A New Serine Protease (AsKSP) with Fibrinolytic Potential Obtained from Aspergillus tamarii Kita UCP 1279: Biochemical, Cytotoxic and Hematological Evaluation

Abstract

This study aimed to characterize and evaluate the fibrinolytic, thrombolytic, hematological, and toxicological aspects of a serine protease (AsKSP) from Aspergillus tamarii Kita UCP 1279. The enzyme was purified using a two-phase aqueous system and assessed for optimal pH (7.0) and temperature (50 °C), stability, and effects of metal ions, inhibitors, and surfactants. AsKSP exhibited stability for up to 120 min at 50 °C and 36 h at pH 7.0. Enzymatic activity was enhanced by Na⁺ and Zn²⁺ and non-ionic surfactants (Tween-80) but inhibited by Cu²⁺, Fe³⁺, Triton X-100, and SDS, reducing activity by up to 62.35%. The highest amidolytic activity was observed for the substrate N-succinyl-Gly–Gly–Phe-p-nitroanilide. SDS-PAGE analysis indicated an approximate molecular mass of 90 kDa. The enzyme showed fibrinolytic activity, degrading 38.81% of fibrin clots in vitro after 90 min, without affecting fibrinogen. Cytotoxicity assays indicated no toxicity (cell viability > 80%). Coagulation assays showed slight prolongation of prothrombin time (PT) and activated partial thromboplastin time (aPTT), with no effect on thrombin time. No red blood cell lysis was observed, and albumin increased enzymatic activity by 31.70%. These findings demonstrate that Aspergillus tamarii Kita UCP 1279 produces a fibrinolytic protease with potential for thrombus treatment, providing a promising foundation for drug development.

1. Introduction

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, recognized as diseases with the highest morbidity and mortality, and therefore considered public health issues [1]. According to the World Health Organization (WHO), more than 17 million people die each year from cardiovascular diseases [2]. In general, these pathologies affect the circulatory system, such as coronary heart disease, peripheral vascular disease, hypertension, arrhythmia, acute myocardial infarction, stroke, and angina pectoris [3].

The formation of fibrin clots is closely involved in the development of CVDs [4], and thus, thrombosis plays an important role in the pathogenesis of various cardiovascular disorders due to the accumulation of clots that block the flow of blood [5]. The main protein component of blood clots, fibrin, is formed from fibrinogen via proteolysis by thrombin and can be hydrolyzed by plasmin to prevent thrombosis in blood vessels. In an imbalanced situation due to some disorders, clots are not hydrolyzed, leading to thrombosis [6,7].

Current therapeutic approaches to combat clot formation include the use of antiplatelet drugs, anticoagulants, and thrombolytic agents [8]. However, many of these drugs have disadvantages, such as high costs, short efficacy when administered intravenously, lack of specificity, potential toxicity, risk of allergic reactions, and a variety of other side effects [9,10]. Therefore, a drug capable of effectively removing blood clots and restoring blood flow is of utmost importance for the effective treatment of thrombotic diseases, and fibrinolytic enzymes present themselves as an alternative in the treatment of these diseases [11].

In this context, the last decade has witnessed an increase in the discovery of thrombolytic agents from numerous microorganisms, leading to the development and characterization of these enzymes with thrombolytic activity and minimal or no side effects [12,13,14,15,16,17,18]. Currently, fibrinolytic proteases produced by microorganisms have the potential to inhibit blood coagulation and can degrade fibrin, playing a key role in the treatment of CVDs [19]. Fungi have proven to be a good source of these enzymes, with the potential for large-scale production in a short period and at lower costs, due to their ability to grow on solid substrates such as agroindustrial waste, facilitating the production and purification of this bioproduct. As a result, they have become attractive targets, since the fibrinolytic enzymes produced by them are generally released extracellularly, making their recovery easier and cheaper [20,21].

The biochemical properties of fungal proteases have great potential for exploration, and there is limited literature on the fibrinolytic potential of these microorganisms. The literature shows that species belonging to the Aspergillus genus have attracted attention as sources of new bioactive compounds [22,23,24,25,26,27], with Aspergillus tamarii being known as a good producer of enzymes of industrial interest, such as collagenolytic, keratinolytic enzymes, fructosyltransferase, proteases, and detergent formulations [28,29,30].

In recent years, particular attention has been given to the production and purification of fibrinolytic proteases, which have the potential to degrade fibrin clots. However, studies on the toxicity of these enzymes and there in vivo effects are scarce. Therefore, the aim of this study was to perform the biochemical characterization and in vitro evaluation of the fibrinolytic, thrombolytic, and toxicity aspects of the protease derived from Aspergillus tamarii Kita UCP 1279, as research in this area may contribute to the development of safer thrombolytic agents for therapeutic use.

2. Results and Discussion

2.1. Biochemical Characterization

2.1.1. Influence of Temperature on the Fibrinolytic Activity of the Protease Produced by Aspergillus tamarii Kita UCP 1279

The effects of temperature on the fibrinolytic activity and thermal stability of the enzyme are illustrated in Figure 1 and Figure 2, respectively. The fibrinolytic protease purified by ATPS showed maximum efficiency (32.86 U/mL) at 50 °C, results that agree with those reported by Dienes et al. [31], Shirasaka et al. [32], Choi et al. [33], and Deng et al. [34] for fibrinolytic enzymes produced by Trichoderma reesei QM9414, Aspergillus oryzae KSK-3, Pleurotus ferulae, and Neurospora sitophila, respectively. Maximum activity at 50 °C was also reported for an enzyme produced by Aspergillus tamarii URM 4634 [28], as was observed in this study for the same species. The residual activity of the protease remained above 80% in the temperature range of 30–50 °C.

However, as the temperature increased (≥60 °C), the protease experienced a reduction in activity due to denaturation, following similar results reported for other fibrinolytic enzymes produced by Aspergillus ochraceus [35], Xylaria curta [15], Mucor subtilissimus UCP 1262 [36], and Virgibacillus halodenitrificans [37], which also observed a similar loss of activity in this temperature range. Regarding thermal stability, the enzyme remained virtually unchanged in its enzymatic activity during 120 min of incubation at 50 °C, as shown in Figure 2.

