Triple Action of Lignosulfonic Acid Sodium: Anti-protease, Antioxidant, and Anti-inflammatory Effects of a Polymeric Heparin Mimetic

Abstract

Background:

Heparins are sulfated glycosaminoglycans that are used as anticoagulants to treat thrombosis. Heparins exhibit other potential therapeutic effects, such as anti-inflammatory, anti-viral, and anti-malarial effects. However, heparins’ strong anticoagulant activity poses a risk of life-threatening bleeding, limiting their therapeutic use for other diseases beyond thrombosis. To exploit the other effects of heparins and eliminate the bleeding risk, we explored an alternative polymer called lignosulfonic acid sodium (LSAS), which acts as a sulfonated heparin mimetic. LSAS targets factor XIa to exert anticoagulant effect, and thus unlike heparins, it is unlikely to cause bleeding.

Methods:

This study investigated the multiple effects of LSAS to identify potential leads for complex pathologies treatment. A series of chromogenic substrate hydrolysis assays was used to evaluate the inhibition of three inflammation-related proteases by LSAS. Its chemical antioxidant activity against the system of ABTS/hydrogen peroxide/metmyoglobin was also determined. Lastly, the LSAS’s effect on TNFα-induced activation of the NF-κB pathway in HEK-293 cells was also tested to determine its cellular anti-inflammatory activity.

Results:

The results showed that LSAS effectively inhibited human neutrophil elastase, cathepsin G, and plasmin, with IC₅₀ values ranging from 0.73 to 212.5 μg/mL. Additionally, LSAS demonstrated a significant chemical antioxidant effect, with an IC₅₀ value of 44.1 μg/mL. Furthermore, at a concentration of approximately 530 μg/mL, LSAS inhibited the TNFα-induced activation of the NF-κB pathway in HEK-293 cells, indicating a substantial anti-inflammatory effect. An essential advantage of LSAS is its high-water solubility and virtual non-toxicity, making it a safe and readily available polymer.

Conclusion:

Based on these findings, LSAS is put forward as a polymeric heparin mimetic with multiple functions, serving as a potential platform for developing novel therapeutics to treat complex pathologies.

Introduction

Heparins are a type of sulfated glycosaminoglycans known for their diverse therapeutic effects, including anti-viral, anti-inflammatory, and anti-malarial properties, among others. However, their clinical utility has been limited due to the potent anticoagulant activity, which poses a risk of severe internal bleeding. In contrast, lignosulfonic acid sodium (LSAS) is a non-saccharide heparin mimetic derived from sulfonated lignin. It achieves its anticoagulant effect by targeting factor XIa (FXIa) [1], an enzyme known to play a significant role in pathological clotting rather than physiological clotting. This crucial distinction means that LSAS is less likely to cause significant bleeding complications, setting the stage for the exploration of the non-saccharide heparin mimetic in various therapeutic applications beyond blood clotting disorders.

LSAS is a light-yellow brown to brown powder, amorphous in nature, obtained from the sulfite pulping of softwood. It is derived from lignin, which constitutes the second largest component of wood. LSAS is water-soluble but does not dissolve in common organic solvents. The industry has utilized LSAS in various applications. In this study, the LSAS used has an average molecular weight of approximately 52,000 and an average molecular number of about 7,000. Unlike unfractionated heparins, which are linear copolymers of glucosamine and uronic acid linked by a (1–4)-glycosidic linkage and are sulfated, LSAS is a polymer of phenylpropane monomers that are randomly sulfonated (Figure 1). Due to its macromolecular structure, LSAS exhibits a range of polydispersity, enabling interactions with biomolecules through hydrogen bonding, anionic, and hydrophobic interactions. Safety and clinical relevance are essential for therapeutic development, and LSAS demonstrates low in vitro cytotoxicity to human genital tract epithelial cells. It does not stimulate NF-κB activation and does not significantly up-regulate IL-1α/β and IL-8. Additionally, LSAS has no adverse effects on epithelial integrity and junctional protein expression [2]. Furthermore, LSAS is virtually non-toxic when administered orally, with an LD₅₀ (lethal dose for 50% of the test population) greater than 40 g/kg. It has a history of use as an animal feed additive, known for its anti-pepsin activity and its protective effects against the development of gastric ulcers [3]. Interestingly, LSAS also exhibits intriguing biological activities, such as in vitro inhibition of the human immunodeficiency virus [4].

