Oxytetracycline has been successfully used to treat flexural limb deformities in foals. However, its antimicrobial properties and potential adverse side effects warrant the investigation of alternatives. Inhibition of matrix metalloproteinases (MMPs) or collagen cross-linking have been suggested as possible mechanisms of action. This study assessed the influence of potential alternative substances lacking antimicrobial properties on collagen gel contraction by juvenile equine myofibroblasts in vitro. Myofibroblasts were isolated from forelimb SDFTs and inferior check ligaments from foals euthanised for reasons unrelated to this study, and seeded in collagen gels containing oxytetracycline, MMP-inhibitors incyclinide, ilomastat, aprotinin, and pentoxifylline, β-aminopropionitrile fumarate (BAPN), and control medium. The gels were measured in photographs up to 96 h after release, and differences in contraction were evaluated. Compared to control, BAPN and incyclinide induced significant inhibition (p < 0.05) of collagen gel contraction by myofibroblasts at all time points, and the inhibition was more potent than that observed with oxytetracycline. Consequently, the inhibitory capacity of BAPN and incyclinide on myofibroblast-mediated collagen gel contraction, which demonstrates their effect on tractional structuring of collagen, makes them promising candidates as potential substitutes for oxytetracycline. Further investigations into their side effects, pharmacodynamics, pharmacokinetics, and application methods in juvenile equine patients are needed.

Nanomedicine strategies leveraging bio-derived nano-carriers offer promising avenues for precision chemotherapy. Here, we introduce a microfluidics-assisted nanoconjugate platform that repurposes Aprotinin (Apr); a naturally occurring protein, as an enhanced anti-cancer nano-carrier for Valproic Acid (VPA), a histone deacetylase inhibitor with anticancer properties. The resulting Aprotinin-Valproic Acid (Apr-VPA) nanoconjugates synthesized via a flow-controlled microfluidic process were characterized by various physioco-chemical methods confirming the stable binding. When tested on in vitro studies in 2D cell culture followed by custom designed 3D tumor spheroid models, the Apr-VPA nanoconjugates demonstrated dose-dependent inhibition of spheroid growth and viability, supporting the nanoconjugate's therapeutic potential. Preliminary in vitro comparisons with healthy endothelial cells suggest selective cytotoxicity, warranting further investigation. Interestingly, the designed nanoconjugates also exhibited pH-responsive release behavior under acidic conditions, indicating potential for envirnment-triggered drug release. Thus, it was expected that a novel high technology-controlled synthesis and stimuli sensitive release system, increasing the bioavailability of drug when conjugated (Apr-VPA) as compared to free VPA may perhaps be tranlated to the clinics when validated in vivo. Therefore, plasma profile of VPA and Apr-VPA nanoconjugates by LC-MS/MS and serum enzymatic markers were assessed in wild-type balb/c mice, depicting significantly enhanced availability of drug when conjugated with a carrier. These findings highlight the feasibility of using aprotinin as a scalable protein-based nanocarrier for tumor-preferential therapeutic response in tested cancer models and support its future development in advanced nanomedicine applications.

Prostate cancer (PCa) metastasis is reliant on the activity of proteases, such as matrix metalloproteinase-2 (MMP-2). While increased extracellular heat shock protein 90α (eHSP90α) has been linked to increased MMP-2 activity, this has not been examined in the context of cellular stress. We examined stress-induced eHSP90α in human prostate cell lines by immunoblot. Fluorometric gelatin dequenching and zymography assays measured MMP activity. Wound healing and Matrigel drop invasion assays were used to quantify cell motility. HSP90α knockout (KO) cells were established with CRISPR/Cas9. Proteases were profiled with molecular inhibitors and protein arrays and validated by siRNA knockdown, immunoblot, and motility assays. Stress increased eHSP90 in four out of four human prostate cell lines examined. Surprisingly, it concurrently decreased MMP-2 activity. The functional relevance of this was demonstrated when conditioned media from stressed cells decreased the motility of non-stressed cells. Screening for protease inhibitors that would rescue stress-induced decreases in MMP-2 activity identified a single serine protease inhibitor: aprotinin. Yet rescue with aprotinin was lost in HSP90α KO cells. A protease array identified stress-induced increases in kallikrein-related peptidase 6 (KLK6). Knockdown of KLK6 rescued stress-induced MMP-2 activity and cell motility. In conclusion, we identify a novel stress-induced extracellular network that regulates MMP-2 activity and cell motility. We identified KLK6 as a stress-induced extracellular protease leading to decreased MMP-2 activity and cellular invasion, while eHSP90α is required for the rescue of MMP-2 activity once KLK6 is neutralized.