2.1.2. Influence of pH on the Fibrinolytic Protease Activity Produced by Aspergillus tamarii Kita UCP 1279

Regarding the pH effect, the enzyme activity produced by Aspergillus tamarii Kita UCP 1279 reached its maximum value of 53.06 U/mL at pH 7.0 in the presence of Tris-HCl buffer, suggesting that the enzyme functions well in the human physiological pH range (Figure 3). Similar results have also been reported by other authors for a fibrinolytic protease produced by Streptomyces parvulus DPUA 1573 [21], Bacillus subtilis DC27 [38], Brevibacillus brevis [39], and fungal species: Aspergillus fumigatus [40], Hericium erinaceum [41], Rhizopus microsporus var. Tuberosus [42], Neurospora sitophila [43], and Cordyceps militaris [16,44].

The protease activity remained stable within the pH range from 7.0 to 11.0, a result like Silva et al. [28] and Amaral et al. [29]. However, at acidic pH values below 6, there was a significant decrease in enzymatic activity, classifying it as a neutral to alkaline protease. The pH stability of the enzyme is important for enzymatic characterization before commercialization. Alkaline proteases can be used in leather, detergent, and pharmaceutical industries [45]. Additionally, the protease showed stability after 36 h of exposure, with residual activity close to 100%, as observed in Figure 4.

Moreover, the data obtained in this study agree with Clementino et al. [46], where the fibrinolytic protease produced by Mucor subtilissimus UCP 1262 using ATPS showed optimal temperature and pH of 50 °C and 7.0, respectively. In other studies, using a fibrinolytic protease produced by Petasites japonicus, the authors found an optimal pH around 7.0, and the highest efficiency was achieved at 60 °C with stability between 30 °C and 60 °C [47]. Patel, Kawale, and Sharma [48] demonstrated that a serine protease with fibrinolytic activity from Euphorbia hirta exhibited ideal pH and temperature for enzymatic activity at pH 7.2 and 50 °C, respectively. These physicochemical characteristics are like those of the fibrinolytic protease from Aspergillus tamarii Kita UCP 1279.

2.1.3. Effect of Metal Ions

The effect of metal ions on enzymatic activity was evaluated by observing the residual activity of the purified fibrinolytic protease using ATPS, aiming to obtain information about the possible action of these ions as positive or negative modulators of the protease. It was found that the enzyme activity was slightly increased by the addition of Na⁺ and Zn²⁺, while an inhibitory effect on protease activity was observed in the presence of Cu²⁺ and Fe³⁺ ions (

Table 1. Effect of metal ions (5 mM) on the fibrinolytic activity of the purified protease produced by Aspergillus tamarii Kita UCP 1279 purified by ATPS.

Metal Ions (5 mM)	Residual Activity (%)
Control	100.00
Cu2+	87.82 * ± 0.84
Mn2+	103.74 ± 0.74
K+	102.99 ± 0.34
Zn2+	106.17 * ± 0.96
Mg2+	105.61 ± 3.25
Na+	107.67 * ± 1.28
Ca2+	98.87 ± 1.38
Fe2+	100.37 ± 1.27
Fe3+	87.07 * ± 0.06

* Statistically significant effects (p value < 0.05).

).

These data are in line with studies on the fibrinolytic protease of Mucor subtilissimus UCP 1262, which showed increased enzymatic activity in the presence of Na⁺, Zn²⁺ and Mg²⁺, at a concentration of 5 mM [6] and a partial decrease in activity with Cu²⁺ and Fe³⁺ ions [46]. Silva, Alves, and Porto [49] found similar results with a protease from Aspergillus tamarii URM 4634, obtained by two-phase aqueous systems, where Na⁺ and Cu²⁺ acted as activators and inhibitors of enzymatic activity, respectively. Furthermore, this result is consistent with other researchers who observed inhibitory effects of Cu²⁺ on the activity of other fibrinolytic enzymes [15,17,50,51,52,53,54]. Cu²⁺ ions have been identified as oxidizing agents of thiol groups, and the suppression of enzymatic activity by this ion may indicate the presence of (-SH) groups at or near the active site. Thus, Cu²⁺ can directly attack cysteine residues to inhibit the protease [55,56,57].

2.1.4. Effect of Inhibitors

The effects of different protease inhibitors were investigated (

Table 2. Effect of inhibitors on the fibrinolytic activity of the purified protease produced by Aspergillus tamarii Kita UCP 1279.

Inhibitors (5 mM)	Residual Activity (%)
Control	100.00
β-mercaptoethanol	99.60 ± 2.38
EDTA	94.88 * ± 0.08
Iodoacetic acid	102.95 ± 0.74
PMSF	15.95 * ± 1.55
TPCK	13.58 * ± 1.87
TLCK	69.29 * ± 1.99

* Statistically significant effects (p value < 0.05).

). Enzymatic activity was strongly reduced by typical serine protease inhibitors, PMSF, TPCK, and TLCK, showing residual activities of 15.95%, 13.58%, and 69.29%, respectively. However, enzymatic activity was not affected by EDTA (94.88%), a metalloprotease inhibitor, or by β-mercaptoethanol (99.6%), a cysteine protease inhibitor. The results of this study identify the protease obtained from Aspergillus tamarii Kita and purified by ATPS as a serine protease, like those produced by Bacillus pumilus MCAS8 [58], Bacillus cereus FF01 [59], Neurospora sitophila [43], Cordyceps militaris [16], Mucor subtilissimus UCP 1262 [36,46], Bacillus subtilis DC27 [38], Streptomyces parvulus DPUA 1573 [21], and Paenibacillus graminis [60].