Figure 1.

Representative chemical structures of reported active chemical species in the mixture of LSAS. Generally, LSAS is a randomly sulfonated polymer of phenylpropane monomers.

This report focuses on the in vitro evaluation of LSAS’s anti-protease, chemical-based antioxidant, and anti-inflammatory effects. The aim is to identify a versatile lead that can guide the discovery and development of effective therapeutics for diseases characterized by complex pathologies, including emphysema, COVID-19, disseminated intravascular coagulation, and others. For instance, the challenges in treating and preventing emphysema stem from its multifaceted pathogenic complexity, which is not yet fully understood [5]. Major pathophysiological mechanisms proposed for emphysema include induced elastolysis, oxidative stress, and inflammation in lung cells [5]. Although the benefits of single-mechanism inhibitors have been demonstrated in cells and animal studies, these therapies have failed to progress to clinical applications. This failure partly results from preclinical studies that focused on testing inhibitors in disease states induced by single mechanism-based agents. Therefore, while the current studies have only provided preliminary evidence and require further support from animal studies and human trials, they reasonably demonstrate that the development of agents capable of targeting the multiple pathophysiological mechanisms involved in diseases like emphysema holds the potential for powerful and long-lasting therapeutic strategies.

Methods and materials

Materials.

LSAS (Mw~52,000) was purchased from Sigma Aldrich (St. Louis, MO, USA). For enzyme kinetics studies, HNE was purchased from Elastin Products Company (Owensville, MO, USA). Human plasmin was purchased from Haematologic Technologies (Essex Junction, VT, USA). Human CatG was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The chromogenic substrates S-1384 for HNE and S-7388 for CatG were from Sigma Aldrich. The chromogenic substrate for thrombin plasmin was purchased from Biomedica Diagnostics (Windsor, NS, Canada). Stock solutions of CatG and plasmin were prepared in 50 mM Tris-HCl buffer, pH 7.4, containing 0.02% Tween80, 150 mM NaCl, and 0.1% PEG8000. Stock solution of HNE was prepared by reconstituting 1 mg with 100 μL of 1:1 200 mM Na acetate:glycerol buffer of pH 5, which was then diluted using HEPES buffer, pH 7.4, containing 125 mM HEPES, 0.125% Triton-X 100, and 100 mM NaCl. For the clotting time (activated partial thromboplastin time (APTT) and prothrombin time (PT)) testing, deficient and pooled normal human plasma was obtained from George King Bio-Medica (Overland Park, KS, USA). APTT reagent containing ellagic acid, PT reagent containing thromboplastin-D, and 0.025 M solution of CaCl₂ were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Effects of LSAS on proteases: HNE, CatG, and plasmin.

The inhibition potentials of LSAS against HNE, CatG, and plasmin were also studied using the chromogenic tripeptide substrate hydrolysis assays, similar to our previous reports [6–9]. Briefly, 5 μL of LSAS (0–5.0 mg/mL in the well) (or the vehicle) and 5 μL of the enzyme were added at 37 °C to each well of a 96-well microplate containing 85–185 μL of 20–50 mM Tris-HCl buffer, pH 7.4, containing, 0.02% Tween80, 0.1% PEG8000, and 100–150 mM NaCl. The final enzyme concentrations were 20 nM for HNE and plasmin and 30 nM for CatG. After 5-min incubation, 5 μL of S-7388 (HNE; final conc. 2 mM), S-7388 (CatG; 2 mM), or Spectrozyme PL (plasmin; 50 nM) was added and the residual enzyme activity was measured from the initial rate of increase in A₄₀₅ nm. We then used Equation (1) to calculate the inhibition parameters if LSAS inhibited 50% or more of the enzyme activity. The dose-dependent inhibition of human enzyme activity was plotted using the Logistic Equation (1) to determine the efficacy (ΔY%; y-axis) and potency (IC₅₀; x-axis) of the inhibition.