The intestine is considered a habitat for both bacteria and parasites. In this study, many fecal bacterial isolates and the protozoan Blastocystis sp. were recovered from stool samples of individuals with gastrointestinal conditions. Isolated bacteria were tested for extracellular protease production, and the most potent producer was identified by 16SrDNA gene sequencing as P. aeruginosa FZM498. The enzyme was extracted and purified to electrophoretic homogeneity by the DEAE-Sepharose ion-exchanger and SDS-PAGE revealed a major band at 42.15 KDa. It exhibited maximal activity at 35 °C with thermostability at 60 °C (T1/2 = 200.04 min). It was most active at pH 8.0 and stable at 5.0-9.5. Enzymatic activity was greatly stimulated in the presence of Fe[2+] ions, but was repressed by Zn[2+] and Hg[2+] ions. Inhibition by PMSF, TLCK, aprotinin, benzamidine, and SBTI protease reagents suggests a serine protease family. The Vmax and Km dynamic constants against azocasein were 36.232 U/mL and 0.0072 mM, respectively. It exhibited the lowest Km value against the synthetic substrate D-Val-Leu-Lys-pNA among all substrates, indicating a plasmin-like activity. Interestingly, when tested against Blastocystis sp., cysts appeared progressively shrunken, ruptured, and mycelial-like, indicating complete structural collapse with leakage of intracellular contents. The importance of this research is that it is the first study to test the anti-Blastocystis activity of an extracted bacterial serine protease from the gut. This could be a promising, eco-friendly, natural alternative as an anti-Blastocystis agent. The objective of this study was to isolate, purify, and biochemically characterize an extracellular serine protease produced by gut-associated bacteria, as well as to assess its in vitro anti-Blastocystis efficacy as a potential natural and ecologically friendly antiparasitic therapy.

CELA3A and CELA3B are the primary elastases secreted by the human pancreas that are digestive enzymes and are markers for pancreatic exocrine insufficiency. CELA1, another family member, is not expressed in the human pancreas but has been detected in the mouse lung and human keratinocytes. However, the enzymatic activity and mechanism of function of human CELA1 has not been previously demonstrated. Here, we show using purified, recombinant pro-hCELA1, that it is activated by trypsin and has pancreatic elastase-like activity. We show that pro-hCELA1 cleavage by trypsin is blocked by aprotinin. We have determined the enzyme kinetics of the active fraction of recombinant hCELA1 and show that it follows steady-state kinetics with a higher substrate affinity than commercial porcine pancreatic elastase.

The stability of protein secondary structure is the basis for the realization of biological functions, and temperature induces conformational changes in proteins by disrupting the equilibrium of the internal hydrogen bond network. This study systematically elucidates the temperature-induced unfolding mechanism of aprotinin based on one-dimensional infrared spectroscopy (1D IR) and two-dimensional infrared spectroscopy (2D IR) simulated by molecular dynamics (MD) simulations. The simulations showed that the characteristic absorption peaks of the amide I band in the IR spectra of the aprotinin were blueshifted and weakened with increasing temperature, indicating that the hydrogen bond breaking leads to the secondary structure transformation of the aprotinin. The 2D IR simulations of aprotinin reveal that when the temperature rose to 333 K, the vibrational coupling peak of the β-sheet at (1625.4 cm[-1], 1653.4 cm[-1]) disappears. At 353 K, new coupling peaks corresponding to random coil and β-turn appear at (1639.7 cm[-1], 1672.2 cm[-1]), and these signals disappear at 373 K, indicating complete unfolding of aprotinin. During the heating process, the 2D IR spectral signal of the aprotinin shifted from excited-state absorption (ESA) to ground-state bleaching (GSB), reflecting the energy transfer process within the protein, and its dynamics process and spectral broadening rate were greatly affected by temperature. The unfolding pathway of aprotinin was again obtained by MD simulation and analyzed in comparison with infrared spectroscopy, and the unfolding behavior of the aprotinin was resolved at the atomic level.