Several studies demonstrate proteases from Aspergillus identified as serine proteases based on their inhibition profiles: Aspergillus nidulans HA-10 [61], Aspergillus oryzae [32,62], Aspergillus flavus MTCC 9952 [52], Aspergillus terreus [63], and Aspergillus ochraceus [35]. Silva et al. [28,45] and Amaral et al. [29] confirmed that fibrinolytic proteases from Aspergillus tamarii were strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting them as serine proteases, as observed in this study for the same species. According to Sun et al. [53], most fibrinolytic enzymes of microbial origin belong to this class. The serine protease is a group of proteolytic enzymes that possess the active group (OH) from the amino acid serine in the catalytic site, and PMSF is known to irreversibly sulfonate the essential serine residue at the active site of a protease, resulting in the loss of enzymatic activity [17,64]. There is a connection between this class of proteases and their affinity for fibrin, the main component of blood clots, which is naturally degraded by plasmin, a serine protease [65].

2.1.5. Effect of Surfactants

According to Wang, Wu, and Liang [66], enzymes are typically deactivated by the introduction of surfactants into the reaction solution, as these surfactants act on the enzyme, promoting a modification in its three-dimensional structure, leading to a reduction in substrate availability. As shown in

Table 3. Effect of surfactants on the fibrinolytic activity of the purified protease produced by Aspergillus tamarii Kita UCP 1279.

Surfactants	Residual Activity (%)
Control	100.00
Tween 20	103.10 ± 2.38
Tween 80	110.70 * ± 0.98
SDS	37.65 * ± 1.47
Triton X-100	62.34 * ± 0.98

* Statistically significant effects (p value < 0.05).

, the activity of the Aspergillus tamarii Kita UCP 1279 protease was moderately inhibited by Triton X-100 and SDS, with a decrease of 37.66% and 62.35%, respectively. However, non-ionic surfactants such as Tween-80 slightly increased enzymatic activity.

Alencar et al. [21] reported a decrease in residual activity for Triton X-100 and SDS in a study with fibrinolytic enzyme from Streptomyces parvulus DPUA 1573. Non-ionic surfactants, in most cases, do not induce conformational changes in proteins, unlike anionic and cationic surfactants, which have denaturing characteristics [67]. SDS, a strong anionic surfactant, is known to be a potent protein denaturant, including for proteases. The ability of this surfactant to reduce enzymatic activity was also verified by other authors [6,35,65,68]. Its effect can be attributed to the denaturing action related to its negative net charge, which interacts with the enzyme molecules, preventing their activity [66]. Furthermore, it is understood that this surfactant can unfold most proteins through interactions between the negatively charged main group of SDS and the positively charged amino acid side chains of proteins [69].

2.1.6. Amidolytic Activity

The amidolytic activity of the fibrinolytic protease from A. tamarii UCP 1279 was evaluated using three different chromogenic substrates (

Table 4. Amidolytic activity of the fibrinolytic activity of the purified protease produced by Aspergillus tamarii Kita UCP 1279 using typical chromogenic substrates after 30 min of incubation.

Substrate	Enzyme	Amidolytic Activity
N-Succinyl-Gly–Gly–Phe-p-nitroanilide	Chymotrypsin	+++
N-Succinyl-L-phenylalanine-p-nitroanilide	Chymotrypsin	+
Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride	Trypsin	−

+++ (high affinity with the substrate). + (low affinity with the substrate). − (no affinity with the substrate).

). The enzyme showed greater specificity for the substrate S-1899 (N-succinyl-Gly–Gly–Phe-p-nitroanilide), a typical chymotrypsin substrate, and did not exhibit affinity for the substrate B-4875 (Nα-benzoyl-DL-arginine 4-nitroanilide). This result suggests that the enzyme can be classified as a serine protease like chymotrypsin, which is consistent with other fibrinolytic enzymes [47,53,60]. In a study conducted by Nascimento et al. [36], a fibrinolytic serine protease from Mucor subtilissimus, purified using biphasic aqueous systems, exhibited amidolytic activity like chymotrypsin. Similar characteristics were also reported for fibrinolytic proteases obtained from fungi, such as Mucor subtilissimus UCP 1262 [6], Lyophyllum shimeji [70], and Neurospora sitophila [34].

2.2. SDS-PAGE and Fibrin Zymogram

SDS-PAGE and fibrin zymogram were used to assess the purity of the fibrinolytic protease produced by Aspergillus tamarii Kita UCP 1279, which was preferentially partitioned into the salt-rich phase after extraction by ATPS, as reported by Alencar et al. [71]. As shown in Figure 5, the molecular mass of this protease was approximately 90 kDa, a result that is consistent with other fibrinolytic enzymes produced by fungi, such as those reported for Mucor subtilissimus UCP 1262 with a molecular weight of 97 kDa [36], and for proteases from Schizophyllum commune BL23 (66–97 kDa) [72] and Bionectria sp. (80–173 kDa) [73], but significantly higher than those reported for fibrinolytic enzymes from Fusarium sp., with molecular masses ranging from 27 kDa to 32 kDa [74], Rhizopus chinensis 12 (17–18 kDa) [75], Pleurotus eryngii (14 kDa) [76], and Schizophyllum commune (21 kDa) [77].

Silva et al. [28], using SDS-PAGE electrophoresis analysis, estimated the molecular weight of a protease from Aspergillus tamarii URM4634 to be 49.3 kDa. Other Aspergillus species, such as A. terreus, A. oryzae LK-101, and A. nidulans HA-10, showed values in the range of 16–42 kDa [61,62,63]. Fibrinolytic proteases with lower molecular weights have also been identified in bacteria such as Paenibacillus graminis (50 kDa) [60], Streptomyces parvulus DPUA 1573 (42.65 kDa) [21], and Bacillus tequilensis (27 kDa) [78]. Baehaki et al. [79] identified a protease with an approximate molecular weight of 124 kDa, purified from Bacillus licheniformis F11.4. The fibrinolytic activity of the protease purified in the saline phase was confirmed by the fibrin zymogram shown in Figure 5. The enzyme was purified in the saline phase, showing only one band, which confirms the capability and effectiveness of extracting this fibrinolytic protease. The molecular weight value found is virtually identical to that observed in the fibrin zymogram of the enzyme partitioned in the salt-rich phase.