$$\text{Y}={\text{Y}}_0+\frac{{\text{Y}}_{\text{M}}-{\text{Y}}_0}{1+{10}^{\left(\text{log}{\left[\text{Inhibitor}\right]}_0-\text{log }{\text{IC}}_{50}\right)\times \text{HS}}}$$ (1)

The equation used in the study relates to the ratio of residual enzyme activity in the presence of LSAS compared to its absence. In the equation, Y represents this ratio, while Y₀ and Y_M correspond to the minimum and maximum values of the fractional residual enzyme activity, respectively. The IC₅₀ denotes the concentration of LSAS at which the enzyme activity is inhibited by 50%, and HS represents the Hill slope. To determine the values of Y₀, Y_M, IC₅₀, and HS, the data was subjected to nonlinear curve fitting during the analysis process.

Chemical antioxidative activity of LSAS.

The antioxidative activity of LSAS was evaluated using the chemical antioxidant assay kit (Cayman Chemical Co.) in a 96-well plate format, following both the manufacturer’s protocol and a previously established procedure [5]. The assay relied on the generation of a chromogenic radical cation ABTS^•+ from ABTS by hydrogen peroxide in the presence of metmyoglobin at room temperature. The increase in absorbance at 405 nm (ΔAbs₄₀₅) was continuously monitored using a microplate reader. A water-soluble tocopherol analog, Trolox^®, was used as a reference antioxidant and tested under the same conditions. The initial rate of ABTS^•+ production (Y) was determined by measuring the linear absorbance increase in the first 5 minutes (ΔAbs_{405,5 min}), and these values were plotted against the logarithmic concentrations of LSAS or Trolox^®. The IC₅₀ and HS values were then calculated through nonlinear regression curve-fitting using the equation mentioned earlier. The curve-fitting analysis demonstrated high “goodness-of-fit” parameters, with coefficient of determination and model selection criterion values equal to or exceeding 0.992 and 3.5, respectively, indicating a satisfactory fit for the data.

Cellular anti-inflammatory effect of LSAS.

The NF-kB Luciferase Reporter HEK293 Stable Cell Line SL-0012 was obtained from Signosis Inc (Santa Clara, CA), and the cells were grown maintained, and plated in 96 well plates according to Signosis protocols. These cells are stably transfected with a Luciferase reporter gene that has a promoter regulated by NF-κB signaling such that Luc activity is only observed if the cells are treated with an activator of NF-κB signaling such as TNFα. In these experiments, bioassays involved cells seeded in 96 well plates, and all treatments were repeated on the plate in four wells with results reported as mean, SD of the quadruplicate wells. Recombinant human TNFα was obtained from R&D Systems and the stimulation of the NF-κB-Luc activity of the SL-0012 cells was measured over the range of 3, 6, 12.5, 25, 20, and 100 nM/ml cell culture media. To measure the ability of LSAS to block NF-κB signaling, the SL-0012 cells were treated with LSAS (0.526–526 μg/mL) combined with 6 nM/ml TNFα, a concentration that was shown to stimulate an intermediate response of NF-κB-Luc activity in the steep part of the dose-response curve that is most sensitive to the inhibition by antagonists. The experiment was repeated four times. Normalized data was calculated for each experiment and then multiple experiments were tabulated to determine means and SE. Statistics comparing all treatments to each other were performed using GraphPad Prizm (multiple T-tests, 2-way ANOVA).

Effect of LSAS on clotting times of normal human plasma.

