Search Results (77)

Blood-sucking organisms produce various anticoagulant proteins that prevent blood clotting in their prey. Even in well-studied species like Hirudo medicinalis, many such proteins remain unidentified. We previously described a novel cysteine-rich anticoagulant (CRA), a distant homolog of antistasin. Later, we discovered another, much larger homolog in the medicinal leech. Its amino acid sequence is also highly cysteine-rich. Analysis of cysteine patterns showed four antistasin-like domain motifs, with one of them strongly disrupted. Since both antistasin and CRA contain two such domains, the new protein represents a duplicated antistasin-like structure. We cloned its cDNA, expressed the recombinant protein in Escherichia coli, purified it by metal-chelate chromatography, refolded it, and tested its anticoagulant properties. Using standard clinical assays—activated partial thromboplastin time, prothrombin time, and thrombin time—we found that the protein inhibited coagulation in all tests, though to varying degrees. These findings suggest that different antistasin-like anticoagulants in the leech enable it to block both intrinsic and extrinsic coagulation pathways, while hirudin inhibits the final step of clot formation. The combination of different anticoagulant proteins allows the leech to effectively prevent the prey’s blood from clotting during feeding. Full article

Platelet-rich fibrin (PRF) is widely used in regenerative dentistry and oral surgery for its ability to promote tissue healing and modulate cellular responses. However, PRF contains not only platelets but also leukocytes and plasma components, complicating efforts to define the specific contribution of platelets to its biological activity. To address this, we used washed, leukocyte-depleted platelets activated with thrombin to generate platelet-released supernatant (PRS), which was applied to gingival fibroblasts. RNA sequencing identified 147 upregulated and 39 downregulated genes (|log₂ fold change| ≥ 2, FDR < 0.001), including cytokines IL11 and CXCL8 previously associated with PRF, as well as mitosis-related genes such as centromere-associated proteins, cell division cycle proteins, kinesin-like proteins, and shugoshins, consistent with gene ontology analyses. Validation by RT-PCR and immunoassays confirmed robust upregulation of IL11 and CXCL8. Functionally, PRS activated TGF-β signaling, indicated by Smad2/3 nuclear translocation, but did not induce NF-κB signaling. These findings demonstrate that platelets are major contributors to PRF’s biological effects, independent of leukocytes and plasma, and elicit a pronounced mitogenic and TGF-β-dominant response in gingival fibroblasts. They also provide insight into the cellular mechanisms underlying PRF-mediated tissue regeneration. Full article

Bothrops species are responsible for the majority of envenomations in Argentina. In particular, Bothrops diporus is among the main species responsible for the majority of envenomations in Argentina and causes significant injury and coagulopathy. Given the significance of this venom, the authors sought to define the toxin responsible for coagulopathy with specialized spectrophotometric and thromboelastographic methods. Utilizing clotting time, spectrophotometry, and thromboelastography, it was determined that B. diporus venom has potent, procoagulant activity in human plasma and buffer milieu. Calcium-dependent and -independent activities consistent with serine protease activity were identified. The activity included both thrombin-generating and thrombin-like enzymatic activity. The venom cleaved the serine protease-specific chromogenic substrate β-Ala-Gly-Arg-p-nitroanilide diacetate, and its activity was inhibited in plasma by antithrombin after addition of heparin. Further, venom exposed in isolation to RuCl₃, a known inhibitor of serine protease-containing venoms, demonstrated decreased activity in human plasma. In conclusion, the present study contributes to a better understanding of B. diporus venom and may have implications for the rational design of inhibitors, antivenom formulations, or preclinical models to study venom-induced coagulopathies. Full article