2.3. Cytotoxicity

Although many studies have been published describing the purification and biochemical characterization of microbial fibrinolytic enzymes, few efforts have been directed towards investigating their toxicity. However, this assay is essential to detect the toxicological profile in different cell lines in a controlled manner. To evaluate the cytotoxic potential of the fibrinolytic protease, the tetrazolium salt reduction method was used in this study, in which metabolically active cells convert water-soluble MTT into an insoluble purple product [80]. The MDA-MB-231 and J774.A1 cell lines are widely used to evaluate the initial cytotoxicity profiles of drug candidate molecules, as they provide preliminary information about potential alterations or toxicity to the kidneys and the immune system, even before conducting in vivo tests. The results demonstrated that the fibrinolytic protease extracted from Aspergillus tamarii Kita UCP 1279 showed no toxicity, and cell viability remained above 80% during the 24 h time frame in the tested cell lines (Figure 6).

Other studies corroborate the findings of this article, which also obtained cell viability results above 80% when evaluating the cytotoxicity of a fibrinolytic protease produced by Streptomyces sp. CC5 in human umbilical vein endothelial cells [53]. Similarly, Da Silva et al. [80] found that the fibrinolytic protease produced by Mucor subtilissimus UCP 1262 was non-toxic to the J774A.1 cell line, with cell viability at the same level. A similar result was also reported by Yeon et al. [81], who found that a serine fibrinolytic protease extracted from Lumbrineris nipponica showed no cytotoxicity to endothelial cells.

The use of cancer cell lines to assess cytotoxicity can be relevant for evaluating the safety of substances in cancer patients. This is because there is a direct relationship between thrombosis and neoplasia, where neoplastic cells promote the activation of coagulation pathways, leading to blood clot formation [5,7]. Therefore, investigating cytotoxicity in cancer cells can help determine whether a substance, such as a fibrinolytic enzyme, is safe for use in cancer patients, considering the potential thrombotic risks associated with this medical condition [82].

Additionally, evaluating cytotoxicity in tumor cell lines can assess selectivity and impact on cell proliferation. Studies with tumor cell lines have already been employed to investigate the cytotoxicity of fibrinolytic enzymes, such as human colon adenocarcinoma [12], human cervical epithelial carcinoma [59], and mouse mammary carcinoma [80]. None of these tumor cell lines showed toxicity, as observed in this study. Furthermore, in the MDA-MB-231 cell line, no tumor cell proliferation occurred when exposed to the fibrinolytic enzyme extracted from Aspergillus tamarii Kita UCP 1279.

2.4. Influence of the Purified Fibrinolytic Protease on Coagulation Times (PT, APTT, and TT)

The anticoagulant activity was evaluated through coagulation times: prothrombin time (PT) for the evaluation of the extrinsic and common pathways (

Table 5. Prothrombin time (PT) as a function of the concentration of the fibrinolytic activity of the purified protease produced by Aspergillus tamarii Kita UCP 1279.

TP/Time	Control	2.5 mg/mL	5 mg/mL	10 mg/mL
15 min	12.80 ± 0.30	12.95 ± 0.35	13.40 ± 0.14	14.15 ± 0.92
45 min	13.40 ± 0.10	13.40 ± 0.07	13.55 ± 0.21	13.85 ± 1.48

), activated partial thromboplastin time (aPTT), used to assess the intrinsic and common pathways of coagulation (

Table 6. Activated partial thromboplastin time (aPTT) as a function of the concentration of the fibrinolytic activity of the purified protease produced by Aspergillus tamarii Kita UCP 1279.

TP/Time	Control	2.5 mg/mL	5 mg/mL	10 mg/mL
15 min	27.10 ± 0.90	27.80 ± 1.70	28.45 ± 0.07	28.55 ± 1.34
45 min	29.00 ± 0.20	30.00 ± 1.98	32.10 ± 0.14	34.50 ± 0.42

), and thrombin time (TT), which reflects the time required for the conversion of fibrinogen into fibrin protein. When it comes to fibrinolytic enzymes, it is important to evaluate not only their action on fibrin but also to investigate whether these proteases impact the enzymes involved in the blood coagulation cascade. The results of these assays are determined by the clot recognition method and the assay kits used.

As shown in Table 5, there was a slight prolongation of the PT when compared to the control group at both tested time points. This prolongation was 1.10 times longer compared to the control (12.8 ± 0.3) at 15 min for the concentration of 10 mg/mL. Regarding the aPTT (Table 6), the prolongation of the coagulation time was observed as the enzyme concentration increased, particularly evident after 45 min of exposure of the fibrinolytic protease to human plasma, prolonging the aPTT by 1.18 times when comparing the control group (29.0 ± 0.2) and the highest tested concentration.

In the thrombin time (TT), we evaluated whether the fibrinolytic enzyme produced by A. tamarii Kita UCP 1279 altered the time required for clot formation after the addition of thrombin. It was observed that there was no interference with thrombin or fibrinogen action in thrombus formation, as clots were formed in less than 1 min. Thus, the obtained results suggest that the fibrinolytic protease slightly prolongs the extrinsic, intrinsic, and common coagulation pathways but does not affect the final step of coagulation, which is the conversion of fibrinogen into fibrous protein. Few studies have been conducted to investigate the complete anticoagulant effect of fibrinolytic enzymes. Alhawiti [83] examined anticoagulant activity using only the activated partial thromboplastin time (aPTT). Miranda et al. [84] obtained similar results for a serine fibrinolytic protease obtained from Mucor subtillissimus UCP 1262, where they observed a significant prolongation of the PT and aPTT coagulation times as the enzyme concentration increased. A similar result was found in another study, where velefibrinase, a fibrinolytic enzyme from Bacillus velezensis Z01, affected PT, aPTT, and TT in a dose-dependent manner [85]. The activity of anticoagulants involves the prevention of blood clot formation, while antiplatelet and thrombolytic agents act in the reduction in blood clots [86]. Thrombosis and bleeding are prevented under normal conditions by the balance between coagulation and fibrinolysis. Any imbalance favors coagulation, leading to thrombosis, platelet aggregation, fibrin production, and the entrapment of red blood cells in arteries or veins [87].

