Suramin, a drug for the treatment of trypanosomiasis, reduces the prothrombotic and metastatic phenotypes of colorectal cancer cells by inhibiting hepsin.

Recently, our group identified serine-protease hepsin from primary tumor as a biomarker of metastasis and thrombosis in patients with localized colorectal cancer. We described hepsin promotes invasion and thrombin generation of colorectal cancer cells in vitro and in vivo and identified venetoclax as a hepsin inhibitor that suppresses these effects. Now, we aspire to identify additional hepsin inhibitors, aiming to broaden the therapeutic choices for targeted intervention in colorectal cancer. METHODS: We developed a virtual screening based on molecular docking between the hepsin active site and 2000 compounds from DrugBank. The most promising drug was validated in a hepsin activity assay. Subsequently, we measured the hepsin inhibitor effect on colorectal cancer cells with basal or overexpression of hepsin via wound-healing, gelatin matrix invasion, and plasma thrombin generation assays. Finally, a zebrafish model determined whether hepsin inhibition reduced the invasion of colorectal cancer cells overexpressing hepsin. RESULTS: Suramin was the most potent hepsin inhibitor (docking score: -11.9691 Kcal/mol), with an IC50 of 0.66 µM. In Caco-2 cells with basal or overexpression of hepsin, suramin decreased migration and significantly reduced invasion and thrombin generation. Suramin did not reduce the thrombotic phenotype in the hepsin-negative colorectal cancer cells HCT-116 and DLD-1. Finally, suramin significantly reduced the in vivo invasion of Caco-2 cells overexpressing hepsin. CONCLUSION: Suramin is a novel hepsin inhibitor that reduces its protumorigenic and prothrombotic effects in colorectal cancer cells. This suggests the possibility of repurposing suramin and its derivatives to augment the repertoire of molecular targeted therapies against colorectal cancer.

Venetoclax is a potent hepsin inhibitor that reduces the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells.

Introduction: Hepsin is a type II transmembrane serine protease and its expression has been linked to greater tumorigenicity and worse prognosis in different tumors. Recently, our group demonstrated that high hepsin levels from primary tumor were associated with a higher risk of metastasis and thrombosis in localized colorectal cancer patients. This study aims to explore the molecular role of hepsin in colorectal cancer. Methods: Hepsin levels in plasma from resected and metastatic colorectal cancer patients were analyzed by ELISA. The effect of hepsin levels on cell migration, invasion, and proliferation, as well as on the activation of crucial cancer signaling pathways, was performed in vitro using colorectal cancer cells. A thrombin generation assay determined the procoagulant function of hepsin from these cells. A virtual screening of a database containing more than 2000 FDA-approved compounds was performed to screen hepsin inhibitors, and selected compounds were tested in vitro for their ability to suppress hepsin effects in colorectal cancer cells. Xenotransplantation assays were done in zebrafish larvae to study the impact of venetoclax on invasion promoted by hepsin. Results: Our results showed higher plasma hepsin levels in metastatic patients, among which, hepsin was higher in those suffering thrombosis. Hepsin overexpression increased colorectal cancer cell invasion, Erk1/2 and STAT3 phosphorylation, and thrombin generation in plasma. In addition, we identified venetoclax as a potent hepsin inhibitor that reduced the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells. Interestingly, pretreatment with Venetoclax of cells overexpressing hepsin reduced their invasiveness in vivo. Discussion: Our results demonstrate that hepsin overexpression correlates with a more aggressive and prothrombotic tumor phenotype. Likewise, they demonstrate the antitumor role of venetoclax as a hepsin inhibitor, laying the groundwork for molecular-targeted therapy for colorectal cancer.

Identification of Kukoamine A, Zeaxanthin, and Clexane as New Furin Inhibitors.

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.

Anti-Tumor Functions of Prelatent Antithrombin on Glioblastoma Multiforme Cells.

Antithrombin, the main physiological inhibitor of the coagulation cascade, exerts anti-tumor effects on glioblastoma multiforme cells. Antithrombin has different conformations: native, heparin-activated, prelatent, latent, and cleaved. The prelatent form has an intermediate affinity between latent and native antithrombin, although it is the most antiangiogenic form. Herein, we investigate the effect of this conformation on the tumorigenic processes of glioblastoma multiforme cells. Antithrombin forms were purified by chromatography. Chromogenic/fluorogenic assays were carried out to evaluate enteropeptidase and hepsin inhibition, two serine proteases involved in these processes. Wound healing, Matrigel invasion and BrdU incorporation assays were performed to study migration, invasion and proliferation. E-cadherin, Vimentin, VEGFA, pAKT, STAT3, pSTAT3, and pERK1/2 expression was assessed by Western blot and/or qRT-PCR. Prelatent antithrombin inhibited both enteropeptidase and hepsin, although it was less efficient than the native conformation. Exposure to prelatent antithrombin significantly reduced migration and invasion but not proliferation of U-87 MG, being the conformation most efficient on migration. Prelatent antithrombin down-regulated VEGFA, pSTAT3, and pERK1/2 expression in U-87 MG cells. Our work elucidates that prelatent antithrombin has surprisingly versatile anti-tumor properties in U-87 MG glioblastoma multiforme cells. This associates with resistance pathway activation, the decreased expression of tumorigenic proteins, and increased angiogenesis, postulating the existence of a new, formerly unknown receptor with potential therapeutic implications.