Plasma clotting assays of APTT and PT are commonly used to study the anticoagulant activity of enzyme inhibitors. APTT measures the effect of the new potential therapeutic entity on the contact/intrinsic pathway-driven clotting which involves FXIIa, FXIa, and FIXa. On the other hand, PT measures the effect of the new potential therapeutic entity on the extrinsic pathway of coagulation which involves FVIIa. These experiments were conducted using the BBL Fibrosystem fibrometer (Becton−Dickinson, Sparles, MD, USA), as documented in earlier studies [1,10]. For the APTT assay, 90 μL of citrated human plasma was mixed with 10 μL of LSAS (or the vehicle) and 100 μL of prewarmed 0.2% ellagic acid. After incubation for 4 min at 37 °C, clotting was provoked by adding 100 μL of prewarmed 0.025 M CaCl₂, and the time to clotting was noted. For the PT assay, thromboplastin-D was prepared by adding 4 mL of distilled water, and then, the resulting mixture was warmed to 37 °C. A 90 μL volume of citrated human plasma was then mixed with 10 μL of LSAS (or the vehicle) and was subsequently incubated for 30 s at 37 °C. Following the addition of 200 μL of prewarmed thromboplastin-D reagent, the time to clotting was noted. In the two assays, about 9 or more concentrations of LSAS were used to construct concentration vs. effect profiles. The data were plotted to a quadratic trendline, which was used to estimate the LSAS concentration of LSAS needed to double the clotting time. Clotting times using 10 μL of highly purified water (negative control) were also measured in a similar manner. The positive control used in APTT and PT assays were (1) UFH, an antithrombin activator; (2) argatroban, a clinically used thrombin inhibitor; (3) rivaroxaban, a clinically used FXa inhibitor, (4) anti-F11, an experimental FXIa inhibitor; and (5) C6B7, an experimental FXIIa inhibitor.

Results and discussion

Effects of LSAS on proteinases: human neutrophil elastase (HNE), cathepsin G (CatG), and plasmin.

To investigate the in vitro anti-protease activity of LSAS, we tested its inhibitory effects against three proteases, namely HNE, CatG, and plasmin. HNE is a serine proteinase of the chymotrypsin family. Excessive HNE activity results in severe cardiopulmonary diseases. Moreover, excessive activity of HNE also appears to contribute to psoriasis and inflammatory skin diseases, rheumatoid arthritis, diabetes type I, chronic kidney disease, neuropathic pain, and the development and progression of cancer. Moreover, severe pneumonia, atherosclerosis, inflammatory bowel disease, Crohn’s disease, and neurological diseases have been found to also be related to exacerbated activity of HNE [11]. Recently, HNE inhibitors have been claimed to treat COVID-19-related acute respiratory distress syndrome [6].

Likewise, CatG is a member of the cationic serine proteinases family. CatG was first identified in the granules of neutrophil leukocytes. Given its dual trypsin- and chymotrypsin-like specificity, CatG has been reported to be a contributing factor in several diseases including coronary artery disease, ischemic reperfusion injury, rheumatoid arthritis, and bone metastasis. It is also implicated in cystic fibrosis, chronic obstructive pulmonary disease, and acute respiratory distress syndrome [7,8]. Along these lines, plasmin is another member of the serine proteinases family. Plasmin is the primary mediator of natural fibrinolysis by cleaving cross-linked fibrin in blood clots. Plasmin can also be generated at the cell surface via plasminogen activation. Localized generation of plasmin has been found to contribute to tumor metastasis and chronic inflammation. Therefore, plasmin inhibition in these conditions can potentially be beneficial as observed in post-ischemic neutrophil migration, colitis, and metastasis of prostate carcinoma cells [8,9]. Thus, a single inhibitor of HNE, CatG, and plasmin can serve as a powerful anti-protease agent which can be useful for diseases with complex pathologies in which the level or the activity of several proteases are elevated.

Using chromogenic substrate hydrolysis assays under near-physiological conditions, we found that LSAS inhibits the three enzymes in a concentration-dependent manner, albeit with different potencies. Figure 2A shows that LSAS inhibits HNE with an IC₅₀ value of 4.7 ± 0.2 μg/mL (~90 nM) and an efficacy of ~100%. Likewise, figure 2B shows that LSAS inhibits CatG with an IC₅₀ value of 0.73 ± 0.11 μg/mL (~14 nM) and an efficacy of ~90%. Figure 2C also demonstrates that LSAS inhibits plasmin with an IC₅₀ value of 212.5 ± 25.8 μg/mL (4.1 μM) and an efficacy of ~90%. These results suggest that LSAS possesses potent in vitro anti-protease activity against multiple disease-related proteases. Importantly, LSAS does not inhibit other related serine proteases such as thrombin (>125 μg/mL), factor IXa (>670 μg/mL), factor Xa (>125 μg/mL), factor XIIa (>710 μg/mL), factor XIIIa (>12.5 μg/mL), trypsin (>600 μg/mL), and chymotrypsin (>2000 μg/mL), at the highest concentrations tested [1].