A prominent anticoagulant effect was observed in fibrinolytic enzymes from Streptomyces sp. CC5 [68], Bacillus subtilis C142 [88], and the CFR15 protease produced by Bacillus amyloliquefaciens MCC2606 [89] due to the in vitro increase in activated partial thromboplastin time and prothrombin time in a time- and dose-dependent manner. The likely biochemical mechanism for this is the inhibition of prothrombin conversion to thrombin, a critical step in the formation of insoluble fibrin filaments and the catalysis of other coagulation factors [90]. The data obtained in this study indicate that the fibrinolytic protease from A. tamarii Kita UCP 1279 has a slight potential to block the activation of the coagulation cascade by inhibiting interaction with coagulation factors or even directly hydrolyzing fibrin. Further investigations may be needed to clarify the detailed mechanisms of action of these effects.

2.5. Hemolytic Activity

Another assay conducted to evaluate the toxicity of the fibrinolytic enzyme produced by Aspergillus tamarii Kita UCP 1279 and purified by ATPS was the hemolytic activity. Hemolysis is a clinical condition that can potentially be severe. In summary, there are four main causes of cell lysis in the body: immunological, mechanical, toxic, and infectious. Therefore, the evaluation of blood compatibility is of utmost importance, as it is essential to analyze the implications on cells before employing a biopharmaceutical [91,92].

The determination of hemolytic activity was carried out according to the guidelines established by ASTM (American Society for Testing and Materials). According to ASTM standards, substances that show a hemolysis rate greater than 5% are categorized as hemolytic, those with a rate between 2% and 5% are considered slightly hemolytic, while those with a rate less than 2% are not classified as hemolytic. The fibrinolytic enzyme from A. tamarii exhibited 0.82%, 1.02%, and 1.07% hemolysis at concentrations of 2.5, 5, and 10 mg/mL, respectively, demonstrating that it is not hemolytic. The results of the hemolytic test can be seen in Figure 7.

Other researchers have also investigated the ability of fibrinolytic enzymes to induce hemolysis. The results obtained in this study were like the work of Majumdar et al. [39], who did not observe hemolytic activity against erythrocytes at the tested concentrations (0–0.5 μM) of the enzyme produced by Brevibacillus brevis FF02B. In another study, Majumdar et al. [59] also found that the fibrinolytic enzyme obtained from Bacillus cereus FF01 was not hemolytic. Costa e Silva et al. [17] found that the partially purified enzyme from Chlorella vulgaris did not show a significant hemolysis rate, causing less than 4% erythrocyte lysis. Other microbial-derived fibrinolytic enzymes also showed similar results, such as the enzyme from Mucor subtilissimus, which caused 2.8% hemolysis [80] and was slightly hemolytic at 10 mg/mL, while at lower concentrations, the degree of hemolysis was negligible. Mukherjee et al. [12] observed that the Bacillus sp. AS-S20-I strain induced 3% hemolysis of the tested erythrocytes, while the assay with Bacillus subtilis LD-8547 [93] caused 5% erythrocyte lysis.

2.6. Fibrinogenolytic Activity

To verify the mode of action of the fibrinolytic enzyme from Aspergillus tamarii Kita UCP 1279 against human fibrinogen, degradation products of fibrinogen were analyzed by SDS-PAGE. Figure 8 shows the degradation of fibrinogen at 0, 15, 30, 60, and 120 min, indicating that the protease did not hydrolyze any of the fibrinogen chains. Fibrinogen is a plasma glycoprotein with a molecular weight of 340 kDa, synthesized by the liver, and composed of three distinct subunits: Aα, Bβ, and γ, with molecular masses of 64, 55, and 47 kDa, respectively. This molecule plays a crucial role in the coagulation cascade, being the last step before conversion into fibrin through the action of thrombin. Its essential function lies in promoting clot formation, contributing significantly to the coagulation process [94,95].

Several studies show fibrinolytic enzymes that act by degrading the fibrinogen chains at different times. The degradation pattern of the chains is not uniform, with proteases hydrolyzing only one [60,80,96], two [88], or even all three subunits of fibrinogen [34]. When fibrinogen hydrolysis occurs, there is a reduction in the fibrinogen supply “in vivo”, causing an anticoagulant effect, inhibiting platelet aggregation, which consequently reduces blood viscosity and increases blood flow, leading to a hemorrhagic effect [97]. Thus, the protease from A. tamarii UCP 1279 appears to be a fibrinolytic agent acting directly on fibrin, as it did not degrade fibrinogen and, therefore, has no anticoagulant effect. Moreover, this result aligns with the data obtained in the coagulation time determinations, evidenced by the low or no anticoagulant effect and no alteration of thrombin time (TT), which is influenced by fibrinogen concentration and the presence of inhibitors of fibrin formation. This characteristic provides an advantage over plasminogen activators, such as Streptokinase, Urokinase, and tissue plasminogen activator (tPA), which promote a hemorrhagic effect.

2.7. In Vitro Thrombolytic Degradation

Coagulation and fibrinolysis are distinct mechanisms that coexist and develop simultaneously, providing a continuous dynamic balance between both [98]. The formation of clots plays a key role in controlling excessive bleeding from wounds and injuries, acting as a protective mechanism. This process can be concisely summarized in three fundamental stages: initially, the rupture of the blood vessel triggers the formation of a complex of substances called the prothrombin activator; then, the prothrombin activator catalyzes the transformation of prothrombin into thrombin; finally, thrombin converts fibrinogen into fibrin strands, which surround platelets, erythrocytes, and plasma, resulting in the formation of the blood clot [99].