To express and purify staphylococcal enterotoxin M (SEM) using immobilized metal affinity chromatography (IMAC), a signal peptide-truncated (ΔNsp) wild-type SEM (SEM_WT) was N-terminally fused in pET-28a(+) to a polyhistidine tag (His_6×-) and thrombin cleavage site (TCS; LVPR↓GS), generating His_6×-TCS-ΔNspSEM_WT. Unexpectedly, 4 °C desalting reduced the fusion protein’s molecular weight by ~2.0 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequencing and mass spectrometry identified cleavage specifically at the arginine (R) and glycine (G) peptide bond (R–G bond) within the TCS motif. AlphaFold 3 revealed an exposed serine protease catalytic triad: histidine 172, serine 178, and aspartic acid 212 (H₁₇₂/S₁₇₈/D₂₁₂) in the β-grasp domain, suggesting intrinsic thrombin-like activity (TLA). Sequential IMAC and size-exclusion high-performance liquid chromatography (SE-HPLC) purification eliminated contaminant concerns, while chromogenic substrate S-2238 (S-2238) assays demonstrated increasing specific activity and purification fold, supporting intrinsic TLA. Critically, the mutation of serine at position 178 to alanine (His_6×-TCS-ΔNspSEM_S178A) abolished TLA but preserved the secondary/tertiary structure, confirming the activity’s origin within the wild-type construct. Molecular dynamics (MD) simulations probed the atomistic mechanism for specific R–G bond cleavage. This work establishes a foundation for understanding ΔNspSEM_WT’s TLA. Full article

Proteases constitute one of the largest sub-classes of enzymes, accounting for ca. 2% of the proteins encoded in the human genome. They play a key role in protein degradation and signaling, regulating a variety of physiological processes. Dysregulation of their activity is associated with various pathological conditions like cancer, neurodegenerative disorders, inflammatory or cardiovascular diseases. Protease activity can be controlled by regulating enzyme concentrations, but also by inhibitors, molecules that modulate enzyme function, inspiring the development of small molecule protease inhibitors for therapeutic purposes. Protease inhibitors can be designed from the corresponding substrates by isostere replacement at the scissile bond. This process yields a first-generation of inhibitors that usually exhibit poor drug-like profiles that need subsequently be improved to generate a second-generation, by smoothing their peptide-like features. This process is reviewed in the present report and exemplified in the successful discovery stories of different inhibitors that correspond to four types of proteases, including the angiotensin converting enzyme (metalloprotease); HIV protease (aspartate protease); thrombin (serine protease) and the proteasome (threonine protease). A detailed description of the stories behind their design from their initial discovery to the final product is described in this report. Moreover, despite successful discovery stories, the challenges associated with designing novel protease inhibitors are examined. Finally, the relevance of these drugs in the present drug market is also reported. Full article

Background/Objectives: Vitamin C is frequently used in several dietary supplements due to its proposed health-promoting properties, while phenolic compounds and especially flavonoids have been suggested to provide synergistic antioxidant and cardiovascular benefits. However, the specific interactions between these compounds and their individual contributions to biological activity remain underexplored. This study aimed to evaluate the antioxidant potential and anti-inflammatory and antiplatelet biological effects of a high-dose (1 g) vitamin C–low-dose (50 mg) bioflavonoid (VCF)-based supplement using both in vitro and in vivo approaches in human platelets. Methods: Total phenolic content was quantified and antioxidant capacity was assessed using DPPH, FRAP, and ABTS assays and compared to individual phenolic standard compounds, including (simple phenolics like gallic acid, flavonoids like quercetin and catechin, and polyphenols like curcumin and tannin), and a standard supplement containing only high-dose vitamin C (VC). ATR-FTIR spectroscopy was used to assess molecular interactions between vitamin C and flavonoids. In vitro anti-inflammatory and antiplatelet activities of all supplements and standards were assessed by quantifying their IC₅₀ values against ADP, PAF, and thrombin-induced platelet aggregation. The in vivo evaluation of the efficacy and synergy of VCF supplement versus VC was achieved by a two-arm clinical study in healthy volunteers by quantifying their platelet reactivity, which was measured via EC₅₀ values on the aforementioned platelet agonists (PAF, ADP, and Thrombin) before (t = 0) and after receiving either solely VC or VCF supplementation for four weeks. Results: From all phenolic standards, the flavonoids and especially a mixture of flavonoids (catechin + quercetin) showed higher in vitro antioxidant capacity and anti-inflammatory and antiplatelet efficacy, followed by polyphenols and then simple phenolics. The VCF supplement showed the most potent antioxidant capacity, but also the strongest anti-inflammatory and antiplatelet activities too, in comparison to the VC and the mixture of flavonoids, suggesting higher synergy and thus bio-efficacy as a result of the co-presence of flavonoids and vitamin C in this supplement. Platelet reactivity decreased over time for PAF and thrombin in both arms of the trial, but no significant differences were observed between treatment groups, suggesting that the number of flavonoids used was not sufficient to translate the in vitro findings to the in vivo setting. Conclusions: VC-containing supplements provide antioxidant, anti-inflammatory, and antiplatelet benefits, while the incorporation of flavonoids may provide synergistic health benefits, but more in vivo assessment is needed to fully evaluate the dose efficacy. Full article

