Cc3 -SPase (30 kDa-proteinase; pI 5.98) was isolated from Cerastes cerastes venom. Its sequence of 271 residues yielded from LC-MALDI-TOF showed high degrees of homology when aligned with other proteinases. Cc3 -SPase cleaved natural and synthetic proteins such as casein and fibrinogen leaving fibrin clots unaffected. Cc3 -SPase was fully abolished by ion chelators, whereas aprotinin, antithrombin III (Sigma Aldrich, Saint-Louis, Missouri, USA), and heparin were ineffective. Affinity of Cc3 -SPase to benzamidine indicated the presence of an aspartate residue in the catalytic site as confirmed by three-dimensional structure consisting of 14 β-strands and four α-helices. Molecular mechanisms revealed that Cc3 -SPase is capable of promoting dysfunctional platelet aggregation via two signaling pathways mediated by the G-coupled protein receptors and αIIbβ3 integrin. Cc3 -SPase is involved in both extrinsic/intrinsic coagulation pathways in deficient plasmas by replacing defective/lacking factors FII, FVII, and FVIII but not FX. Cc3 -SPase could substitute missing factors in blood diseases related to plasma factor deficiencies.

Proteins that account for the hemolytic activity found in scorpaeniform fish venoms are responsible for the majority of the effects observed upon envenomation, for instance, neurotoxic, cardiotoxic and inflammatory effects. These multifunctional toxins, described as protein lethal factors and referred to as cytolysins, are known to be extremely labile molecules. In the present work, we endeavored to overcome this constraint by determining optimal storage conditions for Sp-CTx, the major bioactive component from the scorpionfish Scorpaena plumieri venom. This cardiotoxic hemolytic cytolysin is a large dimeric glycoprotein (subunits of ≈65 kDa) with pore-forming ability. We were able to establish storage conditions that allowed us to keep the toxin partially active for up to 60 days. Stability was achieved by storing Sp-CTx at -80 and -196 °C in the presence of glycerol 10% in a pH 7.4 solution. It was demonstrated that the hemolytic activity of Sp-CTx is calcium dependent, being abolished by EDTA and zinc ions. Furthermore, the toxin exhibited its maximal hemolytic activity at pH between 8 and 9, displaying typical N- and O- linked glycoconjugated residues (galactose (1-4) N-acetylglucosamine and sialic acid (2-3) galactose in N- and/or O-glycan complexes). The hemolytic activity of Sp-CTx was inhibited by phosphatidylglycerol and phosphatidylethanolamine, suggesting a direct electrostatic interaction lipid - toxin in the pore-formation mechanism of action of this toxin. In addition, we observed that the hemolytic activity was inhibited by increasing doses of cholesterol. Finally, we were able to show, for first time, that Sp-CTx is at least partially responsible for the pain and inflammation observed upon envenomation. However, while the edema induced by Sp-CTx was reduced by pre-treatment with aprotinin and HOE-140, pointing to the involvement of the kallikrein-kinin system in this response, these drugs had no significant effect in the toxin-induced nociception. Taken together, our results could suggest that, as has been already reported for other fish cytolysins, Sp-CTx acts mostly through lipid-dependent pore formation not only in erythrocytes but also in other cell types, which could account for the pain observed upon envenomation. We believe that the present work paves the way towards the complete characterization of fish cytolysins.

Matriptase-2 is a type II transmembrane serine protease and a key regulator of systemic iron homeostasis. Since the activation mechanism and several features of the physiological role of matriptase-2 are not fully understood, there is strong need for analytical tools to perform tasks such as distinguishing active and inactive matriptase-2. For this purpose we present a short biotinylated peptide derivative with a chloromethyl ketone group, biotin-RQRR-CMK, as an activity-based probe for matriptase-2. Biotin-RQRR-CMK was kinetically characterized and exhibited a second-order rate constant of inactivation (kinac/Ki) of 10,800 M−1 s−1 towards the matriptase-2 activity in the supernatant of transfected human embryonic kidney (HEK) cells. Biotin-RQRR-CMK was able to label active matriptase-2, as visualized in western blot experiments. Pretreatment with aprotinin, an active-site directed inhibitor of serine proteases, protected matriptase-2 from the reaction with biotin-RQRR-CMK.

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its γ-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Various toxicants, such as drugs and their metabolites, can cause potential ovarian toxicity. As the functional units of the ovary, ovarian follicles are susceptible to this type of damage at all developmental stages. Studying the effects of toxicants on ovarian follicles is an important task. Three-dimensional (3D) hydrogels, such as fibrin alginate interpenetrating networks (FA-IPNs), can support ovarian follicle culture in vitro for extended periods of time and serve as a suitable tool for studying ovotoxicity. Growing follicles encapsulated in the FA-IPN can proteolytically degrade the fibrin component in the FA-IPN. The degradation of fibrin mirrors the follicle growth and serves as a surrogate reporter for follicle health. The speed of fibrin degradation can be further controlled by aprotinin, a small molecule that inhibits plasmin-driven proteolytic degradation, which further expands the application of the described system. In this chapter, we describe methods to (1) isolate and encapsulate mouse ovarian follicles in FA-IPN, (2) follow follicle growth and development in vitro, and (3) evaluate the effects of toxicants on folliculogenesis using fibrin degradation.