The removal of fibrin and blood clots is carried out by the fibrinolytic system. However, the accumulation of fibrin in blood vessels can lead to obstruction of blood flow and is associated with the onset of cardiovascular diseases, resulting in severe clinical complications [44]. Unfortunately, the blood coagulation system can also lead to unwanted clots in blood vessels (pathological thrombosis), which is one of the leading causes of death and disability in many patients [100]. The results of this study showed that the fibrinolytic enzyme produced by Aspergillus tamarii Kita UCP 1279 degraded part of the thrombus in vitro after 90 min of contact with the enzyme. Thrombolytic degradation by both the crude extract and the protease purified by ATPS resulted in degradation rates of 53.44% and 38.81%, respectively, confirming the reduction in the volume and weight of the thrombi.

Similar studies have also demonstrated the effectiveness of fibrinolytic enzymes in the degradation of blood clots, such as the enzyme produced by Paenibacillus graminis, which showed a blood clot degradation of 36.72% by the action of the crude extract and 46.96% by the action of the enzyme purified by SDFA [44]. Ahamed et al. [101] evaluated the thrombolytic potential through an in vitro experiment of the fibrinolytic enzyme produced by Brevibacterium sp., confirming the hydrolysis of the fibrin blood clot. Similar activity was also observed in the serine fibrinolytic protease produced by the Bacillus sp. AS-S20-I strain, which demonstrated thrombolytic properties by promoting clot lysis in an in vitro study [12]. Additionally, the hydrolysis of the clot in vitro by the action of fibrinolytic enzymes produced by microorganisms of the Bacillus genus has also been reported in other studies [93,102,103].

In this regard, it can be observed that the thrombus degradation in vitro observed in this study was superior to the reduction in clot volume observed in an assay performed by Nighat et al. [104], who, using extracts of Bacillus clausii KP10, reported a percentage of 35.16%. Similarly, the fibrinolytic protease extracted from Mucor subtilissimus, which achieved 32.18% degradation [105], and the protease from Chlorella vulgaris, which presented 25.6% clot degradation in vitro over 90 min [17]. According to Prasad et al. [106], in vitro clot lysis is a reliable method to evaluate the thrombolytic activities of fibrinolytic agents produced by microorganisms. Therefore, the potential of the fibrinolytic protease from A. tamarii UCP 1279 found in this study may have useful clinical applications for preventing thrombosis and other related diseases.

2.8. Effects of Serum Albumin on Fibrinolytic Activity

Studies by Anderson and Anderson [107] and Adkins et al. [108] have shown that human blood plasma contains a protein concentration typically around 60–85 mg per mL, among which the following stand out: albumin (~55%), globulin (~38%), fibrinogen (~7%), pro-enzymes, and protease inhibitors (less than 1%). The action of fibrinolytic proteases can be affected by all these components present in blood plasma. Therefore, we decided to analyze the effects of albumin on the enzymatic activity of the fibrinolytic protease produced by Aspergillus tamarii Kita UCP 1279 purified by SDFA. It was observed that albumin influenced the fibrinolytic activity of the purified enzyme, promoting a 31.70% increase in this activity. These results disagree with those presented by Park et al. [109], who, in the presence of BSA, observed a reduction in the enzymatic activity of the three fibrinolytic enzymes studied, with an average reduction of 77.5% compared to the control without albumin.

3. Materials and Methods

3.1. Fibrinolytic Protease Production

The production of the fibrinolytic protease obtained from Aspergillus tamarii Kita (National System for Genetic Heritage and Traditional Knowledge Management/Registration No. AA30B0B) by solid-state fermentation and purified by a two-phase aqueous system (SDFA) was carried out according to Alencar et al. [71]. It is noteworthy that the salt-rich phase, to which the fibrinolytic protease partitions according to the predetermined conditions, was used. These conditions included 12.5% polyethylene glycol 8000 g/mol, 15% sodium phosphate, and pH 8.0.

3.2. Protein Quantification

Protein quantification was performed using the method described by Smith et al. [110], where bovine serum albumin (BSA) was used as the standard substance for the calibration curve.

3.3. Proteolytic Activity Determination

Proteolytic activity was determined as described by Ginther [111]. A 150 µL aliquot of the purified protease was added to 250 µL of 1% azocasein in microtubes, which were incubated at 28 °C in the dark for 60 min. Afterward, 1 mL of 10% trichloroacetic acid (TCA) was added to stop the reaction. The reaction mixture was then centrifuged (14,000× g, 15 min), and the supernatant (800 µL) was transferred to a second tube containing 200 µL of 1.8 M NaOH, which was then vortexed. The absorbance was measured at 420 nm, and the enzymatic activity was defined as an increase of 0.1 in absorbance.

3.4. Fibrinolytic Activity Assay

Fibrinolytic activity was determined by the spectrophotometric method described by Wang, Wu, and Liang [66]. A 0.72% fibrinogen solution in 400 µL of Tris HCl-NaCl 150 mM buffer, pH 7.75, and 100 µL of 245 mM phosphate buffer, pH 7.0, were added to tubes containing 100 µL of thrombin solution (20 U/mL [T9326-150UN—Human thrombin, BioUltra, recombinant, expressed in HEK 293 cells, aqueous solution, ≥95% (SDS-PAGE)—Sigma-Aldrich, St. Louis, MO, USA]) and incubated at 37 °C for 10 min. Subsequently, 100 µL of the purified fibrinolytic protease was added to the tube, and the incubation continued at 37 °C for 60 min. This solution was mixed every 20 min, and after 1 h at 37 °C, the reaction was stopped by adding 700 µL of 0.2 M trichloroacetic acid (TCA). The reaction mixture was centrifuged at 15,000× g for 10 min. Then, 1 mL of the supernatant was collected, and the absorbance of the sample was measured at 275 nm. In this assay, one unit (unit of fibrin degradation) of enzymatic activity was defined as an increase of 0.01 in absorbance at 275 nm.

