b.30711. [6] E.S. Schouten, A.C. van de Pol, A.N.J. Schouten, N.M. Turner, N.J.G. Jansen, C.W. Bollen, The effect of aprotinin, tranexamic acid, and aminocaproic acid on blood loss and use of blood products in major pediatric surgery: a metaanalysis, Pediatr. Crit. Care Med. 10 (2009) 182e190, http://dx.doi.org/ 10.1097/PCC.0b013e3181956d61. [7] M. Sperzel, J. Huetter, Evaluation of aprotinin and tranexamic acid in different in vitro and in vivo models of fibrinolysis, coagulation and thrombus formation, J. Thromb. Haemost. 5 (2007) 2113e2118, http://dx.doi.org/10.1111/ j.1538-7836.2007.02717.x. [8] H.-M. Kang, M.H. Kalnoski, M. Frederick, W.L. Chandler, The kinetics of plasmin inhibition by aprotinin in vivo, Thromb. Res. 115 (2005) 327e340, http://dx.doi.org/10.1016/j.thromres.2004.09.015. [9] S. Westaby, Aprotinin: twenty-five years of claim and counterclaim, J. Thorac. Cardiovasc. Surg. 135 (2008) 487e491, http://dx.doi.org/10.1016/ j.jtcvs.2008.01.002. [10] A.M. Mahdy, Perioperative systemic haemostatic agents, Br. J. Anaesth. 93 (2004) 842e858, http://dx.doi.org/10.1093/bja/aeh227. €th, M. Zühlsdorf, H. Dalichau, P.G. Kirchhoff, H. Kuppe, et al., [11] W. Dietrich, P. Spa Anaphylactic reactions to aprotinin reexposure in cardiac surgery: relation to antiaprotinin immunoglobulin G and E antibodies, Anesthesiology 95 (2001), 64e71e discussion 5Ae6A. [12] A.M. Scheule, W. Beierlein, S. Arnold, F.S. Eckstein, J.M. Albes, G. Ziemer, The significance of preformed aprotinin-specific antibodies in cardiosurgical patients, Anesth. Analgesia 90 (2000) 262e266, http://dx.doi.org/10.1213/ 00000539-200002000-00005. [13] P. Ponte, P. Gonzalez-DeWhitt, J. Schilling, J. Miller, D. Hsu, B. Greenberg, et al., A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors, Nature 331 (1988) 525e527, http://dx.doi.org/10.1038/331525a0. [14] T.R. Hynes, M. Randal, L.A. Kennedy, C. Eigenbrot, X-ray crystal structure of the protease inhibitor domain of Alzheimer's amyloid. beta.-protein precursor, Biochemistry 29 (1990) 10018e10022, http://dx.doi.org/10.1021/ bi00495a002. [15] H. Fritz, G. Wunderer, Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs, Arzneimittelforschung 33 (1983) 479e494. [16] H. Zollner, Handbook of Enzyme Inhibitors, VCH Verlagesgesellschaft, Weinheim, 1993. [17] W.E. Van Nostrand, A.H. Schmaier, R.S. Siegel, S.L. Wagner, W.C. Raschke, Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitztype protease inhibitor domain of the amyloid beta-protein precursor, J. Biol.
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