Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1−/− mice, but not Epac2−/− mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1−/− mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1−/− mouse platelets (thrombocytes) had unbalanced expression of key components of the glycoprotein Ib-IX-V (GPIb-IX-V) complex, with decrease of GP1bβ and no change of GP1bα. This complex is critical for platelet adhesion under arterial shear conditions. Furthermore, Epac1−/− mice have reduced levels of plasma coagulation factors and fibrinogen, increased size of circulating platelets, increased megakaryocytes (the GP1bβ level was decreased also in Epac1−/− bone marrow) and higher abundance of reticulated platelets. Viscoelastic measurement of clotting function revealed Epac1−/− mice with a dysfunction in the clotting process, which corresponds to reduced plasma levels of coagulation factors like factor XIII and fibrinogen. We propose that the observed platelet phenotype is due to deficient Epac1 activity during megakaryopoiesis and thrombopoiesis, and that the defects in blood clotting for Epac1−/− is connected to secondary hemostasis.

Cyanobacteria are an inspiring source of bioactive secondary metabolites. These bioactive agents are a diverse group of compounds which are varying in their bioactive targets, the mechanisms of action, and chemical structures. Cyanobacteria from various environments, especially marine benthic cyanobacteria, are found to be rich sources for the search for novel bioactive compounds. Several compounds with anticancer activities have been discovered from cyanobacteria and some of these have succeeded to enter the clinical trials. Varying anticancer agents are needed to overcome increasing challenges in cancer treatments. Different search methods are used to reveal anticancer compounds from natural products, but cell based methods are the most common. Cyanobacterial bioactive compounds as agents against acute myeloid leukemia are not well studied. Here we examined our new results combined with previous studies of anti-leukemic compounds from cyanobacteria with emphasis to reveal common features in strains producing such activity. We report that cyanobacteria harbor specific anti-leukemic compounds since several studied strains induced apoptosis against AML cells but were inactive against non-malignant cells like hepatocytes. We noted that particularly benthic strains from the Baltic Sea, such as Anabaena sp., were especially potential AML apoptosis inducers. Taken together, this review and re-analysis of data demonstrates the power of maintaining large culture collections for the search for novel bioactivities, and also how anti-AML activity in cyanobacteria can be revealed by relatively simple and low-cost assays.

Aim The cAMP‐mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1−/− mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. Methods In Epac1−/− and Wt C57BL/6J mice, renal papillae, dissected from snap‐frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.‐injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time‐spaced intravenously injected 125I‐ and 131I‐labelled albumin. Results Epac1−/− mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1−/− mice had no increased protein diet‐induced urea‐dependent osmotic diuresis, and expressed Wt levels of aquaporin‐2 (AQP‐2) and urea transporter A1/3 (UT‐A1/3). Epac1−/− mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. Conclusion Our results conform to the hypothesis that Epac1‐dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

More than 40 years ago, the present standard induction therapy for acute myeloid leukemia (AML) was developed. This consists of the metabolic inhibitor cytarabine (AraC) and the cytostatic topoisomerase 2 inhibitor daunorubucin (DNR). In light of the high chance for relapse, as well as the large heterogeneity, novel therapies are needed to improve patient outcome. We have tested the anti-AML activity of 15 novel compounds based on the scaffolds pyrrolo [2,3-a]carbazole-3-carbaldehyde, pyrazolo[3,4-c]carbazole, pyrazolo[4,3-a]phenanthridine, or pyrrolo[2,3-g]indazole. The compounds were inhibitors of Pim kinases, but could also have inhibitory activity against other protein kinases. Ser/Thr kinases like the Pim kinases have been identified as potential drug targets for AML therapy. The compound VS-II-173 induced AML cell death with EC 50 below 5 mmol/L, and was 10 times less potent against nonmalignant cells. It perturbed Pim-kinase-mediated AML cell signaling, such as attenuation of Stat5 or MDM2 phosphorylation, and synergized with DNR to induce AML cell death. VS-II-173 induced cell death also in patients with AML blasts, including blast carrying high-risk FLT3-ITD mutations. Mutation of nucleophosmin-1 was associated with good response to VS-II-173. In conclusion new scaffolds for potential AML drugs have been explored. The selective activity toward patient AML blasts and AML cell lines of the pyrazolo-analogue VS-II-173 make it a promising drug candidate to be further tested in preclinical animal models for AML.

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