3.5. Biochemical Characterization of the Purified Fibrinolytic Protease

All biochemical characterization (optimal pH and temperature, stability, effect of metal ions, inhibitors, and surfactant action) of the fibrinolytic protease activity was carried out as described by Nascimento et al. [6].

3.5.1. Effect of pH and Temperature on Proteolytic Activity

The optimal pH of the fibrinolytic protease was determined through the proteolytic activity assay. To evaluate the effects of pH on activity, the purified enzyme was maintained at room temperature in the following buffer solutions at 0.1 M: sodium acetate-acetic acid buffer (pH 3.0 to 5.0), citrate-phosphate buffer (pH 5.0 to 7.0), Tris-HCl buffer (pH 7.0 to 9.0), and glycine-NaOH buffer (pH 9.0 to 11.0). For pH stability, the enzyme was incubated at its optimal pH for 36 h, and residual enzymatic activity was analyzed at 0, 1, 6, 12, 24, and 36 h. The effects of temperature on activity were determined by incubating the enzyme at various temperatures (10, 20, 30, 37, 40, 50, 60, 70, 80, 90, and 100 °C) for 60 min, followed by measurement of residual activity. Thermal stability was tested at the enzyme’s optimal temperature up to 120 min, and residual enzymatic activity was analyzed at 0, 15, 30, 45, 60, 75, 90, 105, and 120 min. All analyses were performed in triplicate.

3.5.2. Effect of Metal Ions, Inhibitors, and Surfactants

The activity of the fibrinolytic protease was determined in the presence of metal ions (Ca²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Cu²⁺, Mg²⁺, Mn²⁺, Na⁺, and K⁺) by mixing 200 µL of the purified protease with 200 µL of the respective ion solutions, all at a concentration of 5.0 mM, and incubating for 60 min at room temperature. To assess the effect of inhibitors on proteolytic activity, the purified enzyme (200 µL) was exposed to 200 µL of the following inhibitors for 60 min at room temperature: 10 mM phenylmethanesulfonyl fluoride (PMSF), 10 mM β-mercaptoethanol, 10 mM ethylenediaminetetraacetic acid (EDTA), 1% iodoacetamide, and 10 mM tosyl-L-lysine chloromethyl ketone (TLCK) and tosyl-L-phenylalanine chloromethyl ketone (TPCK). Similarly, for surfactant substances, 200 µL of the purified enzyme was incubated with 200 µL of the following surfactants: sorbitan monolaurate ethoxylate (Tween 20), sorbitan monooleate ethoxylate (Tween 80), Triton X-100, and sodium dodecyl sulfate (SDS) for 60 min. Solutions containing 1.0% of the above-mentioned substances were used. After incubation, the residual protease activity was measured, and the results were expressed as a percentage of the control test.

3.5.3. Amidolytic Activity Determination

Amidolytic activity was measured spectrophotometrically using Nα-benzoyl-DL-arginine 4-nitroanilide (catalog number B4875, Sigma-Aldrich, St. Louis, MO, USA), N-succinyl-Gly–Gly–Phe-p-nitroanilide (catalog number S1899, Sigma-Aldrich, St. Louis, MO, USA), and N-succinyl-L-phenylalanine-p-nitroanilide (catalog number S2628, Sigma-Aldrich, St. Louis, MO, USA) as chromogenic substrates. The reaction mixture (200 µL) containing 30 µL of the purified protease solution, 30 µL of the chromogenic substrate, and 140 µL of 20 mM Tris-HCl, pH 7.4, was incubated for 30 min at 37 °C, and the amount of p-nitroaniline was determined by UV–Vis spectrophotometry (BEL Engineering, UV/VIS 190-1000NM, Monza, MB, Italy) at 405 nm. One unit of amidolytic activity (UA) was expressed as the number of micromoles of substrate hydrolyzed by the enzyme per minute per mL, according to Kim et al. [112].

3.6. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-Page)

SDS-PAGE was performed according to the methodology described by Laemmli [113]. Electrophoresis was carried out using a vertical gel electrophoresis system with a 12% polyacrylamide gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA, Life Sciences, Mumbai, India). The molecular weight was determined using a standard marker (250, 150, 100, 75, 50, 37, 25, 20 kDa). After separation, the electrophoresis gels were visualized by staining with 0.02% (v/v) Coomassie Brilliant Blue R-250 in 50% (v/v) methanol and 7.5% (v/v) acetic acid.

3.7. Fibrin Zymography

The fibrinolytic activity of the enzyme was assessed by performing a fibrin zymogram according to the methodology by Kim, Choi and Lee [114] with some modifications. A 0.12% (m/v) fibrinogen solution was mixed in a 12% polyacrylamide gel and incubated in a water bath at 37 °C until complete dissolution of the fibrinogen. Then, a thrombin solution (1 U/mL) was added, and 10 µL of the purified enzyme was used for electrophoresis on the fibrin gel. After electrophoresis, the gel was washed with 2.5% (v/v) Triton X-100 for 10 min, rinsed three times with distilled water, and incubated in reaction buffer (100 mM Tris-HCl pH 7.5) in a water bath at 37 °C for 16 h. After the procedure, the gel was washed again with distilled water and stained with Coomassie Brilliant Blue R-250, following the methodology by Laemmli [36]. The observation of a digested region in the fibrin gel indicated the fibrinolytic activity of the enzyme.

3.8. Cytotoxicity Assay

Human mammary adenocarcinoma cells (MDA-MB-231) and macrophages (J774A.1) were obtained from the Cell Bank of Rio de Janeiro (BCRJ). The MDA-MB-231 cell line was maintained in Leibovitz’s L-15 medium and F-12 medium (50% of each), with 2 mM L-glutamine, without sodium bicarbonate, and 10% fetal bovine serum (FBS), without CO₂. The J774A.1 cell line was maintained in Gibco Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% FBS and 5% CO₂. Cells were counted in a Neubauer chamber for cell viability testing. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine cell viability from the fibrinolytic enzyme of Aspergillus tamarii Kita, following the methodology by Mosmann [115]. The cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/mL, and after 24 h of incubation, the fibrinolytic enzyme was exposed for 24 h at final concentrations of 300, 150, 75, 17.5, and 8.75 µg/mL. After the exposure period, 20 µL of MTT solution (4 mg/mL) was added, and the plates were incubated for 2–3 h. Subsequently, the supernatant was removed, and 100 µL of DMSO (Dimethyl sulfoxide) was added. Absorbance was measured in a Microplate Reader (BioteK Elx808, Basel, Switzerland) at 630 nm. Cytotoxicity was expressed as cell viability: (Absorbance of treated cells × 100/Absorbance of untreated cells), and cytotoxic activity results were expressed as means of the replicates ± standard deviation [80].