Plant latex is a rich source of proteolytic enzymes with potential biomedical applications, particularly in hemostasis. Among them, thrombin-like enzymes (TLEs) have garnered interest in their ability to mimic thrombin by catalyzing the conversion of fibrinogen to fibrin, facilitating clot formation. While TLEs from snake venoms have been well-characterized and applied clinically, their plant-derived counterparts remain underexplored. This review critically examines the structural and functional characteristics of TLEs from plant latex, comparing them to animal-derived TLEs and evaluating their role in both procoagulant and fibrinolytic processes. Emphasis is placed on dual fibrinogenolytic and fibrinolytic activities exhibited by latex proteases, which often vary with concentration, incubation time, and protease type. In vitro coagulation assays and electrophoretic analyses are discussed as critical tools for characterizing their multifunctionality. By addressing the knowledge gaps and proposing future directions, this paper positions plant latex proteases as promising candidates for development in localized hemostatic and thrombolytic therapies. Full article

Beyond haemostasis and thrombosis, platelets are increasingly recognized for playing a crucial role in modulating immunoinflammation. Toll-like receptors (TLRs) constitute the first line of defence against infection and injury, with their engagement stimulating thrombotic and immune responses in platelets. Hence, anti-platelet drugs have been used to treat patients with infections and inflammation. However, due to the increased risk of bleeding with current anti-platelet drugs, alternative therapeutic targets need to be identified to ameliorate the consequences of inflammation-driven platelet hyperactivation. Previously, we demonstrated that resting platelets exhibit a metabolic plasticity that facilitates fuel selection flexibility, while in contrast, thrombin-stimulated platelets become highly glycolytic. Since multiple aspects of platelet activation require energy in terms of ATP, we investigated metabolic alterations in TLR1/TLR2-activated platelets. In this study, we have demonstrated that TLR1/TLR2-induced platelet activation reprogrammed platelets to upregulate glycolysis via CD36-linked mechanisms. In addition, we showed that this glycolytic flux is controlled by hexokinase (HK), which plays a crucial role in TLR1/TLR2-induced platelet aggregation. Targeting platelet metabolism plasticity may offer a novel strategy to inhibit platelet function in TLR-initiated diseases. Full article

Cardiovascular disease remains a leading cause of morbidity and mortality worldwide, frequently arising from platelet hyperactivation and subsequent thrombus formation. Although conventional antiplatelet therapies are available, challenges, such as drug resistance and bleeding complications, require the development of novel agents. In this study, dihydrogeodin (DHG) was isolated from Fennellia flavipes and evaluated using platelets derived from Sprague–Dawley rats. Platelet aggregation induced by collagen, adenosine diphosphate, or thrombin was assessed by light transmission aggregometry; DHG significantly reduced aggregation in a dose-dependent manner. Further assays demonstrated that DHG suppressed intracellular calcium mobilization, adenosine triphosphate release, and integrin αIIbβ₃-dependent fibrinogen binding, thereby impairing clot retraction. Western blot analysis revealed that DHG reduced the phosphorylation of mitogen-activated protein kinases (ERK, JNK, p38) and PI3K/Akt, indicating inhibition across multiple platelet-signaling pathways. Additionally, SwissADME-assisted pharmacokinetics predicted favorable properties without violations of the Lipinski (Pfizer) filter, Muegge (Bayer) filter, Ghose filter, Veber filter, and Egan filter, and network pharmacology revealed inhibition of calcium and MAPK pathways. These results highlight the potential of DHG as a novel antiplatelet agent with broad-spectrum activity and promising drug-like characteristics. Further studies are warranted to assess its therapeutic window, safety profile, and potential for synergistic use with existing antiplatelet drugs. Full article