Figure 2.

Direct inhibition of A) HNE; B) CatG; and C) plasmin by LSAS. The inhibition potential was constructed using the corresponding chromogenic substrate hydrolysis assays. Solid lines represent sigmoidal dose−response fits (equation 1) to the data to obtain the values of IC₅₀, ΔY%, and HS. Error bars represent ± 1 S.E.

Chemical antioxidative activity of LSAS.

Highly reactive oxygen species and free radicals stand accused of assisting just about every human disease. These oxidants and reactive radicals may indirectly injure cells by altering the proteinase/antiproteinase balance that normally exists within the tissue, and even directly via promoting the oxidative degradation of essential cellular components. In addition, reactive oxygen species can also initiate as well as augment inflammation via the upregulation of numerous diverse genes implicated in the inflammatory response, including those that encode for adhesion molecules and proinflammatory cytokines. This may take place by activating certain transcription factors including nuclear transcription factor kB (NF-kB). Therefore, it has long been proposed that antioxidants can help protect against the damaging effects of highly reactive oxygen species and free radicals [12,13].

Along these lines, LSAS’s antioxidative activity was assessed using the ABTS/hydrogen peroxide (H₂O₂) antioxidant assay kit. This assay relies on the production of a chromogenic radical cation ABTS^•+ from 2,2′-azinobis(3-ethyl-benzothiazoline-6-sulphonic acid) (ABTS) triggered by hydrogen peroxide in the presence of metmyoglobin, at room temperature. The increase in absorbance at 405 nm (ΔAbs₄₀₅), measured using a microplate reader, indicates the extent of the antioxidant activity. Figure 3 illustrates the in vitro concentration-dependent chemical anti-oxidative activity of LSAS. The antioxidant assay results were analyzed through curve-fitting to determine the IC₅₀, HS, and efficacy values. LSAS demonstrated potent inhibition of chemical oxidation, with an IC₅₀ value of 44.1 μg/mL (~848 nM) and an efficacy of approximately 92%. In comparison, Trolox^®, a tocopherol analog known as an intermediate antioxidant between vitamins C and E, had an IC₅₀ value of about 290 μM [14]. Hence, LSAS exhibited a remarkable 342-fold higher potency as a radical scavenging and trapping antioxidant than Trolox^®. Additionally, it was previously reported that caffeic acid, a phenylpropanoid monomer present in LSAS, showed a concentration-dependent anti-oxidative activity in the range of 1 to 100 μM, with an IC₅₀ value of 16.8 ± 1.2 μM [15, 16]. This suggests that LSAS is approximately 20-fold more potent as an antioxidant compared to its caffeic acid monomer.

Figure 3.

Dose-dependent in vitro antioxidant activity of LSAS as determined by a chemical assay, which employed the formation of a chromogenic radical cation ABTS^•+ from ABTS by H₂O₂ in the presence of metmyoglobin. This was monitored as absorbance increased at 405 nm. The solid line represents sigmoidal dose−response fits (equation 1) to the data to obtain the values of IC₅₀, ΔY%, and HS. Error bars represent ± 1 S.E.

Cellular anti-inflammatory effect of LSAS.

The transcription factor NF-κB regulates several attributes of immune functions and serves as a crucial mediator of inflammatory responses. For instance, NF-κB promotes the expression of several pro-inflammatory genes, including those encoding for cytokines and chemokines. NF-κB also contributes to inflammasome regulation. In addition, NF-κB plays a critical role in regulating the survival, activation, and differentiation of innate immune cells and inflammatory T cells. Dysregulated NF-κB activation contributes to various inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, atherosclerosis, and multiple sclerosis. Thus, targeting the NF-κB signaling pathway is an attractive method to develop anti-inflammatory therapies [17].