N-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function.

N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.

First exploratory study on the metabolome from plasma exosomes in patients with paroxysmal nocturnal hemoglobinuria.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease in which patients are at increased risk of thrombosis. The mechanisms underlying the associated thrombosis risk are still poorly understood, although it is known that Eculizumab, the drug of choice for symptomatic patients, prevents thrombotic events. Exosomes are extracellular vesicles that can carry and disseminate genetic material, tumor biomarkers and inflammatory mediators. To date, the metabolite cargo of plasma exosomes from PNH patients has not yet been explored. In this pilot trial, we compared the metabolome of plasma exosomes from PNH patients with that of healthy subjects in order to provide further insights into this rare disease. RESULTS: We used a non-targeted metabolomics approach with UPLC-ESI-QTOF-MS/MS and GC-MS platforms. Multivariate analyses revealed the differential occurrence (p < .001) of 78 metabolites in plasma exosomes from PNH patients vs healthy control subjects. Remarkably, prostaglandin F2-alpha (6.1-fold), stearoyl arginine (5.3-fold) and 26-hydroxycholesterol-3-sulfate (11.2-fold) were higher in PNH patients vs healthy controls (p < .001). CONCLUSIONS: This is the first description on the differential metabolite cargo occurring in plasma exosomes from PNH patients. Our results could contribute to the search for possible prognostic biomarkers of thrombotic risk in patients with PNH. Further research in a larger cohort to validate these results is warranted.

Antithrombin is incorporated into exosomes produced by antithrombin non-expressing cells.

Antithrombin is a serine protease inhibitor that exerts a crucial role in hemostasis as the main inhibitor of the coagulation cascade. It plays also critical roles in other processes, such as inflammation and cancer. Here we show that exosomes released by Madin-Darby canine kidney (MDCK) cells cultured in the presence of heparin incorporate antithrombin from the serum. Exosomal antithrombin is found complexed with the serine protease high temperature requirement A1 (HTRA1), whose cellular levels are increased after serum deprival, the condition used to collect exosomes. Although the biological relevance of the presence of antithrombin in exosomes remains to be investigated, our results suggest a functional interplay between antithrombin and HTRA1.

Differential miRNA expression profile and proteome in plasma exosomes from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disease of blood cells caused by the lack of glycosyl phosphatidyl inositol anchored proteins bound to the cell membrane. In consequence, erythrocytes lead to intravascular hemolysis upon complement activation, which promotes high risk of thrombosis, intravascular hemolytic anemia, and bone marrow failure in patients. The mechanisms of thrombosis in PNH are still poorly understood. Treatment with eculizumab reduces intravascular hemolysis and thrombotic risk, but not in all cases. Exosomes are extracellular vesicles released by cells and whose secretion is closely related to the inflammatory status. They participate in cell communication by activating signaling pathways and transferring genetic material and proteins to host cells. In consequence, exosomes may serve as surrogate biomarkers for the prognosis and/or diagnosis of a disease. Isolation of exosomes was carried out from healthy controls and from three groups of PNH patients, i.e. i) with no eculizumab treatment; ii) under treatment with eculizumab that have not suffered thrombosis; and iii) under treatment with eculizumab but that have suffered thrombosis. The miRNAome and proteome was analyzed using plasma focus miRNAs PCR panel and LC-MS analysis respectively. We found differential expression of miRNAs miR-148b-3p, miR-423-3p, miR29b-3p, miR15b-5p, let-7e-5p, miR126-3p, miR-125b-5p and miR-376c-3p as well as hemoglobin, haptoglobin, protein S and C4-binding protein in healthy controls vs PNH patients. Our results warrant further research and provide new information on the content of exosomes that could play a role in the hypercoagulable state in this disease.

Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site.

Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin.

Antithrombin controls tumor migration, invasion and angiogenesis by inhibition of enteropeptidase.

Antithrombin is a key inhibitor of the coagulation cascade, but it may also function as an anti-inflammatory, anti-angiogenic, anti-viral and anti-apoptotic protein. Here, we report a novel function of antithrombin as a modulator of tumor cell migration and invasion. Antithrombin inhibited enteropeptidase on the membrane surface of HT-29, A549 and U-87 MG cells. The inhibitory process required the activation of antithrombin by heparin, and the reactive center loop and the heparin binding domain were essential. Surprisingly, antithrombin non-covalently inhibited enteropeptidase, revealing a novel mechanism of inhibition for this serpin. Moreover, as a consequence of this inhibition, antithrombin was cleaved, resulting in a molecule with anti-angiogenic properties that reduced vessel-like formation of endothelial cells. The addition of antithrombin and heparin to U-87 MG and A549 cells reduced motility in wound healing assays, inhibited the invasion in transwell assays and the degradation of a gelatin matrix mediated by invadopodia. These processes were controlled by enteropeptidase, as demonstrated by RNA interference experiments. Carcinoma cell xenografts in nude mice showed in vivo co-localization of enteropeptidase and antithrombin. Finally, treatment with heparin reduced experimental metastasis induced by HT29 cells in vivo. In conclusion, the inhibition of enteropeptidase by antithrombin may have a double anti-tumor effect through inhibiting a protease involved in metastasis and generating an anti-angiogenic molecule.