Snakebite represents a significant public health challenge in Central and South America, with Lachesis (Bushmaster) species posing unique clinical challenges due to their severe envenomation effects arising from a combination of potent venom and copious venom yields. Using in vitro coagulation assays, we analyzed the coagulotoxic venom effects from four distinct localities: L. muta from Surinam and French Guiana and L. stenophrys from Costa Rica and Panama. This study examined the venom’s impact on human plasma and fibrinogen and evaluated the efficacy of two regionally available antivenoms (PoliVal-ICP and Antivipmyn-Tri) in neutralizing the pathophysiological effects. Our results demonstrated a remarkable consistency in the pseudo-procoagulant venom activity (also known as: thrombin-like) across different species and localities. Antivenom efficacy testing revealed that both the PoliVal-ICP and Antivipmyn-Tri antivenoms effectively neutralized the venom effects across localities for both species, with the ICP antivenom showing the highest neutralization capacity. These toxicology findings highlight the biochemical conservation of venom composition across Lachesis species which underpins effective cross-neutralization in antivenom treatment. Full article

The Philippines has a high diversity of venomous snake species, but there is minimal information on their envenomation effects. This is evidenced by the small number of case reports, the poor reporting of envenomation cases, and the absence of specific antivenoms apart from one against the Philippine cobra (Naja philippinensis). This study sought to profile the action of selected Philippine pit viper venoms on blood coagulation and to investigate whether commercially available non-specific antivenoms can provide adequate protection against these venoms. Venom from the pit vipers Trimeresurus flavomaculatus and Trimeresurus mcgregori were subjected to coagulation assays, antivenom cross-neutralization tests, and thromboelastography. Venoms from both species were able to clot human plasma and isolated human fibrinogen. Consistent with pseudo-procoagulant/thrombin-like activity, the resulting fibrin clots were weak and transient, thereby contributing to net anticoagulation through the depletion of fibrinogen levels. Clotting factors fIXa and fXa were also inhibited by the venoms, further contributing to the net anticoagulant activity. Monovalent and polyvalent antivenoms from the Thai Red Cross Society were effective against both venoms, indicating cross-neutralization of venom toxins; the polyvalent antivenom was able to rescue fibrinogen clotting to a greater degree than the monovalent antivenom. Our findings highlight the coagulopathic effects of these pit viper venoms and suggest the utility of procuring the non-specific antivenoms for areas in the Philippines with a high risk for pit viper envenomation. Full article

Background: The lingering effects of SARS-CoV-2 infection, collectively known as post-COVID syndrome (PCS), affect a significant proportion of recovered patients, manifesting as persistent symptoms like fatigue, cognitive dysfunction, and exercise intolerance. Increasing evidence suggests that endothelial dysfunction and coagulation abnormalities play a central role in PCS pathophysiology. This study investigates hypercoagulability and endothelial dysfunction in PCS through thrombin generation and the von Willebrand factor (VWF)/ADAMTS13 axis. Methods: Plasma samples from 97 PCS patients recruited since October 2020 by the clinical research unit of the Brugmann University Hospital were analyzed. A thrombin generation test was performed on a St-Genesia^® analyzer (Stago) using the Thromboscreen kit; VWF antigen was determined on a CS-2500 analyzer (Siemens); and ADAMTS-13 activity was determined using an ELISA kit (Technozym^®) on an ElX808 plate reader. Results: Thrombin generation testing revealed elevated thrombin production in PCS patients, particularly when thrombomodulin was included. Although most PCS patients showed normalized VWF/ADAMTS13 ratios, 11.3% exhibited elevated ratios (≥1.5), associated with advanced age. Conclusions: Patients with PCS show a consistent pattern of prolonged thrombo-inflammatory dysregulation, highlighted by elevated in vitro thrombin generation and the persistence of abnormal VWF/ADAMTS-13 ratios in a subset of patients. Full article