Figure 4 shows the in vitro cellular anti-inflammatory activity of LSAS, determined by measuring its effect on the TNF_α-induced NFκB-Luc activity in HEK293. Results indicate that 526 μg/mL (~10 μM) has significant cellular anti-inflammatory activity. At this level, LSAS diminished the TNF_α-induced NFκB-Luc activity by ~100%. Importantly, heparin lacked such activity at the highest concentration tested of 100 μM (data not shown). Mechanistically, LSAS does not affect the level of TNFα which induces inflammatory responses via multiple mechanisms. In fact, TNFα is one of the “fast-responding” proinflammatory cytokines and has been shown to activate and translocate NFκB into the nucleus, from which several inflammatory cytokines are released. Although LSAS could act on TNFα and/or TNFα receptors outside the cells, quenching to trigger the subsequent inflammatory cascades, this is unlikely an anti-inflammatory mechanism as it was reported before for CDSO3 [18]. Alternatively, it is possible that LSAS inhibits inflammatory transcription factors including NFκB upon endocytosis into the cells, given its added lipophilicity. Studies to determine the in vivo anti-inflammatory mechanism(s) of LSAS are ongoing and will be reported in due time. Overall, this experiment highlights the promise of LSAS and the approach of designing sulfated glycosaminoglycan mimetics as anti-inflammatory agents with high clinical relevance. It is noteworthy to mention that LSAS did not affect the viability of T-cell leukemia cell line (MT-4), human embryonic kidney cells (HEK293T), and peripheral blood mononuclear cells (PBMCs) at the highest concentration tested of 100 μM [19].

Figure 4.

In vitro, anti-inflammatory activities of four doses of LSAS in the NF-kB Luciferase Reporter HEK293 Stable Cell Line SL-0012 stimulated with 6 nM recombinant human TNFα. At the dose of 526 μg/mL, LSAS demonstrated anti-inflammatory activity by almost completely inhibiting the TNFα-induced NF-kB luciferase activity. Experiments were conducted three times.

Effect of LSAS on clotting times of normal human plasma.

Plasma clotting assays are commonly used to study the anticoagulation potential of new molecules in in vitro settings. The APTT evaluates the effect of potential anticoagulants on the contact/intrinsic pathway-mediated clotting which involves factors IXa, XIa, and XIIa. The PT evaluates the effect of potential anticoagulants on FVIIa in the extrinsic pathway of coagulation. Initially, we evaluated the LSAS effect on APTT using normal human plasma. The effect of different concentrations of LSAS on APTT of normal human plasma (Figure 5) was measured, as described in earlier studies [1]. Results indicated that LSAS concentration-dependently doubled APTT of normal plasma at ~310 ± 24 μg/mL. Furthermore, it was found that >980 μg/mL of LSAS were required to double the PT of human plasma (Figure 5). These results suggest that LSAS is an anticoagulant in human plasma and that it is likely to promote its anticoagulant properties by targeting the intrinsic pathway of coagulation.

Figure 5.

The effects of LSAS on the APTT (intrinsic pathway) and PT (extrinsic pathway) of normal human plasma.

This was further confirmed by comparing the plasma effect profile of LSAS with those of approved and experimental anticoagulants. Under our conditions, unfractionated heparins (UFH) were found to affect both APTT as well as PT at concentrations of 0.7 μg/mL and 2.55 μg/mL, respectively. Likewise, argatroban (thrombin inhibitor) and rivaroxaban (factor Xa inhibitor) both affect APTT and PT at similar concentrations (0.29–0.34 μM and 0.12–0.18 μM, respectively); however, LSAS was found to primarily affect APTT (index of selectivity was >3-fold). In fact, the LSAS plasma profile appears to be similar to that of anti-F11 (FXIa inhibitor) and/or C6B7 (FXIIa inhibitor), which only affected APTT but not PT. Overall, LSAS, similar to heparins, is an anticoagulant, yet it is mechanism is different. While heparins are antithrombin activators and indirect inhibitors of factor Xa and/or thrombin [20–22], LSAS appears to inhibit one or more of clotting factors in the intrinsic/contact activation pathway (factors XIIa, XIa, and/or IXa).