Proteinuria is both a predictor and mediator of chronic kidney disease (CKD) progression, but treatment options targeting its underlying mechanisms are limited. Emerging evidence suggests that aberrantly filtered serine proteases contribute to the pathogenesis of proteinuria and progressive kidney injury through multiple pathways, including podocyte injury, inappropriate activation of the epithelial sodium channel (ENaC), and tubular complement activation. Serine protease inhibitors, such as aprotinin, camostat mesylate, and nafamostat mesylate, as well as off-target effects of amiloride, have shown promise in preclinical and early clinical studies by mitigating these pathological processes. These drugs reduce proteinuria, sodium retention, oxidative stress, inflammation, and fibrosis. However, clinical translation is hindered by limited data from controlled trials, varying pharmacokinetics, and concerns about systemic adverse effects and long-term safety. Endogenous serine protease inhibitors help maintain proteolytic balance in the kidneys, but their capacity may be overwhelmed in proteinuria. Although complete inhibition could disrupt essential functions, pharmacologic modulation of tubular serine protease activity may be a more effective strategy by preserving beneficial activity while limiting pathological effects. This review synthesizes current knowledge on the pathophysiological role of tubular serine proteases and evaluates the therapeutic potential of their inhibition as a potential target in proteinuric diseases. We identify key knowledge gaps, including the need for mechanistic pharmacodynamic trials, biomarker-guided patient selection using urinary serine protease activity, and long-term efficacy and safety studies. Serine protease inhibitors are a promising, underexplored therapeutic strategy in proteinuric conditions that may complement existing treatments by targeting specific pathogenic mechanisms involved in disease progression.

Background/Objectives: Conventional Coagulation Tests (CCTs) fail to accurately reflect Preeclampsia's (PE's) coagulation status, disease progression, and hemostatic alterations. They do not differentiate between the normal hypercoagulability of healthy pregnancies and the pathological hypercoagulability associated with PE. Rotational Thromboelastometry (ROTEM) analyzes clot dynamics from initiation through amplification and propagation to termination and fibrinolysis. However, their application in PE, particularly in neonates born to women with PE, is limited. We aimed to identify the hemostatic alterations in pregnant women with PE using ROTEMs that remain undetected by CCTs and to assess PE's impact on neonatal hemostasis at birth. Methods: This was a single-center observational study (March 2022-March 2024) including 31 women with PE (34 newborns) and 45 pregnancies without PE (47 newborns). Maternal blood was collected intrapartum before placental delivery. Neonatal arterial samples were obtained within the first hour of life before vitamin K administration. ROTEM (Intrinsic (INTEM), Extrinsic (EXTEM), Fibrinogen (FIBTEM), Aprotinin (APTEM)), and CCTs were performed. Subgroup analyses considered PE severity and onset. ROC analyses examined discrimination for persistent maternal thrombocytopenia within 7 days of delivery and association with maternal platelet transfusion. Results: In preeclamptic women, the INTEM and FIBTEM assays were more affected, with higher Actual Clot Firmness (ACF) (p = 0.03, p = 0.04, respectively) and a higher Clot Formation Rate (CFR) (p = 0.03, p = 0.02, respectively). Hyperfibrinolysis was present (CT-APTEM < CT-EXTEM, MCF-APTEM > MCF-EXTEM). Clot Formation Time CFT-EXTEM was an indicator of maternal platelet transfusion (AUC = 0.81). Across EXTEM, INTEM, and APTEM, A10 (Amplitude at 10 min) and CFT showed good discrimination capability for maternal persistent thrombocytopenia within 7 days of delivery (AUCs 0.82-0.95). Neonates of women with PE presented lower ACF across all assays (INTEM p = 0.003; EXTEM p = 0.001; FIBTEM p = 0.01; APTEM p < 0.001), consistent across severity/onset subgroups. Conclusions: In this cohort, ROTEM identified maternal hypercoagulability with hyperfibrinolysis and neonatal hypocoagulability during the first hour of life. Several alterations were not reflected in CCTs. Further prospective studies should evaluate the role and clinical utility of combining ROTEM with CCTs for hemostatic monitoring in women with PE and their neonates.

Bleeding is common after cardiac surgery and is associated with increased morbidity and mortality. The etiology of coagulopathy after cardiopulmonary bypass is complex, involving systemic inflammation, hemodilution, residual heparin effect, platelet activation, hypothermia, and hyperfibrinolysis. Antifibrinolytic agents such as aprotinin and lysine analogs are used to mitigate hyperfibrinolysis. Although epsilon-aminocaproic acid (EACA) is generally considered safe, dosing regimens vary, and thrombotic complications are underreported in national registries. We describe a case of acute intracardiac thrombosis shortly after EACA administration during mitral valve repair in a patient with no known hematologic or hypercoagulable conditions. .