Serine proteases are multifunctional and versatile venom components found in viper snakes, including the Bothrops species, a widely distributed genus notorious for causing the highest number of snakebites across Latin America. These enzymes, representing a significant fraction of Bothrops venom proteomes, exhibit a wide range of biological activities that influence blood coagulation, fibrinolysis, and inflammation. This review provides a comprehensive overview of serine proteases, with a particular focus on those found in the venom of Brazilian Bothrops snakes. The discussion begins with a summary of snake species found in Brazil and their medical relevance. Specifically addressing the Bothrops genus, this review explores the distribution of these species across Brazilian territory and their associated medical importance. Subsequently, the article investigates the biochemistry of Bothrops venoms and the clinical manifestations induced by envenomation. Finally, it offers an in-depth discussion on the serine proteases, highlighting their biochemical properties, mechanisms of action, and potential therapeutic applications. Furthermore, this review provides an in-depth exploration of the diverse serine proteases found in Bothrops venoms and their functional significance, from thrombin-like effects to potent fibrinogenolytic actions, which determine the clinical manifestations of envenomation. This review delves into the evolutionary adaptations and biochemical diversity of serine proteases in Bothrops venoms, emphasizing their critical roles in venom functionality and the resulting pathophysiological effects. Additionally, it opens new avenues for utilizing these enzymes in biomedical applications, underscoring their potential beyond toxinology. Full article

Background: We compared the enzymatic, coagulant, and neuromuscular activities of two variants (yellow—CDRy and white—CDRw) of Crotalus durissus ruruima venom with a sample of C. d. terrificus (CDT) venom and examined their neutralization by antivenom against CDT venom. Methods: The venoms were screened for enzymatic and coagulant activities using standard assays, and electrophoretic profiles were compared by SDS-PAGE. Neutralization was assessed by preincubating venoms with crotalic antivenom and assaying the residual activity. Results: SDS-PAGE showed that the venoms had similar electrophoretic profiles, with the main bands being phospholipase A₂ (PLA₂), serine proteinases, L-amino acid oxidase (LAAO), and phosphodiesterase. CDRy venom had the highest proteolytic and LAAO activities, CDRw venom had greater PLA₂ and esterolytic activities at the highest quantity tested, and CDT had greater PLA₂ activity than CDRy. CDRw and CDT venoms had similar proteolytic and LAAO activities, and CDRy and CDT venoms had comparable esterolytic activity. None of the venoms altered the prothrombin time (PT), but all of them decreased the activated partial thromboplastin time (aPPT); this activity was neutralized by antivenom. The minimum coagulant dose potency was CDRw >> CDRy > CDT. All venoms had thrombin-like activity that was attenuated by antivenom. CDRy and CDRw venoms showed α-fibrinogenolytic activity. All venoms partially cleaved the β-chain. CDRy and CDT venoms caused neuromuscular facilitation (enhanced muscle contractions) followed by complete blockade, whereas CDRw venom caused only blockade. Antivenom neutralized the neuromuscular activity to varying degrees. Conclusions: These findings indicate that while CDR and CDT venoms share similarities, they also differ in some enzymatic and biological activities and in neutralization by antivenom. Some of these differences could influence the clinical manifestations of envenomation by C. d. ruruima and their neutralization by the currently used therapeutic antivenom. Full article

Thrombin binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers that efficiently binds to thrombin, resulting in anticoagulant effects. TBA also possesses promising antiproliferative properties. As with most therapeutic oligonucleotides, chemical modifications are critical for therapeutic applications, particularly to improve thermodynamic stability, resistance in biological environment, and target affinity. To evaluate the effects of nucleobase and/or sugar moiety chemical modifications, five TBA analogues have been designed and synthesized considering that the chair-like G4 structure is crucial for biological activity. Their structural and biological properties have been investigated by Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), native polyacrylamide gel electrophoresis (PAGE) techniques, and PT and MTT assays. The analogue TBAB contains 8-bromo-2′-deoxyguanosine (B) in G-syn glycosidic positions, while TBAL and TBAM contain locked nucleic acid guanosine (L) or 2′-O-methylguanosine (M) in G-anti positions, respectively. Instead, both the two types of modifications have been introduced in TBABL and TBABM with the aim of obtaining synergistic effects. In fact, both derivatives include B in syn positions, exhibiting in turn L and M in the anti ones. The most appealing results have been obtained for TBABM, which revealed an interesting cytotoxic activity against breast and prostate cancer cell lines, while in the case of TBAB, extraordinary thermal stability (T_m approximately 30 °C higher than that of TBA) and an anticoagulant activity higher than original aptamer were observed, as expected. These data indicate TBAB as the best TBA anticoagulant analogue here investigated and TBABM as a promising antiproliferative derivative. Full article