3.9. Coagulation Time Determination

Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured in a semi-automatic coagulometer (Maxcoag, Urit Medical Electronic CO., Guilin, China) using BIOS Diagnostica reagents (Sorocaba, Brazil), following the manufacturer’s instructions. For the assay, a pool of human plasma from 5 healthy individuals was used, provided by the manufacturer. This study was approved by the Research Ethics Committee of UFPE (Opinion No. 63547722.6.0000.5208). Controls for APTT and PT assays were performed with 25 µL of saline solution (NaCl 0.9%) and 100 µL of plasma. To evaluate the anticoagulant effect, purified fibrinolytic protease was used at concentrations of 2.5, 5, and 10 mg/mL, and incubated with standard plasma for 15 and 45 min. After this time, coagulation times were determined by using the appropriate equipment. Thrombin time (TT) was assessed according to the methodology by Miranda et al. [84] with some modifications. First, 50 µL of fibrinolytic protease (1 mg/mL) was incubated with 100 µL of thrombin (20 U/mL) for 45 min. After this period, the solution was transferred tubes (2 mL) containing a mixture of 400 µL Tris-HCl-NaCl 150 mM pH 7.75, 100 µL phosphate buffer 245 mM pH 7.0, and 0.72% bovine fibrinogen, and placed in a water bath at 37 °C until fibrin clot formation was observed.

3.10. Hemolytic Activity

Hemolytic activity was performed using human blood from a healthy male volunteer. This study was approved by the Research Ethics Committee of UFPE with the Certificate of Ethical Consideration Presentation (CAAE: 63547722.6.0000.5208). The assay was conducted following the guidelines of Rajendran et al. [116]. Two milliliters of whole blood were added to 4 mL of PBS (phosphate-buffered saline 0.2 M, pH 7.4) and centrifuged at 5000 rpm for 5 min to isolate red blood cells (RBCs). The erythrocytes were washed twice with 10 mL of PBS and finally diluted in 20 mL of PBS. From the final suspension, 0.4 mL was diluted in 1.6 mL of fibrinolytic enzyme at final concentrations of 2.5, 5, and 10 mg/mL. Control groups were divided into: PBS (negative group), distilled water, and Triton-X (positive group). The tubes were kept at 37 °C for 1 h, then centrifuged for 5 min at 10,000 rpm at 20 °C, and absorbance was measured at 540 nm. The hemolytic degree was expressed by the following formula:

(Test absorbance − Negative absorbance)/(Positive absorbance − Negative absorbance) × 100.

3.11. Fibrinogenolytic Activity and In Vitro Thrombolytic Degradation Assay

3.11.1. Fibrinogen Preparation

Fibrinogen was directly obtained from blood plasma for the fibrin clot degradation assay and fibrinogenolytic activity. The plasma used throughout this study was provided by the Pernambuco Hematology and Hemotherapy Foundation (HEMOPE), and the research partnership between UFPE and HEMOPE was approved by the Research Ethics Committees (Process No. 1.727.579). The serological tests, determined by specific sanitary legislation, were performed by HEMOPE itself to detect potential viral contamination, ensuring the safety of the obtained plasma, with donor information kept confidential by the blood bank.

3.11.2. Fibrinogenolytic Activity

Fibrinogenolytic activity was performed according to Park et al. [109]. For the assay, a mixture of 100 μg of human fibrinogen (from HEMOPE plasma bags) was incubated at 37 °C with 100 μL of the fibrinolytic protease in 25 mM Tris-HCl buffer (pH 7.5). Twenty microliters of this reagent mixture were taken at 0, 15, 30, 60, and 120 min. The reaction was halted by adding 4 μL of SDS-PAGE sample denaturing buffer. The resulting products were analyzed by SDS-PAGE 12% as described by Chang et al. [117].

3.11.3. In Vitro Thrombolytic Degradation Assay

The assay was performed following the methodology described by Couto et al. [60]. In glass tubes, 1 mL of human plasma (from HEMOPE plasma bags) and 200 µL of thrombin solution (20 U/mL) were added and incubated at 37 °C for 10 min. After this period, each fibrin clot formed was weighed using an analytical balance. In each tube containing the already weighed clot, 200 µL of the fibrinolytic protease were added and incubated again at 37 °C for 90 min. The degradation percentage was calculated as described by Da Silva et al. [118]. The entire experiment was performed in triplicate, with the control using 200 µL of 0.15 M saline solution.

3.12. Effects of Serum Albumin on Fibrinolytic Activity

One hundred microliters of fibrinolytic protease were mixed with 100 µL of a 6.7 mg/mL solution of BSA (bovine serum albumin) in 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. After 1 h, a 100 µL aliquot was taken, and the assay was performed as described in Section 2.4.

4. Conclusions

In this study, it was possible to observe that the chymotrypsin-like serine fibrinolytic protease produced by Aspergillus tamarii UCP 1279 and purified by a two-phase aqueous system showed potential to act as a future candidate for a possible thrombolytic agent, as it exhibited no cytotoxicity and did not alter coagulation parameters. All biochemical aspects were characterized, highlighting the stability of this enzyme, which has good fibrinolytic activity, did not promote the lysis of blood cells, and did not exhibit fibrinogenolytic activity. This reinforces the potential use of this enzyme in various medical applications, opening perspectives for its potential application as a new thrombolytic drug.