Conclusion and Future Directions

In this report, we identified LSAS as a non-saccharide heparin mimetic offering a unique polypharmacy opportunity. Using chromogenic substrate hydrolysis assays, we found that LSAS is a potent inhibitor of three inflammation-related serine proteases. LSAS inhibited HNE, CatG, and plasmin with IC₅₀ values of 0.73 – 212.5 μg/mL (0.014 – 4.1 μM). Not only that but LSAS also demonstrated significant chemical anti-oxidant activity at sub-micromolar concentration (~0.85 μM). More importantly, LSAS blocked the TNFα-mediated activation of NF-κB which regulates several attributes of immune functions and serves as a crucial mediator of inflammatory responses. LSAS demonstrated substantial cellular anti-inflammatory activity at low micromolar concentration (~10 μM). As indicated by previous studies [1], LSAS also demonstrates a selective, bleeding-free anti-clotting effect by selectively prolonging the APTT, potentially by inhibiting human FXIa. Remarkably, LSAS does not show significant signs of cellular or animal toxicity, as indicated by various studies [2–4, 23]. In terms of therapeutic applications, LSAS is expected to outperform, in principle, multiple agents that target various diseases. This is because its single-drug poly-pharmacology enhances efficiency while reducing the likelihood of side effects or toxicity. Additionally, due to their anionic nature, LSAS and its derivatives are less likely to cross the blood-brain barrier or placenta, further enhancing their safety profile.

Agents like LSAS present a unique opportunity for the prevention and treatment of a wide range of acute and chronic diseases. Inflammation and oxidative stress are interconnected and closely linked pathophysiological processes. Either of them can occur before or after the other, but when one arises, the likelihood of the other following suit is high. These two factors jointly contribute to the development of numerous chronic diseases, including hypertension, cardiovascular diseases (such as disseminated intravascular coagulation), diabetes and its complications, pulmonary diseases (like emphysema), inflammatory conditions (such as rheumatoid arthritis), neurodegenerative disorders, chronic kidney disease, alcoholic liver disease, infectious diseases (like COVID-19), cancer, and aging. While identifying and treating the primary abnormality hold significant clinical importance, addressing only the primary abnormality may not always yield successful outcomes. This is because once the process is initiated, inflammation and oxidative stress cooperate to amplify each other, leading to progressive damage.

Another potential clinical application for LSAS and its small molecule derivatives lies in the field of anticoagulation for the treatment of venous thromboembolism. LSAS offers several advantages as an anticoagulant: i) availability and economic viability: LSAS is readily accessible, making it economically comparable to heparins. This availability addresses economic concerns; ii) reduced contamination risk: LSAS carries a lower risk of contamination with other super-sulfated polysaccharides, which mitigates the issues seen with heparins and recent supply shortages in the US; and iii) minimal bleeding risk: due to its unique mechanism, which involves selective prolongation of APTT, LSAS is associated with a minimal risk of bleeding compared to heparins. This characteristic enhances its safety for the same clinical applications as heparins [24].

Future directions would include studying the mechanism of inhibition of the proteases by LSAS using enzyme kinetics and mutagenesis studies. Efforts also involve evaluating LSAS efficacy in relevant animal disease models of significance to human health. They would also include the design and development of LSAS-based small molecules with better homogeneity. Given the water solubility of LSAS, these molecules will be suitable for parenteral use. Nevertheless, prodrug strategies will be developed to enable their oral administration. Other pharmacokinetic parameters (distribution, metabolism, and excretion) of LSAS and its small molecule derivatives will be evaluated in appropriate animal models and will be reported in due course.

Lastly, we would like to emphasize that the current in vitro pharmacological study using cell lines can only provide preliminary evidence and should not be extrapolated to any clinical disease model without support from animal studies or human trials.